Note: Descriptions are shown in the official language in which they were submitted.
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MAMMALIAN GENES; RELATED REAGENTS AND METHODS
This filing is a U.S. utility Patent Application claiming
priority to USSN 09/439,735, filed November 15, 1999, which is
incorporated herein by reference.
FIELD OF THE INVENTION
The present invention pertains to compositions related to
proteins which function in controlling biology and physiology
of mammalian cells, e.g., cells of a mammalian immune system.
In particular, it provides purified genes, proteins,
antibodies, and related reagents useful, e.g., to regulate
activation, development, differentiation, and function of
various cell types, including hematopoietic cells.
BACKGROUND OF THE INVENTION
Recombinant DNA technology refers generally to the
technique of integrating genetic information from a donor
source into vectors for subsequent processing, such as through
introduction into a host, whereby the transferred genetic
information is copied and/or expressed in the new environment.
Commonly, the genetic information exists in the form of
complementary DNA (cDNA) derived from messenger RNA (mRNA)
coding for a desired protein product. The carrier is
frequently a plasmid having the capacity to incorporate cDNA
for later replication in a host and, in some cases, actually to
control expression of the cDNA and thereby direct synthesis of
the encoded product in the host.
Cell lineages especially important to the immune response
include two classes of lymphocytes: B-cells, which can produce
and secrete immunoglobulins (proteins with the capability of
recognizing and binding to foreign matter to effect its
removal), and T-cells of various subsets that secrete
lymphokines and induce or suppress the B-cells and various
other cells (including other T-cells) making up the immune
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network. These lymphocytes interact with many other cell
types.
Dendritic cells (DC) are the professional antigen
presenting cells (APC) of the immune system. They have the
unique capability to activate naive T lymphocytes and as such
play an important role in the induction of immune responses.
Multiple studies have indicated that DC are superior in priming
naive T cells when compared to other APC such as B cells and
macrophages. Steinman (1991) Ann. Rev. Immunol. 9:271-296;
Hart (1997) Blood 90:3245-3287; and Levin, et al. (1993) J.
Immunol. 151:6742-6750. Recent experiments suggest that
distinct DC subsets can be recognized that have strikingly
different influences on the type of immune response generated
in vivo. See, e.g., Rissoan, et al. (1999) Science 283, 1183-
1186; Pulendran, et al. (1999) Proc. Natl. Acad. Sci. USA
96:1036-1041; Maldonado-Lopez, et al. (1999) J. Exp. Med.
189:587-592; and Smith and Fazekas de St.Groth (1999) J. Ex~,.
Med. 189:593-598.
DC are bone marrow-derived cells, that in their immature
stage are scattered throughout the body and are particularly
efficient in antigen uptake through a variety of cell surface
receptors specialized in capturing antigens. See Sallusto, et
al. (1995) J. Exp. Med. 182:389-400. Upon inflammation, DC
migrate via lymph or blood to the secondary lymphoid organs.
This migration process seems to be regulated by a coordinated
up- and downregulation of chemokine receptors. See Sallusto,
et al. (1998) Eur. J. Immunol. 28:2760-2769; and Sozzani, et
al. (1998) J. Immunol. 161:1083-1086.
Upon arrival in the T cells areas, DC are mature and fully
stimulatory, well-equipped to attract and interact with naive T
cells. These DC express high levels of MHC class I, MHC class
II, adhesion molecules and co-stimulatory molecules, that
supports the induction of primary T cell responses.
Subsequently, CD40 ligation on the DC after interaction with
CD4+ T helper cells leads to maximal activation of the mature
DC, further increasing their capacity to stimulate naive
cytotoxic T lymphocytes. See Caux, et al. (1994) J. Exp. Med.
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180:1263-1272; Cella, et al. (1996) J. Exp. Med. 184:747-752;
Schoenberger, et al. (1998) Nature 393:480-483; Ridge, et al.
(1998) Nature 393:474-478; and Bennett, et al. (1998) Nature
393:478-480.
Besides T cells, NK cells, and macrophages, another
important cell lineage is the mast cell (which has not been
positively identified in all mammalian species), which is a
granule-containing connective tissue cell located proximal to
capillaries throughout the body. These cells are found in
especially high concentrations in the lungs, skin, and
gastrointestinal and genitourinary tracts. Mast cells play a
central role in allergy-related disorders, particularly
anaphylaxis as follows: when selected antigens crosslink one
class of immunoglobulins bound to receptors on the mast cell
surface, the mast cell degranulates and releases mediators,
e.g., histamine, serotonin, heparin, and prostaglandins, which
cause allergic reactions, e.g., anaphylaxis.
Although the role of DC in a wide variety of immunological
processes has been demonstrated, the molecular mechanisms that
regulate DC differentiation, migration and maturation are still
poorly understood. Research to better understand and treat
various immune disorders has been hampered by the general
inability to maintain cells of the immune system in vitro.
Immunologists have discovered that culturing these cells can be
accomplished through the use of T-cell and other cell
supernatants, which contain various growth factors, including
many of the lymphokines.
From the foregoing, it is evident that the discovery and
development of new surface antigens could contribute to new
therapies for a wide range of degenerative or abnormal
conditions which directly or indirectly involve the immune
system and/or hematopoietic cells. In particular, the
discovery and development of lymphokines which enhance or
potentiate the beneficial activities of known lymphokines would
be highly advantageous. The present invention provides new
compositions and related compounds, and methods for their use.
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SUMMARY OF THE INVENTION
The present invention is directed to mammalian, e.g.,
rodent, canine, feline, primate, proteins designated Dendritic
Cell Specific Transmembrane Protein (DC-STAMP) and DNAX Surface
Protein (DSP-1) and their biological activities. It includes
nucleic acids coding for polypeptides themselves and methods
for their production and use. The nucleic acids of the
invention are characterized, in part, by their homology to
complementary DNA (cDNA) sequences disclosed herein, and/or by
functional assays applied to the polypeptides, which are
typically encoded by these nucleic acids. Methods for
modulating or intervening in the control of surface protein
dependent physiology or an immune response are provided.
The present invention is based, in part, upon the
discovery of novel surface proteins from dendritic cells or
mast cells. In particular, it provides primate, e.g., human,
sequences. Functional equivalents exhibiting significant
sequence homology will be available from other mammalian, e.g.,
cow, horse, rat, mouse, and non-mammalian species, e.g., warm
blooded animals, including birds.
In various protein embodiments, the invention provides: a
substantially pure or recombinant DC-STAMP or DSP-1 polypeptide
exhibiting identity over a length of at least about 12 amino
acids to SEQ ID NO: 2 or 5 or 7; a natural sequence DC-STAMP of
SEQ ID NO: 2; a natural sequence DSP-1 of SEQ ID NO: 5 or 7;
and a fusion protein comprising DC-STAMP or DSP-1 sequence. In
certain embodiments, the segment of identity is at least about
14, 17, or 19 amino acids. In other embodiments, the DC-STAMP
or DSP-1: comprises a mature sequence comprising the sequence
from Tables 1 or 2; or exhibits a post-translational
modification pattern distinct from natural DC-STAMP or DSP-1;
or the polypeptide: is from a warm blooded animal selected from
a mammal, including a primate; comprises at least one
polypeptide segment of SEQ ID NO: 2 or 5 or 7; exhibits a
plurality of fragments; is a natural allelic variant of DC-
STAMP or DSP-1; has a length at least about 30 amino acids;
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exhibits at least two non-overlapping epitopes which are
specific for a primate DC-STAMP or DSP-1; exhibits sequence
identity over a length of at least about 20 amino acids to
primate DC-STAMP or DSP-1; is glycosylated; has a molecular
5 weight of at least 30 kD with natural glycosylation; is a
synthetic polypeptide; is attached to a solid substrate; is
conjugated to another chemical moiety; is a 5-fold or less
substitution from natural sequence; or is a deletion or
insertion variant from a natural sequence. Preferred
embodiments include a composition comprising: a sterile DC-
STAMP or DSP-1 polypeptide; or the DC-STAMP or DSP-1
polypeptide and a carrier, wherein the carrier is: an aqueous
compound, including water, saline, and/or buffer; and/or
formulated for oral, rectal, nasal, topical, or parenteral
administration. In fusion protein embodiments, the protein can
have: mature polypeptide sequence from Tables 1 or 2; a
detection or purification tag, including a FLAG, His6, or Ig
sequence; and/or sequence of another cytokine or chemokine,
including Flt3 ligand.
Kit embodiments include those with a DC-STAMP or DSP-1
polypeptide, and: a compartment comprising the polypeptide;
and/or instructions for use or disposal of reagents in the kit.
In binding compound embodiments, the compound may have an
antigen binding site from an antibody, which specifically binds
to a natural DC-STAMP or DSP-1 polypeptide, wherein: the DC-
STAMP or DSP-1 is a primate protein; the binding compound is an
Fv, Fab, or Fab2 fragment; the binding compound is conjugated
to another chemical moiety; or the antibody: is raised against
a peptide sequence of a mature polypeptide portion from Tables
1 or 2; is raised against a mature DC-STAMP or DSP-1; is raised
to a purified primate DC-STAMP or DSP-1; is immunoselected; is
a polyclonal antibody; binds to a denatured DC-STAMP or DSP-1;
exhibits a Kd of at least 30 ~M; is attached to a solid
substrate, including a bead or plastic membrane; is in a
sterile composition; or is detectably labeled, including a
radioactive or fluorescent label. Kits containing binding
compounds include those with: a compartment comprising the
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binding compound; and/or instructions for use or disposal of
reagents in the kit. Often the kit is capable of making a
qualitative or quantitative analysis. Preferred compositions
will comprise: a sterile binding compound; or the binding
compound and a carrier, wherein the carrier is: an aqueous
compound, including water, saline, and/or buffer; and/or
formulated for oral, rectal, nasal, topical, or parenteral
administration.
Nucleic acid embodiments include an isolated or
LO recombinant nucleic acid encoding a DC-STAMP or DSP-1
polypeptide or fusion protein, wherein: the DC-STAMP or DSP-1
is from a primate; and/or the nucleic acid: encodes an
antigenic peptide sequence of Tables 1 or 2; encodes a
plurality of antigenic peptide sequences of Tables 1 or 2;
exhibits identity to a natural cDNA encoding the segment; is an
expression vector; further comprises an origin of replication;
is from a natural source; comprises a detectable label;
comprises synthetic nucleotide sequence; is less than 6 kb,
preferably less than 3 kb; is from a primate, including a
human; comprises a natural full length coding sequence; is a
hybridization probe for a gene encoding the DC-STAMP or DSP-1;
or is a PCR primer, PCR product, or mutagenesis primer. The
invention also provides a cell, tissue, or organ comprising
such a recombinant nucleic acid, and preferably the cell will
be: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a
yeast cell; an insect cell; a mammalian cell; a mouse cell; a
primate cell; or a human cell.
Kit embodiments include those with such nucleic acids,
and: a compartment comprising the nucleic acid; a compartment
further comprising the DC-STAMP or DSP-1 protein or
polypeptide; and/or instructions for use or disposal of
reagents in the kit. Typically, the kit is capable of making a
qualitative or quantitative analysis.
In certain embodiments, the nucleic acid: hybridizes under
wash conditions of 30° C and less than 2M salt, or of 45° C
and/or 500 mM salt, or 55° C and/or 150 mM salt, to SEQ ID NO:
1 or 4 or 6; or exhibits identity over a stretch of at least
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about 30, 55, or 75 nucleotides, to a primate DC-STAMP or DSP-
1.
The invention embraces a method of modulating physiology
or development of a cell or tissue culture cells comprising
contacting the cell with an agonist or antagonist of a primate
DC-STAMP or DSP-1. The method may be where: the contacting is
in combination with an agonist or antagonist of Flt3 ligand; or
the contacting is with an antagonist, including a binding
composition comprising an antibody binding site which
specifically binds a DC-STAMP or DSP-1.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
All references cited herein are incorporated herein by
reference to the same extent as if each individual publication
or patent application was specifically and individually
indicated to be incorporated by reference.
OUTLINE
I. General
LO II. Purified DC-STAMP or DSP-1
A. physical properties
B. biological properties
III. Physical Variants
A. sequence variants, fragments
B. post-translational variants
1. glycosylation
2. others
IV. Functional Variants
A. analogs, fragments
1. agonists
2. antagonists
B. mimetics
1. protein
2. chemicals
C. species variants
V. Antibodies
A. polyclonal
B. monoclonal
C. fragments, binding compositions
VI. Nucleic Acids
A. natural isolates; methods
B. synthetic genes
C. methods to isolate
VII. Making DC-STAMP or DSP-1, mimetics
A. recombinant methods
B. synthetic methods
C. natural purification
VIII. Uses
A. diagnostic
B. therapeutic
IX. Kits
A. nucleic acid reagents
B. protein reagents
C. antibody reagents
X. Isolating receptors for DC-STAMP or DSP-1
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I. General
The present invention provides amino acid sequences and
DNA sequences encoding various mammalian proteins which are
membrane proteins, e.g., which are surface molecules which may
mediate a signal between immune or other cells. See, e.g.,
Paul (1997) Fundamental Immunoloay (3d ed.) Raven Press, N.Y.
The proteins, and fragments, or antagonists will be useful in
physiological modulation of cells expressing a receptor or
binding partner. It is likely that DC-STAMP or DSP-1 has
LO either stimulatory or inhibitory effects on hematopoietic
cells, including, e.g., lymphoid cells, such as T-cells, B-
cells, natural killer (NK) cells, macrophages, dendritic cells,
hematopoietic progenitors, mast cells, etc. The proteins will
also be useful as antigens, e.g., immunogens, for raising
antibodies to various epitopes on the protein, both linear and
conformational epitopes.
Various cDNAs encoding DC-STAMP or DSP-1 were identified.
The DC-STAMP was identified from cDNA libraries prepared form
human monocyte-derived dendritic cells. The DSP-1 was
identified from a cDNA library derived from a human HEL cell
line.
Along with B lymphocytes and mononuclear phagocytes,
dendritic cells (DC) are the professional antigen presenting
cells (APC). DC are unique in their ability to present antigen
to naive T cells, and play therefore a central role in the
initiation of immune responses. Characterization of DC
specific genes may help to unravel the mechanism underlying
their potent antigen presenting capacity. Here is described
the identification of a novel transcript, isolated by random
sequencing of clones from a cDNA library prepared from
monocyte-derived DC. A 2.3 kb messenger RNA is specifically
expressed by DC, and not in a panel of other leukocytes or non-
hematopoietic cells. In addition, no expression was detected
in tissue of several human organs. The transcript encodes an
approximately 470 amino acid protein, which is comprised of 7
putative transmembrane domains. This novel protein has been
designated Dendritic Cell Specific Transmembrane Protein (DC-
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STAMP). Expression of a DC-STAMP-GFP fusion protein in 293
cells indicates that DC-STAMP is expressed at the cell surface.
No sequence homology was found with another protein or
multimembrane spanning receptor. DC-STAMP appears to be a
5 novel DC-specific multimembrane spanning protein, representing
a new group of transmembrane proteins.
To characterize DC at the molecular level, cDNA libraries
were prepared from human monocyte-derived dendritic cells (DC)
and over 250 cDNA clones were characterized by nucleotide
LO sequence analysis. See Marland, et al. (1997) in Ricciardi-
Castognoli (ed.) Dendritic Cells in Fundamental and Clinical
Immunologv, Vol 3, Plenum Publ. Corp. One of these cDNA clones
was analyzed in further detail as it contained a unique
sequence not present in the GenBank databases and its partial
open reading frame (ORF) appeared to encode a putative
transmembrane (TM) region. To determine the expression pattern
of this novel messenger RNA, Northern blot analysis was
performed using RNA from non-stimulated DC as well as RNA from
a panel of freshly isolated leukocyte populations and several
T, B, and monocytic cell lines. A message of 2.3 kb was
specifically detected in DC but not in any of the other cell
populations tested. Therefore, this novel protein was
designated DC-STAMP (DC-Specific Transmembrane Protein). The
finding that this RNA is enriched in the poly A+ RNA fraction
from DC indicates that the mRNA encoding DC-STAMP is
polyadenylated.
The human DC-STAMP gene will encode a membrane protein, of
about 470 amino acids. See Table 1 and SEQ. ID. NO: 1 and 2.
DC-STAMP exhibits structural motifs characteristic of a member
of multiple membrane spanning proteins, e.g., 7 transmembrane
receptors. Other notable motifs or features include asn168-
thr170, asn187-ser188, and asn357-ser359 (three predicted N-
linked glycosylation sites); and thr286-1ys288 (potential site
for phosphorylation by PKC); and 1ys426-ser429 and arg438-
ser441 (potential sites for cAMP-dependent protein kinases).
The human DSP-1 appears to exist in two forms. One form,
designated the long form, encodes a membrane protein of about
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313 amino acids, and the other, designated the short form,
encodes a membrane protein of about 200 amino acids. The short
form seems to result from deletion of nucleotides 94-433 of the
long form, and the corresponding amino acids of the protein.
Both forms seem to encode type I membrane proteins, with the
transmembrane segment corresponding to long form residues about
1eu172-g1y188. Other notable motifs or features include three
ITIM motifs, corresponding to long form residues 1eu222-1eu227,
va1244-va1251, and 1eu258-va1263. See, e.g., Thomas (1995) J.
LO Exp. Med. 181:1953-xx; and Lanier (1997) Immunity 6:371.
Classically, the Immunoreceptor Tyrosine-based Inhibitory
Motifs (ITIM) recruit intracellular tyrosine phosphatases, and
the receptors provide an inhibitory signal to the cell. This
suggests that the DSP-1 antigen is involved in a negative
regulatory signaling pathway in the expressing cells, e.g.,
monocytes, T, NK, and/or mast cells. Thus, the binding
partner, probably a surface receptor or soluble ligand, might
inhibit monocyte, T, NK, and/or mast cell degranulation,
chemotaxis, or signaling.
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Table 1: Primate, e.g., human, DC-STAMP (SEQ ID NO: 1 and 2):
ggggggtggc atttctgcat tcgaagaaga atctgagaga aacctgacgc agggagc 57
atg ggt atc tgg acc tca ggc act gat atc ttc cta agt ctt tgg gag 105
Met Gly Ile Trp Thr Ser Gly Thr Asp Ile Phe Leu Ser Leu Trp Glu
1 5 10 15
att tac gtg tct cca aga agc ccc gga tgg atg gac ttt atc cag cat 153
LO Ile Tyr Val Ser Pro Arg Ser Pro Gly Trp Met Asp Phe Ile Gln His
20 25 30
ttg gga gtt tgc tgt ttg gtt get ctt att tca gtg ggc ctc ctg tct 201
Leu Gly Val Cys Cys Leu Val Ala Leu Ile Ser Val Gly Leu Leu Ser
35 40 45
gtg gcc gcc tgc tgg ttt ctg cca tca atc ata gcg gcc get gcc tcc 249
Val Ala Ala Cys Trp Phe Leu Pro Ser Ile Ile Ala Ala Ala Ala.Ser
50 55 60
tgg att atc acg tgt gtt ctg ctg tgt tgc tcc aag cat gca cga tgt 297
Trp Ile Ile Thr Cys Val Leu Leu Cys Cys Ser Lys His Ala Arg Cys
65 70 75 80
ttt att ctt ctt gtc ttt ctc tct tgt ggc ctg cgt gaa ggc agg aat 345
Phe Ile Leu Leu Val Phe Leu Ser Cys Gly Leu Arg Glu Gly Arg Asn
85 90 95
get ttg att gca get ggc aca ggg atc gtc atc ttg gga cac gta gaa 393
3 0 Ala Leu Ile Ala Ala Gly Thr Gly Ile Val Ile Leu Gly His Val Glu
100 105 110
aat att ttt cac aac ttt aaa ggt ctc cta gat ggt atg act tgc aac 441
Asn Ile Phe His Asn Phe Lys Gly Leu Leu Asp Gly Met Thr Cys Asn
115 120 125
cta agg gca aag agc ttt tcc ata cat ttt cca ctt ttg aaa aaa tat 489
Leu Arg Ala Lys Ser Phe Ser Ile His Phe Pro Leu Leu Lys Lys Tyr
130 135 140
att gag gca att cag tgg att tat ggc ctt gcc act cca cta agt gta 537
Ile Glu Ala Ile Gln Trp Ile Tyr Gly Leu Ala Thr Pro Leu Ser Val
145 150 155 160
4 5 ttt gat gac ctt gtt tct tgg aac cag acc ctg gca gtc tct ctt ttc 585
Phe Asp Asp Leu Val Ser Trp Asn Gln Thr Leu Ala Val Ser Leu Phe
165 170 175
agt ccc agc cat gtc ctg gag gca cag cta aat gac agc aaa ggg gaa 633
Ser Pro Ser His Val Leu Glu Ala Gln Leu Asn Asp Ser Lys Gly Glu
180 185 190
gtc ctg agc gtc ttg tac cag atg gca aca acc aca gag gtg ttg tcc 681
Val Leu Ser Val Leu Tyr Gln Met Ala Thr Thr Thr Glu Val Leu Ser
195 200 205
tcc ctg ggt cag aag cta ctt gcc ttt gca ggg ctt tcg ctc gtc ctg 729
Ser Leu Gly Gln Lys Leu Leu Ala Phe Ala Gly Leu Ser Leu Val Leu
210 215 220
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ctt ggc act ggc ctc ttc atg aag cga ttt ttg ggc cct tgt ggt tgg 777
Leu Gly Thr Gly Leu Phe Met Lys Arg Phe Leu Gly Pro Cys Gly Trp
225 230 235 240
aag tat gaa aac atc tac atc acc aga caa ttt gtt cag ttt gat gaa 825
Lys Tyr Glu Asn Ile Tyr Ile Thr Arg Gln Phe Val Gln Phe Asp Glu
245 250 255
LO agg gag aga cat caa cag agg ccc tgt gtg ctc ccg ctg aat aag gag 873
Arg Glu Arg His Gln Gln Arg Pro Cys Val Leu Pro Leu Asn Lys Glu
260 265 270
gaa agg agg aag tat gtc atc atc ccg act ttc tgg ccg act cct aaa 921
L5 Glu Arg Arg Lys Tyr Val Ile Ile Pro Thr Phe Trp Pro Thr Pro Lys
275 280 285
gaa agg aaa aac ctg ggg ctg ttt ttc ctc ccc ata ctt atc cat ctc 969
Glu Arg Lys Asn Leu Gly Leu Phe Phe Leu Pro Ile Leu Ile His Leu
20 290 295 300
tgc atc tgg gtg ctg ttt gca get gta gat tat ctg ctg tat cgg ctc 1017
Cys Ile Trp Val Leu Phe Ala Ala Val Asp Tyr Leu Leu Tyr Arg Leu
305 310 315 320
att ttc tca gtg agc aag cag ttt caa agc ttg cca ggg ttt gag gtt 1065
Ile Phe Ser Val Ser Lys Gln Phe Gln Ser Leu Pro Gly Phe Glu Val
325 330 335
3 cac ttgaaactg cacggagag aaacaagga actcaagat attatc cat 1113
0
His LeuLysLeu HisGlyGlu LysGlnGly ThrGlnAsp IleIle His
340 345 350
gat tcttccttt aatatatct gtgtttgaa cccaactgt atccca aaa 1161
3 Asp SerSerPhe AsnIleSer ValPheGlu ProAsnCys IlePro Lys
5
355 360 365
cca aaattcctt ctatctgag acctgggtt cctctcagt gttatt ctt 1209
Pro LysPheLeu LeuSerGlu ThrTrpVal ProLeuSer ValIle Leu
40 370 375 380
ttg atattagtg atgctggga ctgttgtcc tctatcctt atgcaa ctt 1257
Leu IleLeuVal MetLeuGly LeuLeuSer SerIleLeu MetGln Leu
385 390 395 400
45
aaa atcctggtg tcagcatct ttctacccc agcgtggag aggaag cgc 1305
Lys IleLeuVal SerAlaSer PheTyrPro SerValGlu ArgLys Arg
405 410 415
5 atc caatatctg catgcaaag ctgcttaaa aaaagatca aagcag ccg 1353
0
Ile GlnTyrLeu HisAlaLys LeuLeuLys LysArgSer LysGln Pro
420 425 430
ctg ggagaagtc aaaagacgg ctgagtctc tatcttaca aagatt cat 1401
55 Leu GlyGluVal LysArgArg LeuSerLeu TyrLeuThr LysIle His
435 440 445
ttc tgg ctt cca gtc ctg aaa atg att agg aag aag caa atg gac atg 1449
Phe Trp Leu Pro Val Leu Lys Met Ile Arg Lys Lys Gln Met Asp Met
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450 455 460
gca agt gca gac aag tca tgagagaccc cgactactcc tcagccacat 1497
Ala Ser Ala Asp Lys Ser
465 470
LO
cgcaccaaca attctcttca ggtctaggat ggcagtcact attcatgccg gataatagag 1557
ttatgcctcc tttcatctca aagccaaaga gctgccaggt aaatggttat gtggtctatg 1677
aactatgtga cgcagtcctc tcaggagtct gagtttacag agccaacttg cagcacctgg 1617
ttccaaacaa accacatgat cttgcctgtg tcacaatgta acaagactct agctgggtcc 1737
L5 cctggtgatg agtttcagca tagaataatg ttcaaggaaa agaaaacgaa aacagtttaa 1797
atctctacca cagcctcaca agcaaatgct aaggggaaca tacatgtaaa aagccagcaa 1857
actatcttca aactcttccg tccttaatgt cttccatggc tattgccccc acaatggtct 1917
cttttctccc tgctccctta ttaaagaact ctttctgaaa ccc 1960
MGIWTSGTDIFLSLWEIYVSPRSPGWMDFIQHLGVCCLVALISVGLLSVAACWFLPSIIAAAASWIITCVLLCC
SKHARCFILLVFLSCGLREGRNALIAAGTGIVILGHVENIFHNFKGLLDGMTCNLRAKSFSIHFPLLKKYIEAI
QWIYGLATPLSVFDDLVSWNQTLAVSLFSPSHVLEAQLNDSKGEVLSVLYQMATTTEVLSSLGQKLLAFAGLSL
VLLGTGLFMKRFLGPCGWKYENIYITRQFVQFDERERHQQRPCVLPLNKEERRKYVIIPTFWPTPKERKNLGLF
FLPILIHLCIWVLFAAVDYLLYRLIFSVSKQFQSLPGFEVHLKLHGEKQGTQDIIHDSSFNISVFEPNCIPKPK
FLLSETWVPLSVILLILVMLGLLSSILMQLKILVSASFYPSVERKRIQYLHAKLLKKRSKQPLGEVKRRLSLYL
TKIHFWLPVLKMIRKKQMDMASADKS
Reverse translation, e.g., nucleic acids encoding polypeptides
(N can be A, C, G, or T; SEQ ID NO: 3):
3 5 ATGGGNATHTGGACNWSNGGNACNGAYATHTTYYTNWSNYTNTGGGARATHTAYGTNWSNCCNMGNWSNCCNGG
NTGGATGGAYTTYATHCARCAYYTNGGNGTNTGYTGYYTNGTNGCNYTNATHWSNGTNGGNYTNYTNWSNGTNG
CNGCNTGYTGGTTYYTNCCNWSNATHATHGCNGCNGCNGCNWSNTGGATHATHACNTGYGTNYTNYTNTGYTGY
WSNAARCAYGCNMGNTGYTTYATHYTNYTNGTNTTYYTNWSNTGYGGNYTNMGNGARGGNMGNAAYGCNYTNAT
HGCNGCNGGNACNGGNATHGTNATHYTNGGNCAYGTNGAR.AAYATHTTYCAYAAYTTYAARGGNYTNYTNGAYG
4 O GNATGACNTGYAAYYTNMGNGCNAARWSNTTYWSNATHCAYTTYCCNYTNYTNAARAARTAYATHGARGCNATH
CARTGGATHTAYGGNYTNGCNACNCCNYTNWSNGTNTTYGAYGAYYTNGTNWSNTGGAAYCARACNYTNGCNGT
NWSNYTNTTYWSNCCNWSNCAYGTNYTNGARGCNCARYTNAAYGAYWSNAARGGNGARGTNYTNWSNGTNYTNT
AYCARATGGCNACNACNACNGARGTNYTNWSNWSNYTNGGNCARAARYTNYTNGCNTTYGCNGGNYTNWSNYTN
GTNYTNYTNGGNACNGGNYTNTTYATGAARMGNTTYYTNGGNCCNTGYGGNTGGAARTAYGARAAYATHTAYAT
4 5 HACNMGNCARTTYGTNCARTTYGAYGARMGNGARMGNCAYCARCARMGNCCNTGYGTNYTNCCNYTNAAYAARG
ARGARMGNMGNAARTAYGTNATHATHCCNACNTTYTGGCCNACNCCNAARGARMGNAAR.AAYYTNGGNYTNTTY
TTYYTNCCNATHYTNATHCAYYTNTGYATHTGGGTNYTNTTYGCNGCNGTNGAYTAYYTNYTNTAYMGNYTNAT
HTTYWSNGTNWSNAARCARTTYCARWSNYTNCCNGGNTTYGARGTNCAYYTNAARYTNCAYGGNGARAARCARG
GNACNCARGAYATHATHCAYGAYWSNWSNTTYAAYATHWSNGTNTTYGARCCNAAYTGYATHCCNAARCCNAAR
50 TTYYTNYTNWSNGARACNTGGGTNCCNYTNWSNGTNATHYTNYTNATHYTNGTNATGYTNGGNYTNYTNWSNWS
NATHYTNATGCARYTNAARATHYTNGTNWSNGCNWSNTTYTAYCCNWSNGTNGARMGNAARMGNATHCARTAYY
TNCAYGCNAARYTNYTNAARAARMGNWSNAARCARCCNYTNGGNGARGTNAARMGNMGNYTNWSNYTNTAYYTN
ACNAARATHCAYTTYTGGYTNCCNGTNYTNAARATGATHMGNAARAARCARATGGAYATGGCNWSNGCNGAYAA
RWSN
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Table 2: Primate, e.g., human, DSP-1 forms, both long and short forms:
DSP-1L (SEQID and5):
NO:
4
5 atg gcctta ccagtg accgccttg ctcctgccg ctagccttg ctgctc 48
Met AlaLeu ProVal ThrAlaLeu LeuLeuPro LeuAlaLeu LeuLeu
-20 -15 -10
cac gccgcc aggccg gattacaag gacgatgac gacaagatc gatctg 96
LO His AlaAla ArgPro AspTyrLys AspAspAsp AspLysIle AspLeu
-5 -1 1 5 10
agc aaatgc aggacc gtggcgggc cccgtgggg ggatccctg agtgtg 144
Ser LysCys ArgThr ValAlaGly ProValGly GlySerLeu SerVal
15 15 20 25
cag tgtccc tatgag aaggaacac aggaccctc aacaaatac tggtgc 192
Gln CysPro TyrGlu LysGluHis ArgThrLeu AsnLysTyr TrpCys
30 35 40
aga ccaccacag attttc ctatgtgacaag attgtg gagaccaaa ggg 240
Arg ProProGln IlePhe LeuCysAspLys IleVal GluThrLys Gly
45 50 55
2 tca gcaggaaaa aggaac ggccgagtgtcc atcagg gacagtcct gca 288
5
Ser AlaGlyLys ArgAsn GlyArgValSer IleArg AspSerPro Ala
60 65 70 75
aac ctcagcttc acagtg accctggagaat ctcaca gaggaggat gca 336
3 Asn LeuSerPhe ThrVal ThrLeuGluAsn LeuThr GluGluAsp Ala
0
80 85 90
ggc acctactgg tgtggg gtggatacaccg tggctc cgagacttt cat 384
Gly ThrTyrTrp CysGly ValAspThrPro TrpLeu ArgAspPhe His
35 95 100 105
gat cccgttgtc gaggtt gaggtgtccgtg ttcccg gcatcaacg tca 432
Asp ProValVal GluVal GluValSerVal PhePro AlaSerThr Ser
110 115 120
40
atg acacctgca agtatc actgcggccaag acctca acaatcaca act 480
Met ThrProAla SerIle ThrAlaAlaLys ThrSer ThrIleThr Thr
125 130 135
45 gca tttccacct gtatca tccactaccctg tttgca gtgggtgcc acc 528
Ala PheProPro ValSer SerThrThrLeu PheAla ValGlyAla Thr
140 145 150 155
cac agtgccagc atccag gaggaaactgag gaggtg gtgaactca cag 576
50 His SerAlaSer IleGln GluGluThrGlu GluVal ValAsnSer Gln
160 165 170
ctc ccgctgctc ctctcc ctgctggcattg ttgctg cttctgttg gtg 624
Leu ProLeuLeu LeuSer LeuLeuAlaLeu LeuLeu LeuLeuLeu Val
55 175 180 185
ggg gcctccctg ctagcc tggaggatgttt cagaaa tggatcaaa get 672
Gly AlaSerLeu LeuAla TrpArgMetPhe GlnLys TrpIleLys Ala
190 195 200
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ggt gac cat tca gag ctg tcc cag aac ccc aag cag get gcc acg cag 720
Gly Asp His Ser Glu Leu Ser Gln Asn Pro Lys Gln Ala Ala Thr Gln
205 210 215
agt gag ctg cac tac gca aat ctg gag ctg ctg atg tgg cct ctg cag 768
Ser Glu Leu His Tyr Ala Asn Leu Glu Leu Leu Met Trp Pro Leu Gln
220 225 230 235
LO gaa aag cca gca cca cca agg gag gtg gag gtg gaa tac agc act gtg 816
Glu Lys Pro Ala Pro Pro Arg Glu Val Glu Val Glu Tyr Ser Thr Val
240 245 250
gcc tcc ccc agg gaa gaa ctt cac tat gcc tcg gtg gtg ttt gat tct 864
L5 Ala Ser Pro Arg Glu Glu Leu His Tyr Ala Ser Val Val Phe Asp Ser
255 260 265
aac acc aac agg ata get get cag agg cct cgg gag gag gaa cca gat 912
Asn Thr Asn Arg Ile Ala Ala Gln Arg Pro Arg Glu Glu Glu Pro Asp
20 270 275 280
tca gat tac agt gtg ata agg aag aca tag 942
Ser Asp Tyr Ser Val Ile Arg Lys Thr
285 290
MALPVTALLLPLALLLHAARPDYKDDDDKIDLSKCRTVAGPVGGSLSVQCPYEKEHRTLNKYWCRPPQIFLCDK
IVETKGSAGKRNGRVSIRDSPANLSFTVTLENLTEEDAGTYWCGVDTPWLRDFHDPWEVEVSVFPASTSMTPA
SITAAKTSTITTAFPPVSSTTLFAVGATHSASIQEETEEWNSQLPLLLSLLALLLLLLVGASLLAWRMFQKWI
3 O KAGDHSELSQNPKQAATQSELHYANLELLMWPLQEKPAPPREVEVEYSTVASPREELHYASWFDSNTNRIAAQ
RPREEEPDSDYSVIRKT
DSP-1S form (SEQ ID N0: 6 and 7):
atg gccttacca gtgaccgcc ttgctc ctgccgcta gccttgctg ctc 48
Met AlaLeuPro ValThrAla LeuLeu LeuProLeu AlaLeuLeu Leu
-20 -15 -10
4 cac gccgccagg ccggattac aaggac gatgacgac aagatcgat atg 96
0
His AlaAlaArg ProAspTyr LysAsp AspAspAsp LysIleAsp Met
-5 -1 1 5 10
aca cctgcaagt atcactgcg gccaag acctcaaca atcacaact gca 144
4 Thr ProAlaSer IleThrAla AlaLys ThrSerThr IleThrThr Ala
5
15 20 25
ttt ccacctgta tcatccact accctg tttgcagtg ggtgccacc cac 192
Phe ProProVal SerSerThr ThrLeu PheAlaVal GlyAlaThr His
50 30 35 40
agt gccagcatc caggaggaa actgag gaggtggtg aactcacag ctc 240
Ser AlaSerIle GlnGluGlu ThrGlu GluValVal AsnSerGln Leu
50 55
ccg ctgctcctc tccctgctg gcattg ttgctgctt ctgttggtg ggg 288
Pro LeuLeuLeu SerLeuLeu AlaLeu LeuLeuLeu LeuLeuVal Gly
65 70 75
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gcc tcc ctgctagcc tggaggatg tttcagaaa tggatcaaa getggt 336
Ala Ser LeuLeuAla TrpArgMet PheGlnLys TrpIleLys AlaGly
80 85 90
gac cat tcagagctg tcccagaac cccaagcag getgccacg cagagt 384
Asp His SerGluLeu SerGlnAsn ProLysGln AlaAlaThr GlnSer
95 100 105
gag ctg cactacgca aatctggag ctgctgatg tggcctctg caggaa 432
LO Glu Leu HisTyrAla AsnLeuGlu LeuLeuMet TrpProLeu GlnGlu
110 115 120
aag cca gcaccacca agggaggtg gaggtggaa tacagcact gtggcc 480
Lys Pro AlaProPro ArgGluVal GluValGlu TyrSerThr ValAla
L5 125 130 135
tcc ccc agggaagaa cttcactat gcctcggtg gtgtttgat tctaac 528
Ser Pro ArgGluGlu LeuHisTyr AlaSerVal ValPheAsp SerAsn
140 145 150 155
20
acc aac aggataget getcagagg cctcgggag gaggaacca gattca 576
Thr Asn ArgIleAla AlaGlnArg ProArgGlu GluGluPro AspSer
160 165 170
2 gat tac agtgtgata aggaagaca tag 603
5
Asp Tyr SerValIle ArgLysThr
175
MAL PVTALLLP LALLLHAARPDY KDDDDKIDMTPASITAAKTSTITTAFPP
VSSTTLFAVGATHSASIQEETEE
3 VVN SQLPLLLS LLALLLLLLVGA SLLAWRMFQKWI KAGDHSELSQNPKQAATQSE
LHYANLELLMWPLQEKPAP
O
PRE VEVEYSTVASPR EELHYASWFDS NTNRIAAQRPRE EEPDSDYSVIRKT
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Alignment of long and short forms:
DSP-1L 1 MALPVTALLLPLALLLHAARPDYKDDDDKIDLSKCRTVAGPVGGSLSVQC 50
DSP-1S 1 MALPVTALLLPLALLLHAARPDYKDDDDKID------------------- 31
r, *******************************
LO
DSP-1L 51 PYEKEHRTLNKYWCRPPQIFLCDKIVETKGSAGKRNGRVSIRDSPANLSF 100
DSP-1S 32 --__-____-________--____-___--___-_______--_____-_ 31
DSP-1L 101 TVTLENLTEEDAGTYWCGVDTPWLRDFHDPWEVEVSVFPASTSMTPASI 150
DSP-1S 32 --________-___-____-____-____--___-_________MTPASI 37
******
L5 DSP-1L 151 TAAKTSTITTAFPPVSSTTLFAVGATHSASIQEETEEVVNSQLPLLLSLL 200
DSP-1S 38 TAAKTSTITTAFPPVSSTTLFAVGATHSASIQEETEEVVNSQLPLLLSLL 87
**************************************************
DSP-1L 201 ALLLLLLVGASLLAWRMFQKWIKAGDHSELSQNPKQAATQSELHYANLEL 250
20 DSP-1S 88 ALLLLLLVGASLLAWRMFQKWIKAGDHSELSQNPKQAATQSELHYANLEL 137
**************************************************
DSP-1L 251 LMWPLQEKPAPPREVEVEYSTVASPREELHYASWFDSNTNRIAAQRPRE 300
DSP-1S' 138 LMWPLQEKPAPPREVEVEYSTVASPREELHYASWFDSNTNRIAAQRPRE 187
25 **************************************************
DSP-1L 301 EEPDSDYSVIRKT 313
DSP-1S 188 EEPDSDYSVIRKT 200
*************
Reverse translation, e.g., nucleic acids encoding polypeptides (N can be
A, C, G, or T)
3 5 long (SEQ ID N0: 8); N may be A, C, G, or T:
ATGGCNYTNCCNGTNACNGCNYTNYTNYTNCCNYTNGCNYTNYTNYTNCAYGCNGCNMGNCCNGAYTAYAARGAY
GAYGAYGAYAARATHGAYYTNWSNAARTGYMGNACNGTNGCNGGNCCNGTNGGNGGNWSNYTNWSNGTNCARTGY
CCNTAYGARAARGARCAYMGNACNYTNAAYAARTAYTGGTGYMGNCCNCCNCARATHTTYYTNTGYGAYAARATH
GTNGARACNAARGGNWSNGCNGGNAARMGNAAYGGNMGNGTNWSNATHMGNGAYWSNCCNGCNAAYYTNWSNTTY
4 O
ACNGTNACNYTNGARAAYYTNACNGARGARGAYGCNGGNACNTAYTGGTGYGGNGTNGAYACNCCNTGGYTNMGN
GAYTTYCAYGAYCCNGTNGTNGARGTNGARGTNWSNGTNTTYCCNGCNWSNACNWSNATGACNCCNGCNWSNATH
ACNGCNGCNAARACNWSNACNATHACNACNGCNTTYCCNCCNGTNWSNWSNACNACNYTNTTYGCNGTNGGNGCN
ACNCAYWSNGCNWSNATHCARGARGARACNGARGARGTNGTNAAYWSNCARYTNCCNYTNYTNYTNWSNYTNYTN
GCNYTNYTNYTNYTNYTNYTNGTNGGNGCNWSNYTNYTNGCNTGGMGNATGTTYCARAARTGGATHAARGCNGGN
4 5
GAYCAYWSNGARYTNWSNCARAAYCCNAARCARGCNGCNACNCARWSNGARYTNCAYTAYGCNAAYYTNGARYTN
YTNATGTGGCCNYTNCARGARAARCCNGCNCCNCCNMGNGARGTNGARGTNGARTAYWSNACNGTNGCNWSNCCN
MGNGARGARYTNCAYTAYGCNWSNGTNGTNTTYGAYWSNAAYACNAAYMGNATHGCNGCNCARMGNCCNMGNGAR
GARGARCCNGAYWSNGAYTAYWSNGTNATHMGNAARACN
5 0 short (SEQ ID NO: 9); N may be A, C, G, or T:
ATGGCNYTNCCNGTNACNGCNYTNYTNYTNCCNYTNGCNYTNYTNYTNCAYGCNGCNMGNCCNGAYTAYAARGAY
GAYGAYGAYAARATHGAYATGACNCCNGCNWSNATHACNGCNGCNAARACNWSNACNATHACNACNGCNTTYCCN
CCNGTNWSNWSNACNACNYTNTTYGCNGTNGGNGCNACNCAYWSNGCNWSNATHCARGARGARACNGARGARGTN
GTNAAYWSNCARYTNCCNYTNYTNYTNWSNYTNYTNGCNYTNYTNYTNYTNYTNYTNGTNGGNGCNWSNYTNYTN
55 GCNTGGMGNATGTTYCARAARTGGATHAARGCNGGNGAYCAYWSNGARYTNWSNCARAAYCCNAARCARGCNGCN
ACNCARWSNGARYTNCAYTAYGCNAAYYTNGARYTNYTNATGTGGCCNYTNCARGARAARCCNGCNCCNCCNMGN
GARGTNGARGTNGARTAYWSNACNGTNGCNWSNCCNMGNGARGARYTNCAYTAYGCNWSNGTNGTNTTYGAYWSN
AAYACNAAYMGNATHGCNGCNCARMGNCCNMGNGARGARGARCCNGAYWSNGAYTAYWSNGTNATHMGNAAR.ACN
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The currently available methods to generate large amounts
of DC in vitro allow detailed molecular analysis of DC. See
Romani, et al. (1994) J. Exp. Med. 180:83-93. cDNA libraries
derived from monocyte-derived DC were analyzed leading to the
identification of several interesting gene products, including
a novel DC-specific chemokine, DC-CK1. See Zhou and Tedder
(1996) Proc. Natl. Acad. Sci. USA.93:2588-2592.
Pairwise protein sequence alignments performed between DC-
STAMP and members of several 7 TM subclasses (ClustalW) showed
LO identities below 20%, suggesting that the DC-STAMP protein
represents a novel protein family. However, the structural
homology of DC-STAMP to members of the superfamily of G-protein
coupled (or linked) receptors (GPCR, or GPLR) suggests related
function of this molecule. As a class, these receptors are
integral membrane proteins characterized by amino acid
sequences which contain seven hydrophobic domains. See, e.g.,
Ruffolo and Hollinger (eds. 1995) G-Protein Coupled
Transmembrane Sianalina Mechanisms CRC Press, Boca Raton, FL;
Watson and Arkinstall (1994) The G-Protein Linked Receptor
FactsBook -Academic Press, San Diego, CA; Peroutka (ed. 1994) G
Protein-Coupled Receptors CRC Press, Boca Raton, FL; Houslay
and Milligan (1990) G-Proteins as Mediators of Cellular
SicLnalinc~ Processes Wiley and Sons, New York, NY; and Dohlman,
et al. (1991) Ann. Rev. Biochem. 60:653-688. These hydrophobic
domains are predicted to represent transmembrane spanning
regions of the proteins. These GPCRs are found in a wide range
of organisms and are typically involved in the transmission of
signals to the interior of the cell, e.g., through interaction,
e.g., with heterotrimeric G-proteins. They respond to a wide
and diverse range of agents including lipid analogs, amino acid
derivatives, small peptides, and other molecules.
A predicted model of the structure of the DC-STAMP protein
has an extracellular N-terminus, a cytoplasmic C-terminus, 3
cytoplasmic loops, and 3 extracellular loops, containing 2
consensus sequences for N-linked glycosylation on the second
and one on the third extracellular loop.
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The serine residues in the C-terminus of DC-STAMP are
putative targets for phosphorylation. For several 7 TM
proteins, it has been shown that phosphorylation of serine and
threonine residues in the C-tail of the receptor by G protein
5 coupled receptor kinases results in uncoupling of the activated
receptor from its G proteins, thereby desensitizing the
receptor. See Bohm, et al. (1997) J. Biol. Chem. 332:1-18.
Further experiments can be performed to determine whether the
DC-STAMP protein can be phosphorylated at these serine
LO residues.
Other characteristics of 7 TM proteins include a signature
of cysteine residues in the first two extracellular loops,
which might form disulphide bridges and stabilize the protein
structure. See Savarese and Fraser (1992) J. Biol. Chem.
15 283:1-19. Also, cysteine residues in the carboxyl tails are
potential sites for palmitoylation and may serve to form a
fourth intracellular loop. See O'Dowd et al. (1989) J. Biol.
Chem. 264:7564-7569; and Strader, et al. (1994) Ann. Rev.
Biochem. 63:101-132. The DC-STAMP protein contains an
20 alternative signature of cysteines in TM1 and TM2, and has no
cysteine residues in its C-terminus. Combined with the absence
of any sequence homology to 7 TM receptors, the described
characteristics of the DC-STAMP protein suggest that this novel
protein does not belong to any of the existing 7 TM subclasses.
DC-STAMP could either form a novel 7 TM protein subclass or be
the first member of a new family of multi-membrane spanning
proteins.
Characteristic of the DC-STAMP terminus is its very basic
amino acid composition. There is some indication that
juxtamembrane clusters of positively charged residues in
cytoplasmic receptor tails can associate with proteins of the
ERM (ezrin, radaxin, moesin) family. See Bretscher (1999)
Curr. Op. Cell Biol. 11:109-116. Since these ERM proteins have
been implicated as membrane cytoskeletal linkers, this might
suggest association of DC-STAMP with the cytoskeleton.
Ligation of DC-STAMP might affect adhesive or migratory
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capacities, essential for proper DC function. These can be
tested.
Structural predictions for the type III membrane protein
suggest hydrophobic transmembrane segments from about va135-
a1a51 (TM1); ser57-ser75 (TM2); asn96-i1e114 (TM3); tyr144-
asp162 (TM4); 1eu214-phe230 (TM5); 1eu295-va1313 (TM6); and
pro379-met398 (TM7). The use of several TM prediction programs
for the hydropathy analysis of DC-STAMP resulted in different
models, regarding the position and number of transmembrane
LO domains. The data suggest a model in which the DC-STAMP
protein contains 7 transmembrane domains. First, the position
of potential glycosylation sites, putative phosphorylation
recognition sites and the intracellular C-terminus favor this
model. Second, based on the presence of charged amino acids,
L5 which generally flank transmembrane regions, the model supports
a type IIIb integral membrane protein, with the N-terminus of
DC-STAMP outside and the C-terminus on the luminal side of the
membrane. Finally, the DC-STAMP protein contains a proline
residue between TM1 and TM2. Prolines are known to disrupt
20 helices and the proline residue at position 56 in DC-STAMP
could help to establish a loop and redirect the protein into
the membrane. This could possibly compensate for the rather
short hydrophobic stretches of TM1 (transmembrane region 1) and
TM2, 17 and 18 amino acids in length, respectively. Also, both
25 TM1 and TM2 contain a pair of cysteine residues, which could
further stabilize this intramembrane loop by a disulphide
bridge near the outer membrane side.
However, since TM2, TM3, and TM4 are rather weak TM
regions, alternative models cannot be excluded comprising 5 or
30 4 TM regions, in which TM1 and TM2 form a single transmembrane
domain and either TM3 or TM4 or both are not present. Only two
5 TM spanning proteins have been described so far, the 865
amino acid AC133 orphan receptor, expressed by hematopoietic
stem cells (Miraglia, et al. (1997) Blood 90:5013-5021), and
35 the CD47 molecule (Lindberg, et al. (1993) J. Cell. Biol.
123:485-496). The TM4 superfamily consists of nearly 20 genes,
encoding proteins which are thought to be involved in the
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grouping and stabilization of cell-surface proteins. The DC-
STAMP protein however, does not show significant homology to
either these TM4 or TM5 proteins, indicating that DC-STAMP
represents a novel protein family.
Transmembrane segments are typically 20-25 amino acids in
length. Based upon models and data on bacteriorhodopsin, these
regions are predicted to be a-helices and be oriented to form a
ligand binding pocket. See, e.g., Findley, et al. (1990)
Trends Pharmacol. Sci. 11:492-499. Other data suggest that the
LO amino termini of the proteins are extracellular, and the
carboxy termini are intracellular. See, e.g., Lodish, et al.
(1995) Molecular Cell Bioloav 3d ed., Scientific American, New
York; and Watson and Arkinstall~(1994) The G-Protein Linked
R_ecentor FactsBook Academic Press, San Diego, CA.
Phosphorylation cascades have been implicated in the signal
transduction pathway of these receptors.
7 TM receptors comprise a family of very heterogeneous
proteins that signal through heterotrimeric G proteins
(Strader, et al. (1994) Ann. Rev. Biochem. 63:101-132),
including chemokine, hormone and photoreceptors. The vast
majority of 7 TM proteins are G-protein coupled. The presence
of an aspartate in the second transmembrane region and a so-
called "DRY or ERY motif", closely following the third
transmembrane region, are both thought to be involved in the
signal transduction via G-proteins. See Savarese and Fraser
(1992) J. Biol. Chem. 283:1-19; and Bourne (1997) Curr. Op.
Cell Biol. 9:134-142. DC-STAMP contains neither of these
sequences, but such motifs may possibly not be recognized as
such. Similarly, other 7 TM proteins, such as the Duffy
antigen receptor (DARC) on erythrocytes and the EGF-7TM
receptors, also lack consensus sequences for G protein coupling
and no signal transduction via these receptors has been proven
as yet. See Horuk, et al. (1996) J. Leukoc. Biol. 59:29-38;
and McKnight and Gordon (1996) Immunol. Today 17:283-287.
Although the full spectrum of biological activities
mediated by these 7 transmembrane receptors has not been fully
determined, chemoattractant effects are recognized. Chemokine
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receptors are notable members of the GPCR family. See, e.g.,
Samson, et al. (1996) Biochemistry 35:3362-3367; and Rapport,
et al. (1996) J. Leukocyte Bioloctv 59:18-23. The best known
biological functions of chemokine molecules relate to
chemoattraction of leukocytes. However, new chemokines and
receptors are being discovered, and their biological effects on
the various cells responsible for immunological responses are
topics of continued study.
DC-STAMP agonists, or antagonists, may also act as
LO functional or receptor antagonists, e.g., which block DC
interactions or physiology, or mediating the opposite actions.
DC are implicated in T cell mediated immunity, which is
important in various diseases. T cell immunity is deficient in
various contexts, e.g., in tumor immunotherapy and allergic
responses. Conversely, it is overactive in autoimmune diseases
and transplantation rejection contexts. Thus, DC-STAMP, or its
antagonists, may be useful in the treatment of abnormal medical
conditions, including immune disorders, e.g., immune
deficiencies, chronic inflammation, or tissue rejection, or
other physiological conditions. The implication of antigen
presentation in initiation of an immune response is a likely
condition to be affected by the use of a DC-STAMP related
reagent. Compositions combining the DC-STAMP and other DC
affecting reagents will often be used. See below.
The DSP-1 forms are highly expressed in mast cells, which
are implicated in allergic responses, particularly in release
of histamine. See, e.g., Kaliner and Metcalfe (eds. 1992) The
Mast Cell in Health and Disease. Reagents related to
activation or deactivation of DSP-1 signaling may be important
in medical conditions mediated by cells expressing the antigen.
Both the long (L) and short (s) forms are type I membrane
proteins, and possess cytoplasmic domains with multiple ITIM
motifs, suggesting an inhibitory receptor signaling role. See,
e.g., Kung, et al. (1999) J. Immunol. 162:5876-87; Carlyle, et
al. (1999) J. Immunol. 162:5917-5923; Nakamura, et al. (1997)
J. Exp. Med. 185:673-684; Olcese, et al. (1996) J. Immunol.
156:4531-4534; and Daeron, et al. (1995) Immunity 3:635-646.
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The transmembrane segments correspond approximately to residues
172-188 (166-198) for the L form, and 59-75 (53-85) of the S
form. However the actual boundaries of transmembrane segments
may vary or depend upon kinetic and other factors.
These natural antigens will be capable of mediating
various biochemical responses which lead to biological or
physiological responses in target cells. The preferred
embodiments characterized herein are from primate, e.g., human,
but other species counterparts will exist in nature.
Additional sequences for proteins in other mammalian species,
e.g., primates, canines, felines, and rodents, should also be
available, particularly the domestic animal species. See
below. The descriptions below are directed, for exemplary
purposes, to a human DC-STAMP or DSP-1, but are likewise
applicable to related embodiments from other species.
II. Purified DC-STAMP or DSP-1
Primate, e.g., human, DC-STAMP or DSP-1 amino acid
sequences, are shown in Tables 1 or 2. Other naturally
occurring nucleic acids which encode the proteins can be
isolated by standard procedures using the provided sequences,
e.g., PCR techniques, or by hybridization. Primer extension or
RACE methods can extend to adjacent sequence, either on message
or genomic. These amino acid sequences, provided amino to
carboxy, are important in providing sequence information for
the proteins allowing for distinguishing the protein antigen
from other proteins and exemplifying numerous variants.
Moreover, the peptide sequences allow preparation of peptides
to generate antibodies to recognize such segments, and
nucleotide sequences allow preparation of oligonucleotide
probes, both of which are strategies for detection or
isolation, e.g., cloning, of genes encoding such sequences.
As used herein, the term "human DC-STAMP" shall encompass,
when used in a protein context, a protein having amino acid
sequence corresponding to a polypeptide shown in SEQ ID NO: 2,
or significant fragments thereof. Preferred embodiments
comprise a plurality of distinct, e.g., nonoverlapping,
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segments of the specified length. Typically, the plurality
will be at least two, more usually at least three, and
preferably 5, 7, or even more. Vdhile the length minima are
provided, longer lengths, of various sizes, may be appropriate,
5 e.g., one of length 7, and two of length 12. Similarly with
the term DSP-1 and SEQ ID NO: 4 and 6.
Binding components, e.g., antibodies, typically bind to an
antigen with high affinity, e.g., at least about 100 nM,
usually better than about 30 nM, preferably better than about
LO 10 nM, and more preferably at better than about 3 nM.
Counterpart proteins will be found in mammalian species other
than human, e.g., other primates, ungulates, or rodents. Non-
mammalian species should also possess structurally or
functionally related genes and proteins, e.g., birds or fish.
15 The term "polypeptide" as used herein includes a
significant fragment or segment, and encompasses a stretch of
amino acid residues of at least about 8 amino acids, generally
at least about 12 amino acids, typically at least about 16
amino acids, preferably at least about 20 amino acids, and, in
20 particularly preferred embodiments, at least about 30 or more
amino acids, e.9., 35, 40, 45, 50, etc. Such fragments may
have ends which begin and/or end at virtually all positions,
e.g., beginning at residues 1, 2, 3, etc., and ending at, e.g.,
150, 149, 148, etc., in all practical combinations.
25 Particularly interesting peptides have ends corresponding to
structural domain boundaries, e.g., transmembrane segments or
identified motifs. See Tables 1 and 2.
The term "binding composition" refers to molecules that
bind with specificity to DC-STAMP or DSP-1, e.g., in an
antibody-antigen interaction. The specificity may be more or
less inclusive, e.g., specific to a particular embodiment, or
to groups of related embodiments, e.g., primate, rodent, etc.
It also includes compounds, e.g., proteins, which specifically
associate with DC-STAMP, including in a natural physiologically
relevant protein-protein interaction, either covalent or non-
covalent. The molecule may be a polymer, or chemical reagent.
A functional analog may be a protein with structural
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modifications, or it may be a molecule which has a molecular
shape which interacts with the appropriate binding
determinants. The compounds may serve as agonists or
antagonists of a receptor binding interaction, see, e.g.,
Goodman, et al. (eds.) Goodman & Gilman's: The Pharmacological
Bases of Therapeutics (current ed.) Pergamon Press.
Substantially pure, e.g., in a protein context, typically
means that the protein is free from other contaminating
proteins, nucleic acids, or other biologicals derived from the
LO original source organism. Purity may be assayed by standard
methods, typically by weight, and will ordinarily be at least
about 40% pure, generally at least about 50% pure, often at
least about 60% pure, typically at least about 80% pure,
preferably at least about 90% pure, and in most preferred
embodiments, at least about 95% pure. Carriers or excipients
will often be added.
Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect
polypeptide solubility, including temperature, electrolyte
environment, size and molecular characteristics of the
polypeptide, and nature of the solvent. Typically, the
temperature at which the polypeptide is used ranges from about
4° C to about 65° C. Usually the temperature at use is greater
than about 18° C. For diagnostic purposes, the temperature
will usually be about room temperature or warmer, but less than
the denaturation temperature of components in the assay. For
therapeutic purposes, the temperature will usually be body
temperature, typically about 37° C for humans and mice, though
under certain situations the temperature may be raised or
lowered in situ or in vitro.
The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a
denatured state. The polypeptide may be associated with other
polypeptides in a quaternary structure, e.g., to confer
solubility, or associated with lipids or detergents.
The solvent and electrolytes will usually be a
biologically compatible buffer, of a type used for preservation
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of biological activities, and will usually approximate a
physiological aqueous solvent. Usually the solvent will have a
neutral pH, typically between about 5 and 10, and preferably
about 7.5. On some occasions, one or more detergents will be
added, typically a mild non-denaturing one, e.g., CHS
(cholesteryl hemisuccinate) or CHAPS (3-[3-
cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a low
enough concentration as to avoid significant disruption of
structural or physiological properties of the protein. In
LO other instances, a harsh detergent may be used to effect
significant denaturation.
III. Physical Variants
This invention also encompasses proteins or peptides
having substantial amino acid sequence identity with the amino
acid sequences of the DC-STAMP or DSP-1 antigens. The variants
include species, polymorphic, or allelic variants.
Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. See also Needleham, et al.
(1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Chapter
One in Time Warps Strina Edits and Macromolecules: The Theorv
and Practice of Seauence Comparison, Addison-Wesley, Reading,
MA; and software packages from IntelliGenetics, Mountain View,
CA; and the University of Wisconsin Genetics Computer Group,
Madison, WI. Sequence identity changes when considering
conservative substitutions as matches. Conservative
substitutions typically include substitutions within the
following groups: glycine, alanine; valine, isoleucine,
leucine; aspartic acid, glutamic acid; asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine,
tyrosine. The conservation may apply to biological features,
functional features, or structural features. Homologous amino
acid sequences are typically intended to include natural
polymorphic or allelic and interspecies variations of a protein
sequence. Typical homologous proteins or peptides will have
from 25-100% identity (if gaps can be introduced), to 50-100%
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identity (if conservative substitutions are included) with the
amino acid sequences of the antigens. Identity measures will
be at least about 35%, generally at least about 40%, often at
least about 50%, typically at least about 60%, usually at least
about 70%, preferably at least about 80%, and more preferably
at least about 90%.
The isolated DC-STAMP or DSP-1 DNA can be readily modified
by nucleotide substitutions, nucleotide deletions, nucleotide
insertions, and inversions of short nucleotide stretches.
LO These modifications may result in novel DNA sequences which
encode these antigens, their derivatives, or proteins having
similar physiological, immunogenic, antigenic, or other
functional activity. These modified sequences can be used to
produce mutant antigens or to enhance expression. Enhanced
expression may involve gene amplification, increased
transcription, increased translation, and other mechanisms.
"Mutant DC-STAMP" encompasses a polypeptide otherwise falling
within the sequence identity definition of the DC-STAMP as set
forth above, but having an amino acid sequence which differs
from that of the antigen as normally found in nature, whether
by way of deletion, substitution, or insertion. This generally
includes proteins having significant identity with a protein
having sequence of SEQ ID NO: 2, and as sharing various
biological activities, e.g., antigenic or immunogenic, with
those sequences, and in preferred embodiments contain most of
the natural full length disclosed sequences. Full length
sequences will typically be preferred, though truncated
versions will also be useful, likewise, genes or proteins found
from natural sources are typically most desired. Similar
concepts apply to different DC-STAMP proteins, particularly
those found in various warm blooded animals, e.g., mammals and
birds. These descriptions are generally meant to encompass
many DC-STAMP proteins, not limited to the particular primate
embodiments specifically discussed.
DC-STAMP or DSP-1 mutagenesis can also be conducted by
making amino acid insertions or deletions. Substitutions,
deletions, insertions, or any combinations may be generated to
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29
arrive at a final construct. Insertions include amino- or
carboxy- terminal fusions. Random mutagenesis can be conducted
at a target codon and the expressed mutants can then be
screened for the desired activity. Methods for making
substitution mutations at predetermined sites in DNA having a
known sequence are well known in the art, e.g., by M13 primer
mutagenesis or polymerase chain reaction (PCR) techniques.
See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987 and
Supplements); and Kunkel, et al. (1987) Methods in Enzvmol.
154:367-382. Preferred embodiments include, e.g., 1-fold, 2-
fold, 3-fold, 5-fold, 7-fold, etc., preferably conservative
substitutions at the nucleotide or amino acid levels.
Preferably the substitutions will be away from the conserved
cysteines, and often will be in the regions away from the
extramembrane domains. Such variants may be useful to produce
specific antibodies, and often will share many or all
biological properties.
The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of
proteins or segments which are naturally not normally fused in
the same manner. A similar concept applies to heterologous
nucleic acid sequences.
In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
target-binding or other segments may be "swapped" between
different new fusion polypeptides or fragments. See, e.g.,
Cunningham, et al. (1989) Science 243:1330-1336; and O'Dowd, et
al. (1988) J. Biol. Chem. 263:15985-15992.
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce
useful synthetic DNA fragments. A double stranded fragment
will often be obtained either by synthesizing the complementary
strand and annealing the strand together under appropriate
conditions or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence, e.g., PCR
techniques.
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Structural analysis can be applied to this gene, in
comparison to members of related gene families, e.g., GPCRs.
In particular, (3-sheet and a-helix residues can be determined
using, e.g., RASMOL program, see Bazan, et al. (1996) Nature
5 379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle and
Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al.
(1991) Protein Engineering 4:263-269. Preferred residues for
substitutions include the surface exposed extramembrane
residues which would be predicted to interact with a
LO counterstructure or ligand. Other residues which should
conserve function will be conservative substitutions,
particularly at a position far from the surface exposed
residues, e.g., an intramembrane residue.
15 IV. Functional Variants
The blocking of physiological response to DC-STAMP or DSP-
1 may result from the competitive inhibition of binding of a
ligand or counterstructure to the antigen.
In vitro assays of the present invention will often use
20 isolated protein, soluble fragments comprising receptor binding
segments of these proteins, or fragments attached to solid
phase substrates. These assays will also allow for the
diagnostic determination of the effects of either binding
segment mutations and modifications, or ligand mutations and
25 modifications.
This invention also contemplates the use of competitive
drug screening assays, e.g., where neutralizing antibodies to
the antigen, or receptor binding fragments compete with a test
compound.
30 "Derivatives" of DC-STAMP or DSP-1 antigens include amino
acid sequence mutants from naturally occurring forms,
glycosylation variants, and covalent or aggregate conjugates
with other chemical moieties. Covalent derivatives can be
prepared by linkage of functionalities to groups which are
found in amino acid side chains or at the N- or C- termini,
e.g., by standard means. See, e.g., Lundblad and Noyes (1988)
Chemical Reagents for Protein Modification, vols. 1-2, CRC
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31
Press, Inc., Boca Raton, FL; Hugli (ed. 1989) Techniques in
Protein Chemistry, Academic Press, San Diego, CA; and Wong
(1991) Chemistry of Protein Conjugation and Cross Linking, CRC
Press, Boca Raton, FL.
In particular, glycosylation alterations are included,
e.g., made by modifying the glycosylation patterns of a
polypeptide during its synthesis and processing, or in further
processing steps. See, e.g., Elbein (1987) Ann. Rev. Biochem.
56:497-534. Also embraced are versions of the peptides with
LO the same primary amino acid sequence which have other minor
modifications, including phosphorylated amino acid residues,
e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
Fusion polypeptides between DC-STAMP or DSP-1 and other
homologous or heterologous proteins are also provided. Many
7TM receptors or other surface proteins are multimeric, e.g.,
homodimeric entities, and a repeat construct may have various
advantages, including lessened susceptibility to proteolytic
cleavage. Typical examples are fusions of a reporter
polypeptide, e.g., luciferase, with a segment or domain of a
protein, e.g., a receptor-binding segment, so that the presence
or location of the fused ligand may be easily determined. See,
e.g., Dull, et al., U.S. Patent No. 4,859,609. Other gene
fusion partners include bacterial f3-galactosidase, trpE,
Protein A, i3-lactamase, alpha amylase, alcohol dehydrogenase,
yeast alpha mating factor, and detection or purification tags
such as a FLAG sequence of His6 sequence. See, e.g., Godowski,
et al. (1988) Science 241:812-816.
Fusion peptides will typically be made by either
recombinant nucleic acid methods or by synthetic polypeptide
methods. Techniques for nucleic acid manipulation and
expression are described generally, e.g., in Sambrook, et al.
(1989) Molecular Clonina~ A Laboratory Manual (2d ed.), vols.
1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.
1993) Current Protocols in Molecular Bioloav, Greene and Wiley,
NY. Techniques for synthesis of polypeptides are described,
e.g., in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156;
Merrifield (1986) Science 232: 341-347; Atherton, et al. (1989)
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32
Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,
Oxford; and Grant (1992) Synthetic Pegtides: A User's Guide,
W.H. Freeman, NY. Refolding methods, e.g., with membranes, may
be applicable to synthetic proteins.
This invention also contemplates the use of derivatives of
DC-STAMP or DSP-1 proteins other than variations in amino acid
sequence or glycosylation. Such derivatives may involve
covalent or aggregative association with chemical moieties or
protein carriers. Covalent or aggregative derivatives will be
LO useful as immunogens, as reagents in immunoassays, or in
purification methods such as for affinity purification of
binding partners, e.g., other antigens. A DC-STAMP or DSP-1
can be immobilized by covalent bonding to a solid support such
as cyanogen bromide-activated SEPHAROSE, by methods which are
well known in the art, or adsorbed onto polyolefin surfaces,
with or without glutaraldehyde cross-linking, for use in the
assay or purification of antibodies or an alternative binding
composition. These proteins can also be labeled with a
detectable group, e.g., for use in diagnostic assays.
Purification of antigen may be effected by an immobilized
antibody or complementary binding partner, e.g., binding
portion of a receptor.
A solubilized fragment of this invention can be used as an
immunogen for the production of antisera or antibodies specific
for binding. Purified antigen can be used to screen monoclonal
antibodies or antigen-binding fragments, encompassing antigen
binding fragments of natural antibodies, e.g., Fab, Fab',
F(ab)2, etc. Purified DC-STAMP or DSP-1 antigens can also be
used as a reagent to detect antibodies generated in response to
the presence of elevated levels of the antigen, which may be
diagnostic of an abnormal or specific physiological or disease
condition. This invention contemplates antibodies raised
against amino acid sequences encoded by nucleotide sequence
shown in SEQ ID NO: 1 or 4 or 6, or fragments of proteins
containing it. In particular, this invention contemplates
antibodies having binding affinity to or being raised against
specific domains, e.g., extracellular segments.
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The present invention contemplates the isolation of
additional closely related species variants. Southern and
Northern blot analysis will establish that similar genetic
entities exist in other mammals. It is likely that antigens
are widespread in species variants, e.g., rodents, lagomorphs,
carnivores, artiodactyla, perissodactyla, and primates.
The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities
in structure, expression, and function. Elucidation of many of
LO the physiological effects of the molecules will be greatly
accelerated by the isolation and characterization of additional
distinct species or polymorphic variants of them. In
particular, the present invention provides useful probes for
identifying additional homologous genetic entities in different
species.
The isolated genes will allow transformation of cells
lacking expression of DC-STAMP or DSP-1, e.g., either species
types or cells which lack corresponding proteins and exhibit
negative background activity. This should allow analysis of
the function of antigen in comparison to untransformed control
cells.
Dissection of critical structural elements which effect
the various physiological functions mediated through these
antigens is possible using standard techniques of modern
molecular biology, particularly in comparing members of the
related class. See, e.g., the homolog-scanning mutagenesis
technique described in Cunningham, et al. (1989) Science
243:1339-1336; and approaches used in O'Dowd, et al. (1988) J.
Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990) EMBO
_J. 9:4381-4390.
Intracellular functions would probably involve receptor
signaling. However, protein internalization may occur under
certain circumstances, and interaction between intracellular
components and ligand or receptor may occur. Specific segments
of interaction of membrane antigen with interacting components
may be identified by mutagenesis or direct biochemical means,
e.g., cross-linking or affinity methods. Structural analysis
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34
by crystallographic or other physical methods will also be
applicable. Further investigation of the mechanism of signal
transduction will include study of associated components which
may be isolatable by affinity methods or by genetic means,
e.g., complementation analysis of mutants.
Further study of the expression and control of DC-STAMP or
DSP-1 will be pursued. The controlling elements associated
with the antigens should exhibit differential physiological,
developmental, tissue specific, or other expression patterns.
LO Upstream or downstream genetic regions, e.g., control elements,
are of interest.
Structural studies of the membrane antigens will lead to
design of new antigens, particularly analogs exhibiting agonist
or antagonist properties on the molecule. This can be combined
with previously described screening methods to isolate antigens
exhibiting desired spectra of activities.
V. Antibodies
Antibodies can be raised to various epitopes of the
membrane proteins, including species, polymorphic, or allelic
variants, and fragments thereof, both in their naturally
occurring forms and in their recombinant forms. Additionally,
antibodies can be raised to the proteins in either their active
forms or in their inactive forms, including native or denatured
versions. Anti-idiotypic antibodies are also contemplated.
Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the antigens can
be raised by immunization of animals with conjugates of the
fragments with immunogenic proteins. Monoclonal antibodies are
prepared from cells secreting the desired antibody. These
antibodies can be screened for binding to normal or defective
proteins, or screened for agonistic or antagonistic activity,
e.g., mediated through a receptor. Antibodies may be agonistic
or antagonistic, e.g., by sterically blocking binding to a
receptor. These monoclonal antibodies will usually bind with
at least a KD of about 1 mM, more usually at least about 300
~.M, typically at least about 100 ~.M, more typically at least
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about 30 ~,M, preferably at least about 10 ~.M, and more
preferably at least about 3 ~M or better.
A DC-STAMP or DSP-1 protein that specifically binds to or
that is specifically immunoreactive with an antibody generated
5 against a defined immunogen, such as an immunogen consisting of
the amino acid sequence of SEQ ID NO: 2, is typically
determined in an immunoassay. The immunoassay typically uses a
polyclonal antiserum which was raised, e.g., to a protein of
SEQ ID NO: 2. This antiserum is selected to have low
10 crossreactivity against other related proteins, e.g., human or
rodent DC-STAMP, preferably from the same species, and any such
crossreactivity is removed by immunoabsorption prior to use in
the immunoassay.
In order to produce antisera for use in an immunoassay,
15 the protein of SEQ ID N0: 2, or a combination thereof, is
isolated as described herein. For example, recombinant protein
may be produced in a mammalian cell line. An appropriate host,
e.g., an inbred strain of mice such as Balb/c, is immunized
with the selected protein, typically using a standard adjuvant,
20 such as Freund's adjuvant, and a standard mouse immunization
protocol (see Harlow and Lane, supra). Alternatively, a
synthetic peptide derived from the sequences disclosed herein
and conjugated to a carrier protein can be used an immunogen.
Polyclonal sera are collected and titered against the immunogen
25 protein in an immunoassay, e.g., a solid phase immunoassay with
the immunogen immobilized on a solid support. Polyclonal
antisera with a titer of 104 or greater are selected and tested
for their cross reactivity against other related family
members, e.g., rodent DC-STAMP, using a competitive-binding
30 immunoassay such as the one described in Harlow and Lane,
supra, at pages 570-573. Preferably at least one other related
family member is used in this determination in conjunction
with, e.g., the primate embodiment. The desired target family
members can be produced as recombinant proteins and isolated
35 using standard molecular biology and protein chemistry
techniques as described herein.
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Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the
protein of SEQ ID NO: 2 can be immobilized to a solid support.
Proteins added to the assay compete with the binding of the
antisera to the immobilized antigen. The ability of the above
proteins to compete with the binding of the antisera to the
immobilized protein is compared to the protein of SEQ ID NO: 2.
The percent crossreactivity for the above proteins is
calculated, using standard calculations. Those antisera with
LO less than 10% crossreactivity with each of the proteins listed
above are selected and pooled. The cross-reacting antibodies
are then removed from the pooled antisera by immunoabsorption
with the above-listed proteins.
The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen protein (e.g., the protein of
SEQ ID NO: 2). In order to make this comparison, the two
proteins are each assayed at a wide range of concentrations and
the amount of each protein required to inhibit 50% of the
binding of the antisera to the immobilized protein is
determined. If the amount of the second protein required is
less than twice the amount of the protein of the selected
protein or proteins that is required, then the second protein
is said to specifically bind to an antibody generated to the
immunogen.
The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing
antibodies, they can be screened for ability to bind to the
antigens without inhibiting binding to a receptor. As
neutralizing antibodies, they can be useful in competitive
binding assays. They will also be useful in detecting or
quantifying DC-STAMP or DSP-1 protein or their receptors. See,
e.g., Chan (ed. 1987) Immunoloav~ A Practical Guide, Academic
Press, Orlando, FL; Price and Newman (eds. 1991) Principles and
_Practice of Immunoassay, Stockton Press, N.Y.; and Ngo (ed.
1988) Nonisotopic Immunoassay, Plenum Press, N.Y. Cross
absorptions, depletions, or other~means will provide
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preparations of defined selectivity, e.g., unique or shared
species specificities. These may be the basis for tests which
will identify various groups of antigens.
Further, the antibodies, including antigen binding
fragments, of this invention can be potent antagonists that
bind to the antigen and inhibit functional binding, e.g., to a
receptor which may elicit a biological response. They also can
be useful as non-neutralizing antibodies and can be coupled to
toxins or radionuclides so that when the antibody binds to
LO antigen, a cell expressing it, e.g., on its surface, is killed.
Further, these antibodies can be conjugated to drugs or other
therapeutic agents, either directly or indirectly by means of a
linker, and may effect drug targeting.
Antigen fragments may be joined to other materials,
L5 particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. An antigen and its
fragments may be fused or covalently linked to a variety of
immunogens, such as keyhole limpet hemocyanin, bovine serum
albumin, tetanus toxoid, etc. See Microbioloay, Hoeber Medical
20 Division, Harper and Row, 1969; Landsteiner (1962) Specificity
of Serological Reactions, Dover Publications, New York;
Williams, et al. (1967) Methods in Immunology and
Immunochemistry, vol. 1, Academic Press, New York; and Harlow
and Lane (1988) Antibodie s A Laboratory Manual, CSH Press,
25 NY, for descriptions of methods of preparing polyclonal
antisera.
In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing
30 such monoclonal antibodies may be found in, e.g., Stites, et
al. (eds.) Basic and Clinical Immunology (4th ed.), Lange
Medical Publications, Los Altos, CA, and references cited
therein; Harlow and Lane (1988) Antibodies: A Laboratory
Manual, CSH Press; Goding (1986) Monoclonal Antibodies:
35 _Principles and Practice (2d ed.), Academic Press, New York; and
particularly in Kohler and Milstein (1975) in Nature 256:495-
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38
497, which discusses one method of generating monoclonal
antibodies.
Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar
vectors. See, Huse, et al. (1989) "Generation of a Large
Combinatorial Library of the Immunoglobulin Repertoire in Phage
Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature
341:544-546. The polypeptides and antibodies of the present
LO invention may be used with or without modification, including
chimeric or humanized antibodies. Frequently, the polypeptides
and antibodies will be labeled by joining, either covalently or
non-covalently, a substance which provides for a detectable
signal. A wide variety of labels and conjugation techniques
are known and are reported extensively in both the scientific
and patent literature. Suitable labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent
moieties, chemiluminescent moieties, magnetic particles, and
the like. Patents, teaching the use of such labels include
U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant,
immunoglobulins may be produced, see Cabilly, U.S. Patent No.
4,816,567; Moore, et al., U.S. Patent No. 4,642,334; and Queen,
et al. (1989) Proc. Nat'1 Acad. Sci. USA 86:10029-10033.
The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can
be prepared where the antibodies are linked to a solid support.
See, e.g., -Wilchek et al. (1984) Meth. Enzymol. 104:3-55. The
converse may be used to purify antibodies.
Antibodies raised against DC-STAMP or DSP-1 will also be
useful to raise anti-idiotypic antibodies. These will be
useful in detecting or diagnosing various immunological
conditions related to expression of the respective antigens.
VI. Nucleic Acids
The described peptide sequences and the related reagents
are useful in detecting, isolating, or identifying a DNA clone
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encoding DC-STAMP or DSP-1, e.g., from a natural source.
Typically, it will be useful in isolating a gene from mammal,
and similar procedures will be applied to isolate genes from
other species, e.g., warm blooded animals, such as birds and
mammals. Cross hybridization will allow isolation of DC-STAMP
from the same, e.g., polymorphic variants, or other species. A
number of different approaches will be available to
successfully isolate a suitable nucleic acid clone.
The purified protein or defined peptides are useful for
LO generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate monoclonal or polyclonal antibodies.
See, e.g., Coligan (1991) Current Protocols in Immunolo4y
Wiley/Greene; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual, Cold Spring Harbor Press.
For example, the specific binding composition could be
used for screening of an expression library made from a cell
line which expresses a DC-STAMP. Screening of intracellular
expression can be performed by various staining or
immunofluorescence procedures. Binding compositions could be
used to affinity purify or sort out cells expressing a surface
fusion protein.
The peptide segments can also be used to predict
appropriate oligonucleotides to screen a library. The genetic
code can be used to select appropriate oligonucleotides useful
as probes for screening. See, e.g., SEQ ID NO: 1 or 4 or 6.
In combination with polymerase chain reaction (PCR) techniques,
synthetic oligonucleotides will be useful in selecting correct
clones from a library. Complementary sequences will also be
used as probes, primers, or antisense strands. Various
fragments should be particularly useful, e.9., coupled with
anchored vector or poly-A complementary PCR techniques or with
complementary DNA of other peptides.
This invention contemplates use of isolated DNA or
fragments to encode an antigenic or biologically active
corresponding polypeptide, particularly lacking the portion
coding an untranslated 5' portion of the described sequence.
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In addition, this invention covers isolated or recombinant DNA
which encodes a biologically active protein or polypeptide and
which is capable of hybridizing under appropriate conditions
with the DNA sequences described herein. Said biologically
5 active protein or polypeptide can be an intact antigen, or
fragment, and have an amino acid sequence disclosed in, e.g.,
SEQ ID NO: 2 or 5 or 7, particularly a mature, secreted
polypeptide. Further, this invention covers the use of
isolated or recombinant DNA, or fragments thereof, which encode
LO proteins which exhibit high identity to membrane DC-STAMP or
DSP-1. The isolated DNA can have the respective regulatory
sequences in the 5' and 3' flanks, e.g., promoters, enhancers,
poly-A addition signals, and others. Alternatively, expression
may be effected by operably linking a coding segment to a
L5 heterologous promoter, e.g., by inserting a promoter upstream
from an endogenous gene.
An "isolated" nucleic acid is a nucleic acid, e.g., an
RNA, DNA, or a mixed polymer, which is substantially separated
from other components which naturally accompany a native
20 sequence, e.g., ribosomes, polymerases, and/or flanking genomic
sequences from the originating species. The term embraces a
nucleic acid sequence which has been removed from its naturally
occurring environment, and includes recombinant or cloned DNA
isolates and chemically synthesized analogs or analogs
25 biologically synthesized by heterologous systems. A
substantially pure molecule includes isolated forms of the
molecule. Generally, the nucleic acid will be in a vector or
fragment less than about 50 kb, usually less than about 30 kb,
typically less than about 10 kb, and preferably less than about
30 6 kb.
An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments,
contain minor heterogeneity. This heterogeneity is typically
found at the polymer ends or portions not critical to a desired
35 biological function or activity.
A "recombinant" nucleic acid is defined either by its
method of production or its structure. In reference to its
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41
method of production, e.g., a product made by a process, the
process is use of recombinant nucleic acid techniques, e.g.,
involving human intervention in the nucleotide sequence,
typically selection or production. Alternatively, it can be a
nucleic acid made by generating a sequence comprising fusion of
two fragments which are not naturally contiguous to each other,
but is meant to exclude products of nature, e.g., naturally
occurring mutants. Thus, e.g., products made by transforming
cells with a nonnaturally occurring vector is encompassed, as
LO are nucleic acids comprising sequence derived using any
synthetic oligonucleotide process. Such is often done to
replace a codon with a redundant codon encoding the same or a
conservative amino acid, while typically introducing or
removing a sequence recognition site.
Alternatively, it is performed to join together nucleic
acid segments of desired functions to generate a single genetic
entity comprising a desired combination of functions not found
in the commonly available natural forms. Restriction enzyme
recognition sites are often the target of such artificial
manipulations, but other site specific targets, e.9.,
promoters, DNA replication sites, regulation sequences, control
sequences, or other useful features may be incorporated by
design. A similar concept is intended for a recombinant, e.g.,
fusion, polypeptide. Specifically included are synthetic
nucleic acids which, by genetic code redundancy, encode
polypeptides similar to fragments of these antigens, and
fusions of sequences from various different species or
polymorphic variants.
A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally
at least about 22 nucleotides, ordinarily at least about 29
nucleotides, more often at least about 35 nucleotides,
typically at least about 41 nucleotides, usually at least about
47 nucleotides, preferably at least about 55 nucleotides, and
in particularly preferred embodiments will be at least about 60
or more nucleotides, e.g., 67, 73, 81, 89, 95, etc.
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42
A DNA which codes for a DC-STAMP or DSP-1 protein will be
particularly useful to identify genes, mRNA, and cDNA species
which code for related or similar proteins, as well as DNAs
which code for homologous proteins from different species.
There will be homologs in other species, including primates,
rodents, canines, felines, birds, and fish. Various DC-STAMP
or DSP-1 proteins should be homologous and are encompassed
herein. However, even proteins that have a more distant
evolutionary relationship to the antigen can readily be
LO isolated under appropriate conditions using these sequences if
they are sufficiently homologous. Primate membrane proteins
are of particular interest.
Recombinant clones derived from the genomic sequences,
e.g., containing introns, will be useful for transgenic
studies, including, e.g., transgenic cells and organisms, and
for gene therapy. See, e.g., Goodnow (1992) "Transgenic
Animals" in Roitt (ed.) Encyclopedia of Immunoloay, Academic
Press, San Diego, pp. 1502-1504; Travis (1992) Science
256:1392-1394; Kuhn, et al. (1991) Science 254:707-710;
Capecchi (1989) Science 244:1288; Robertson (ed. 1987)
Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, IRL Press, Oxford; Rosenberg (1992) J. Clinical
Oncoloav 10:180-199; and Cournoyer and Caskey (1993) Ann. Rev.
Immunol. 11:297-329. Alternatively, expression may be effected
by operably linking a coding segment to a heterologous
promoter, e.9., by inserting a promoter upstream from an
endogenous gene. See, e.g., Treco, et al. W096/29411 or USSN
08/406,030.
Substantial homology, e.g., identity, in the nucleic acid
sequence comparison context means either that the segments, or
their complementary strands, when compared, are identical when
optimally aligned, with appropriate nucleotide insertions or
deletions, in at least about 50% of the nucleotides, generally
at least about 58%, ordinarily at least about 65%, often at
least about 71%, typically at least about 77%, usually at least
about 85%, preferably at least about 95 to 98% or more, and in
particular embodiments, as high as about 99% or more of the
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43
nucleotides. Alternatively, substantial homology exists when
the segments will hybridize under selective hybridization
conditions, to a strand, or its complement, typically using a
sequence of DC-STAMP or DSP-1, e.g., in SEQ ID NO: 1 or 4 or 6.
Typically, selective hybridization will occur when there is at
least about 55% identity over a stretch of at least about 30
nucleotides, preferably at least about 75% over a stretch of
about 25 nucleotides, and most preferably at least about 90%
over about 20 nucleotides. See, Kanehisa (1984) Nuc. Acids
LO Res. 12:203-213. The length of identity comparison, as
described, may be over longer stretches, and in certain
embodiments will be over a stretch of at least about 17
nucleotides, usually at least about 28 nucleotides, typically
at least about 40 nucleotides, and preferably at least about 75
to 100 or more nucleotides.
Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions.
Stringent temperature conditions will usually include
temperatures in excess of about 30° C, usually in excess of
about 37° C, typically in excess of about 55° C, 60° C,
or 65°
C, and preferably in excess of about 70° C. Stringent salt
conditions will ordinarily be less than about 1000 mM, usually
less than about 400 mM, typically less than about 250 mM,
preferably less than about 150 mM, including about 100, 50, or
even 20 mM. However, the combination of parameters is much
more important than the measure of any single parameter. See,
e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.
Hybridization under stringent conditions should give a
background of at least 2-fold over background, preferably at
least 3-5 or more.
For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference
sequences are input into a computer, subsequence coordinates
are designated, if necessary, and sequence algorithm program
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44
parameters are designated. The sequence comparison algorithm
then calculates the percent sequence identity for the test
sequences) relative to the reference sequence, based on the
designated program parameters.
Optical alignment of sequences for comparison can be
conducted, e.9., by the local homology algorithm of Smith and
Waterman (1981) Adv. Appl. Math. 2:482, by the homology
alignment algorithm of Needleman and Wunsch (1970) J. Mol.
Biol. 48:443, by the search for similarity method of Pearson
and Lipman (1988) Proc. Nat'1 Acad. Sci. USA 85:2444, by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, WI), or by
visual inspection (see generally Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP
creates a multiple sequence alignment from a group of related
sequences using progressive, pairwise alignments to show
relationship and percent sequence identity. It also plots a
tree or dendrogram showing the clustering relationships used to
create the alignment. PILEUP uses a simplification of the
progressive alignment method of Feng and Doolittle (1987) J.
Mol. Evol. 35:351-360. The method used is similar to the
method described by Higgins and Sharp (1989) CABIOS 5:151-153.
The program can align up to 300 sequences, each of a maximum
length of 5,000 nucleotides or amino acids. The multiple
alignment procedure begins with the pairwise alignment of the
two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most
related sequence or cluster of aligned sequences. Two clusters
of sequences are aligned by a simple extension of the pairwise
alignment of two individual sequences. The final alignment is
achieved by a series of progressive, pairwise alignments. The
program is run by designating specific sequences and their
amino acid or nucleotide coordinates for regions of sequence
comparison and by designating the program parameters. For
example, a reference sequence can be compared to other test
sequences to determine the percent sequence identity
CA 02391669 2002-05-14
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relationship using the following parameters: default gap weight
(3.00), default gap length weight (0.10), and weighted end
gaps.
Another example of algorithm that is suitable for
5 determining percent sequence identity and sequence similarity
is the BLAST algorithm, which is described Altschul, et al.
(1990) J. Mol. Biol. 215:403-410. Software for performing
BLAST analyses is publicly available through the National
Center for Biotechnology Information
10 (http:www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either
match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence.
15 T is referred to as the neighborhood word score threshold
(Altschul, et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both
directions along each sequence for as far as the cumulative
20 alignment score can be increased. Extension of the word hits
in each direction are halted when: the cumulative alignment
score falls off by the quantity X from its maximum achieved
value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue
25 alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLAST program uses
as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix
(see Henikoff and Henikoff (1989) Proc. Nat'1 Acad. Sci. USA
30 89:10915) alignments (B) of 50, expectation (E) of 10, M=5,
N=4, and a comparison of both strands.
In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and
35 Altschul (1993) Proc. Nat'1 Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication
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46
of the probability by which a match between two nucleotide or
amino acid sequences would occur by chance. For example, a
nucleic acid is considered similar to a reference sequence if
the smallest sum probability in a comparison of the test
nucleic acid to the reference nucleic acid is less than about
0.1, more preferably less than about 0.01, and most preferably
less than about 0.001.
A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the
LO polypeptide encoded by the first nucleic acid is
immunologically cross reactive with the polypeptide encoded by
the second nucleic acid, as described below. Thus, a
polypeptide is typically substantially identical to a second
polypeptide, for example, where the two peptides differ only by
conservative substitutions. Another indication that two
nucleic acid sequences are substantially identical is that the
two molecules hybridize to each other under stringent
conditions, as described below.
DC-STAMP or DSP-1 from other mammalian species can be
cloned and isolated by cross-species hybridization of closely
related species. Homology may be relatively low between
distantly related species, and thus hybridization of relatively
closely related species is advisable. Alternatively,
preparation of an antibody preparation which exhibits less
species specificity may be useful in expression cloning
approaches.
VII. Making DC-STAMP or DSP-1; Mimetics
DNA which encodes the DC-STAMP or DSP-1 or fragments
thereof can be obtained by chemical synthesis, screening cDNA
libraries, or screening genomic libraries prepared from a wide
variety of cell lines or tissue samples. See, e.g., Okayama
and Berg (1982) Mol. Cell. Biol. 2:161-170; Gubler and Hoffman
(1983) Gene 25:263-269; and Glover (ed. 1984) DNA Cloning: A
Practical Approach, IRL Press, Oxford. Alternatively, the
sequences provided herein provide useful PCR primers or allow
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47
synthetic or other preparation of suitable genes encoding a DC-
STAMP or DSP-1; including naturally occurring embodiments.
This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length DC-STAMP or DSP-1 or
fragments which can in turn, e.g., be used to generate
polyclonal or monoclonal antibodies; for binding studies; for
construction and expression of modified molecules; and for
structure/function studies.
Vectors, as used herein, comprise plasmids, viruses,
LO bacteriophage, integratable DNA fragments, and other vehicles
which enable the integration of DNA fragments into the genome
of the host. See, e.g., Pouwels, et al. (1985 and Supplements)
Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.; and
Rodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular
L5 C_lonina Vectors and Their Uses, Buttersworth, Boston, MA.
For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
20 participates in directing the polypeptide to the cell membrane
or in secretion of the polypeptide. A promoter is operably
linked to a coding sequence if it controls the transcription of
the polypeptide; a ribosome binding site is operably linked to
a coding sequence if it is positioned to permit translation.
25 Usually, operably linked means contiguous and in reading frame,
however, certain genetic elements such as repressor genes are
not contiguously linked but still bind to operator sequences
that in turn control expression. See, e.g., Rodriguez, et al.,
Chapter 10, pp. 205-236; Balbas and Bolivar (1990) Methods in
30 Enzymoloav 185:14-37; and Ausubel, et al. (1993) Current
_Protocols in Molecular Bioloav, Greene and Wiley, NY.
Representative examples of suitable expression vectors
include pCDNAI; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell
35 51:503-512; and a baculovirus vector such as pAC 373 or pAC
610. See, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199.
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48
It will often be desired to express a DC-STAMP or DSP-1
polypeptide in a system which provides a specific or defined
glycosylation pattern. See, e.g., Luckow and Summers (1988)
Bio/Technoloay 6:47-55; and Kaufman (1990) Meth. Enzymol.
185:487-511.
The DC-STAMP or DSP-1, or a fragment thereof, may be
engineered to be phosphatidyl inositol (PI) linked to a cell
membrane, but can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl
LO inositol phospholipase-C. This releases the antigen in a
biologically active form, and allows purification by standard
procedures of protein chemistry. See, e.g., Low (1989)
Biochim. BioQhys. Acta 988:427-454; Tse, et al. (1985) Science
230:1003-1008; and Brunner, et al. (1991) J. Cell Biol.
114:1275-1283.
Now that the DC-STAMP or DSP-1 has been characterized,
fragments or derivatives thereof can be prepared by
conventional processes for synthesizing peptides. These
include processes such as are described in Stewart and Young
(1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,
Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of
Peptide Synthesis, Springer-Verlag, New York; Bodanszky (1984)
The Principles of Peptide Synthesis, Springer-Verlag, New York;
'and Villafranca (ed. 1991) Techniaues in Protein Chemistry II,
Academic Press, San Diego, Ca.
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VIII. Uses
The present invention provides reagents which will find
use in diagnostic applications as described elsewhere herein,
e.g., in DC, T, NK, monocyte, or mast cell mediated conditions,
or below in the description of kits for diagnosis. The gene
may be useful in forensic sciences, e.g., to distinguish rodent
from human, or as a marker to distinguish between different
cells exhibiting differential expression or modification
patterns.
This invention also provides reagents with significant
commercial and/or therapeutic potential. The DC-STAMP or DSP-1
(naturally occurring or recombinant), fragments thereof, and
antibodies thereto, along with compounds identified as having
binding affinity to DC-STAMP or DSP-1, should be useful as
reagents for teaching techniques of molecular biology,
immunology, or physiology. Appropriate kits may be prepared
with the reagents,~e.g., in practical laboratory exercises in
production or use of proteins, antibodies, cloning methods,
histology, etc.
The reagents will also be useful in the treatment of
conditions associated with abnormal physiology or development,
including immunological conditions. They may be.useful in
vitro tests for presence or absence of interacting components,
which may correlate with success of particular treatment
strategies. In particular, modulation of physiology of
various, e.g., hematopoietic or lymphoid, cells will be
achieved by appropriate methods for treatment using the
compositions provided herein. See, e.9., Thomson (ed. 1998)
_The Cytokine Handbook (3d ed.) Academic Press, San Diego;
Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating
Factors Cambridge University Press; and Aggarwal and Gutterman
(1991) Human Cytokines Blackwell Pub.
For example, a disease or disorder associated with
abnormal expression or abnormal signaling by a DC-STAMP should
be a likely target for an agonist or antagonist. The new
membrane proteins should play a role in regulation or
development of hematopoietic cells, e.g., lymphoid cells, which
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affect immunological responses, e.g., inflammation and/or
autoimmune disorders. Alternatively, it may affect vascular
physiology or development, or neuronal effects.
In particular, the DC-STAMP is likely to be important in
5 mediating DC function. DC are the professional antigen
presenting cells to T and B cells, and should be important in T
cell mediated immune responses. Increases in T cell immunity
should be important in, e.g., tumor immunotherapy, allergic
conditions, and vaccine adjuvants. Important tumors include,
LO e.g., carcinomas, including lung, colon, prostate, and breast,
and melanomas. See, e.g., Bertino, et al. (eds. 1996)
Encyclopedia of Cancer Academic Press; Devita, et al. (eds.
1997) Cancer' Principles & Practice of Oncology Lippincott,
Williams and Wilkins; Devita (1997) Principles and Practice of
15 Oncology Lippincott Williams and Wilkins; Cavalli, et al.
(1996) Textbook of Medical Oncology Dunitz Martin Ltd; Horwich
(ed. 1995) Oncoloay~ A Multidisciplinary Textbook Lippincott-
Raven; Peckham, et al. (eds. 1995) Oxford Textbook of Oncology
Oxford Univ. Press; Mendelsohn, et al. (1995) The Molecular
20 Basis of Cancer Saunders, Philadelphia; and McArdle (1990)
Surgical Oncoloay~ Current Concepts and Practice Butterworth-
Heinemann. Among the allergic conditions, e.g., where a shift
from Th2 humoral responses to Thl cellular responses may be
indicated, include asthma, pollen rhinitis, medicament
25 allergies, food allergies, house dust mite allergies, etc.
See, e.g., See, e.g., Lockey and Bukantz (eds. 1998) Allergen
Immunotherapv; and Patterson (ed. 1997) Allergic Diseases:
Diagnosis and Management. Conversely, decreases in T cell
immunity should be important in, e.g., autoimmune conditions or
30 transplantation rejection circumstances. Autoimmune diseases
include, e.g., diabetes melitis, psoriasis, and multiple
sclerosis. See, e.g., Morrow (ed. 1999) Autoimmune Rheumatic
Disease, Weetman (ed. 1998) Endocrine Autoimmunity and
Associated Conditions; Rose and Mackay (eds. 1998) The
35 Autoimmune Diseases (3d ed.) Academic Press, San Diego; Kay
(ed. 1997) Allerav and Allergic Diseases Blackwell Science,
Malden MA; Samter, et al. (eds.) Immunoloaical Diseases vols. 1
CA 02391669 2002-05-14
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51
and 2, Little, Brown and Co.; and Coutinho and Kazatchkine
(eds. 1993) Autoimmunity: PhysiolocLv and Disease. Transplant
rejection and treatment are an established branch of medicine
as described, e.g., in Racusen (ed. 1998) Kidnev Transplant
Rejection; Kelso and Clouston (1996) Cytokines in
Transplantation ; and Solez, et al. (eds. 1996) Solid Or a~n
Transplant Rejection. Combination treatments might be used,
combining a therapeutic related to the DC-STAMP or DSP-1
signaling with another therapeutic used to treat symptoms of
LO the conditions, e.g., with Flt3 ligand, G, CSF, radiation or
chemotherapy, antihistamines, IL-10, Tregl cells, cyclosporin,
or interferons.
Likewise, the DSP-1 therapeutic reagents may be useful to
modulate function of monocyte, T, NK, or mast cell mediated
conditions. It should be useful as a mast cell marker, being
present on those cells, and likely modulates signaling with or
by those cells.
Various abnormal conditions are known in different cell
types which will produce DC-STAMP, e.g., as evaluated by mRNA
expression by Northern blot analysis. See Berkow (ed.) The
Merck Manual of Diagnosis and Therapv, Merck & Co., Rahway,
N.J.; Thorn, et al. Harrison's Principles of Internal Medicine,
McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford
Textbook of Medicine, Oxford University Press, Oxford. Many
other medical conditions and diseases involve activation by T
cells, and many of these will be responsive to treatment by an
agonist or antagonist provided herein. See, e.g., Stites and
Terr (eds.; 1991) Basic and Clinical Immunoloay Appleton and
Lange, Norwalk, Connecticut; and Samter, et al. (eds.)
Immunoloaical Diseases Little, Brown and Co. These problems
should be susceptible to prevention or treatment using
compositions provided herein.
DC-STAMP or DSP-1, antagonists, antibodies, etc., can be
purified and then administered to a patient, veterinary or
human. These reagents can be combined for therapeutic use with
additional active or inert ingredients, e.g., in conventional
pharmaceutically acceptable carriers or diluents, e.g.,
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immunogenic adjuvants, along with physiologically innocuous
stabilizers, excipients, or preservatives. These combinations
can be sterile filtered and placed into dosage forms as by
lyophilization in dosage vials or storage in stabilized aqueous
preparations. This invention also contemplates use of
antibodies or binding fragments thereof, including forms which
are not complement binding.
Drug screening using DC-STAMP or DSP-1 or fragments
thereof can be performed to identify compounds having binding
LO affinity to or other relevant biological effects on DC-STAMP or
DSP-1 functions, including isolation of associated components.
Subsequent biological assays can then be utilized to determine
if the compound has intrinsic stimulating activity and is
therefore a blocker or antagonist in that it blocks the
activity of DC-STAMP or DSP-1 signaling. Likewise, a compound
having intrinsic stimulating activity can activate the signal
pathway and is thus an agonist in that it simulates the
activity of DC-STAMP or DSP-1 signaling. Antibodies may be
used to mediate, e.g., antigen dependent cell-mediated
cytotoxicity, complement fixation, to localize enzymes or other
means of activating inert pro-toxins, as diagnostic labels, or
conjugated to compounds which will absorb energy to eliminate
cells in proximity to where the antibody binds.
This invention further contemplates the therapeutic use of
blocking antibodies to these antigens as antagonists and of
stimulatory antibodies as agonists. This approach should be
particularly useful with other DC-STAMP or DSP-1 species
variants.
The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the
patient, and other medicants administered. Thus, treatment
dosages should be titrated to optimize safety and efficacy.
Typically, dosages used in vitro may provide useful guidance in
the amounts useful for in situ administration of these
reagents. Animal testing of effective doses for treatment of
particular disorders will provide further predictive indication
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53
of human dosage. Various considerations are described, e.g.,
in Gilman, et al. (eds.) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, latest Ed., Pergamon
Press; and Reminaton's Pharmaceutical Sciences, latest ed.,
Mack Publishing Co., Easton, Penn. Methods for administration
are discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal
diffusion, and others. Pharmaceutically acceptable carriers
will include water, saline, buffers, and other compounds
LO described, e.g., in the Merck Index, Merck & Co., Rahway, New
Jersey. Dosage ranges would ordinarily be expected to be in
amounts lower than 1 mM concentrations, typically less than
about 10 ~,M concentrations, usually less than about 100 nM,
preferably less than about 10 pM (picomolar), and most
preferably less than about 1 fM (femtomolar), with an
appropriate carrier. Slow release formulations, or a slow
release apparatus will often be utilized for continuous or long
term administration. See, e.g., Langer (1990) Science
249:1527-1533.
DC-STAMP or DSP-1, fragments thereof, and antibodies to it
or its fragments, antagonists, and agonists, may be
administered directly to the host to be treated or, depending
on the size of the compounds, it may be desirable to conjugate
them to carrier proteins such as ovalbumin or serum albumin
prior to their administration. Therapeutic formulations may be
administered in many conventional dosage formulations. While
it is possible for the active ingredient to be administered
alone, it is preferable to present it as a pharmaceutical
formulation. Formulations typically comprise at least one
active ingredient, as defined above, together with one or more
acceptable carriers thereof. Each carrier should be both
pharmaceutically and physiologically acceptable in the sense of
being compatible with the other ingredients and not injurious
to the patient. Formulations include those suitable for oral,
rectal, nasal, topical, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration.
The formulations may conveniently be presented in unit dosage
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form and may be prepared by any methods well known in the art
of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and
Gilman's~ The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Reminaton's Pharmaceutical Sciences, 17th
ed. (1990), Mack Publishing Co., Easton, Penn.; Avis, et al.
(eds. 1993) Pharmaceutical Dosage Forms: Parenteral
Medications, Dekker, New York; Lieberman, et al. (eds. 1990)
Pharmaceutical Dosage Forms: Tablets, Dekker, New York; and
Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms:
Disperse Systems, Dekker, New York. The therapy of this
invention may be combined with or used in association with
other agents, e.9., other therapeutics for treatment of
symptoms of the indications described.
Both naturally occurring and recombinant forms of the DC-
STAMPS or DSP-is of this invention are particularly useful in
kits and assay methods which are capable of screening compounds
for binding activity to the proteins. Several methods of
automating assays have been developed in recent years so as to
permit screening of tens of thousands of compounds in a short
period. See, e.9., Fodor, et al. (1991) Science 251:767-773,
which describes means for testing of binding affinity by a
plurality of defined polymers synthesized on a solid substrate.
The development of suitable assays can be greatly facilitated
by the availability of large amounts of purified, soluble
antigens as provided by this invention.
Other methods can be used to determine the critical
residues in DC-STAMP or DSP-1 counterreceptor or ligand
interactions. Mutational analysis can be performed, e.g., see
Somoza, et al. (1993) J. Exptl. Med. 178:549-558, to determine
specific residues critical in the interaction and/or signaling.
PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (King
and Sternberg (1996) Protein Sci. 5:2298-2310) can provide
secondary structure predictions of a-helix (H), (3-strand (E),
or coil (L). Surface exposed residues would affect ligand or
receptor binding, while embedded residues would affect general
structure.
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For example, antagonists can normally be found once the
antigen has been structurally defined, e.g., by tertiary
structure data. Testing of potential interacting analogs is
now possible upon the development of highly automated assay
5 methods using a purified DC-STAMP or DSP-1. In particular, new
agonists and antagonists will be discovered by using screening
techniques described herein. Of particular importance are
compounds found to have a combined binding affinity for a
spectrum of DC-STAMP molecules, e.g., compounds which can serve
10 as antagonists for species variants of DC-STAMP.
One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing a DC-STAMP or DSP-1.
Cells may be isolated which express an antigen in isolation
15 from other molecules. Such cells, either in viable or fixed
form, can be used for standard binding partner binding assays.
See also, Parce, et al. (1989) Science 246:243-247; and Owicki,
et al. (1990) Proc. Nat'1 Acad. Sci. USA 87:4007-4011, which
describe sensitive methods to detect cellular responses.
20 Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to wn antigen and is described in
detail in Geysen, European Patent Application 84/03564,
published on September 13, 1984. First, large numbers of
25 different small peptide test compounds are synthesized on a
solid substrate, e.g., plastic pins or some other appropriate
surface, see Fodor, et al. (1991). Then all the pins are
reacted with solubilized, unpurified or solubilized, purified
DC-STAMP, and washed. The next step involves detecting bound
30 DC-STAMP.
Rational drug design may also be based upon structural
studies of the molecular shapes of the DC-STAMP or DSP-1 and
other effectors or analogs. Effectors may be other proteins
which mediate other functions in response to binding, or other
35 proteins which normally interact with the membrane proteins,
e.g., a receptor. One means for determining which sites
interact with specific other proteins is a physical structure
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determination, e.g., x-ray crystallography or 2 dimensional NMR
techniques. These will provide guidance as to which amino acid
residues form molecular contact regions, as modeled, e.g.,
against other cytokine-receptor models. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson (1976) Protein Crvstalloqraphv, Academic
Press, New York.
IX. Kits
LO This invention also contemplates use of DC-STAMP or DSP-1
proteins, fragments thereof, peptides, and their fusion
products in a variety of diagnostic kits and methods for
detecting the presence of another DC-STAMP or DSP-1 or binding
partner. Typically the kit will have a compartment containing
either a defined DC-STAMP or DSP-1 peptide or gene segment or a
reagent which recognizes one or the other, e.g., DC-STAMP or
DSP-1 fragments or antibodies.
A kit for determining the binding affinity of a test
compound to a DC-STAMP would typically comprise a test
compound; a labeled compound, for example a binding partner or
antibody having known binding affinity for DC-STAMP; a source
of DC-STAMP (naturally occurring or recombinant); and a means
for separating bound from free labeled compound, such as a
solid phase for immobilizing the molecule. Compartments
containing reagents, and instructions, will normally be
provided. Once test compounds are screened, those having
suitable binding affinity to the antigen can be evaluated in
suitable biological assays, as are well known in the art, to
determine whether they act as agonists or antagonists to DC-
STAMP signaling pathway. The availability of recombinant DC-
STAMP polypeptides also provides well defined standards for
calibrating such assays.
Antibodies, including antigen binding fragments, specific
for the DC-STAMP or DSP-1 or fragments are useful in diagnostic
applications to detect the presence of elevated levels of
antigen and/or its fragments. Such diagnostic assays can
employ lysates, live cells, fixed cells, immunofluorescence,
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cell cultures, body fluids, and further can involve the
detection of antigens related to the antigen in serum, or the
like. Diagnostic assays may be homogeneous (without a
separation step between free reagent and antigen-binding
partner complex) or heterogeneous (with a separation step).
Various commercial assays exist, such as radioimmunoassay
(RIA), enzyme-linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), enzyme-multiplied immunoassay technique
(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and
LO the like. See, e.g., Van Vunakis, et al. (1980) Meth Enzvmol.
70:1-525; Harlow and Lane (1980) Antibodies: A Laboratory
Manual, CSH Press, NY; and Coligan, et al. (eds. 1993) Current
Protocols in Immunoloay, Greene and Wiley, NY.
Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against a DC-STAMP or DSP-1, as such may
be diagnostic of various abnormal states. For example,
overproduction of DSP-1 may result in production of various
immunological reactions which may be diagnostic of abnormal
physiological states, particularly in allergic conditions.
Frequently, the reagents for diagnostic assays are
supplied in kits, so as to optimize the sensitivity of the
assay. For the subject invention, depending upon the nature of
the assay, the protocol, and the label, either labeled or
unlabeled antibody or binding partner, or labeled antigen is
provided. This is usually in conjunction with other additives,
such as buffers, stabilizers, materials necessary for signal
production such as substrates for enzymes, and the like.
Preferably, the kit will also contain instructions for proper
use and disposal of the contents after use. Typically the kit
has compartments for each useful reagent. Desirably, the
reagents are provided as a dry lyophilized powder, where the
reagents may be reconstituted in an aqueous medium providing
appropriate concentrations of reagents for performing the
assay.
Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways. For
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example, labeling may be achieved by covalently or non-
covalently joining a moiety which directly or indirectly
provides a detectable signal. In any of these assays, the
binding partner, test compound, antigen, or antibodies thereto
can be labeled either directly or indirectly. Possibilities
for direct labeling include label groups: radiolabels such as
125I~ enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and
alkaline phosphatase, and fluorescent labels (U.S. Pat. No.
3,940,475) capable of monitoring the change in fluorescence
LO intensity, wavelength shift, or fluorescence polarization.
Possibilities for indirect labeling include biotinylation of
one constituent followed by binding to avidin coupled to one of
the above label groups.
There are also numerous methods of separating the bound
from the free antigen, or alternatively the bound from the free
test compound. The antigen can be immobilized on various
matrixes followed by washing. Suitable matrixes include
plastic such as an ELISA plate, filters, and beads. See, e.g.,
Coligan, et al. (eds. 1993) Current Protocols in ImmunoloQV,
Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitable
separation techniques include, without limitation, the
fluorescein antibody magnetizable particle method described in
Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double
antibody magnetic particle separation as described in U.S. Pat.
No. 4,659,678.
Methods for linking proteins or their fragments to the
various labels are well known. Many of the techniques involve
the use of activated carboxyl groups either through the use of
carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin
such as maleimide, for linkage, or the like. Fusion proteins
will also find use in these applications.
Another diagnostic aspect of this invention involves use
of oligonucleotide or polynucleotide sequences taken from the
sequence of a DC-STAMP or DSP-1. These sequences can be used
as probes for detecting levels of the antigen message in
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59
samples from patients suspected of having an abnormal
condition, e.g., inflammatory or autoimmune. The preparation
of both RNA and DNA nucleotide sequences, the labeling of the
sequences, and the preferred size of the sequences has received
ample description and discussion in the literature. See, e.g.,
Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79:4381-
4385; Caskey (1987) Science 236:962-967; and Wilchek et al.
(1988) Anal. Biochem. 171:1-32.
Diagnostic kits which also test for the qualitative or
quantitative expression of other molecules are also
contemplated. Diagnosis or prognosis may depend on the
combination of multiple indications used as markers. Thus,
kits may test for combinations of markers. See, e.g., Viallet,
et al. (1989) Progress in Growth Factor Res. 1:89-97. Kits may
contain additional reagents to evaluate other cell subsets.
X. Isolating a DC-STAMP or DSP-1 Ligand or Receptor
Both DC-STAMP and DSP-1 are cell surface antigens, which
may be receptor for a ligand or another surface antigen.
Having isolated one component of such an interaction, methods
exist for isolating a ligand or binding receptor partner. See,
Gearing, et al. (1989) EMBO J. 8:3667-3676. For example, means
to label the antigen without interfering with the binding to
its partner can be determined. For example, an affinity label
can be fused to either the amino- or carboxyl-terminus of the
ligand. Such label may be a FLAG epitope tag, or, e.g., an Ig
or Fc domain. An expression library can be screened for
specific binding of the antigen, e.9., by cell sorting, or
other screening to detect subpopulations which express such a
binding component. See, e.g., Ho, et al. (1993) Proc. Nat'1
Acad: Sci. USA 90:11267-11271; and Liu, et al. (1994) J.
Immunol. 152:1821-29. Alternatively, a panning method may be
used. See, e.g., Seed and Aruffo (1987) Proc. Nat'1 Acad. Sci.
USA 84:3365-3369.
Protein cross-linking techniques with label can be applied
to isolate binding partners of the DC-STAMP or DSP-1. This
would allow identification of proteins which specifically
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interact with the antigen, e.g., in a ligand-receptor or
receptor-receptor manner.
Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
5 apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
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EXAMPLES
I. General Methods
Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning,
A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring
Harbor Press, NY; Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed.) Vols. 1-3, CSH Press, NY; Ausubel,
et al., Bioloay Greene Publishing Associates, Brooklyn, NY; or
Ausubel, et al. (1987 and Supplements) Current Protocols in
LO Molecular Bioloav Wiley/Greene, NY; Innis, et al. (eds. 1990)
PCR Protocols A Guide to Methods and Applications Academic
Press, NY. Methods for protein purification include such
methods as ammonium sulfate precipitation, column
chromatography, electrophoresis, centrifugation,
crystallization, and others. See, e.g., Ausubel, et al. (1987
and periodic supplements); Deutscher (1990) "Guide to Protein
Purification," Methods in Enzymoloay vol. 182, and other
volumes in this series; Coligan, et al. (1995 and supplements)
Current Protocols in Protein Science John Wiley and Sons, New
York, NY; P. Matsudaira (ed. 1993) A Practical Guide to
Protein and Peptide Purification for MicroseQUencina, Academic
Press, San Diego, CA; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway, NJ,
or Bio-Rad, Richmond, CA. Combination with recombinant
techniques allow fusion to appropriate segments (epitope tags),
e.g., to a FLAG sequence or an equivalent which can be fused,
e.g., via a protease-removable sequence. See, e.g., Hochuli
(1989) Chemische Industrie 12:69-70; Hochuli (1990)
"Purification of Recombinant Proteins with Metal Chelate
Absorbent" in Setlow (ed.) Genetic Engineering, Principle and
Methods 12:87-98, Plenum Press, NY; and Crowe, et al. (1992)
QIAexpress~ The Hicih Level Expression & Protein Purification
System QUIAGEN, Inc., Chatsworth, CA.
Standard immunological .techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunoloay vols. 1-4, Blackwell Science; Coligan (1991) Current
Protocols in Immunoloay Wiley/Greene, NY; and Methods in
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Enzymoloay vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132,
150, 162, and 163. Cytokine assays are described, e.g., in
Thomson (ed. 1998) The Cytokine Handbook (3d ed.) Academic
Press, San Diego; Mire-Sluis and Thorpe (1998) Cvtokines
Academic Press, San Diego; Metcalf and Nicola (1995) The
Hematopoietic Colony Stimulatinct Factors Cambridge University
Press; and Aggarwal and Gutterman (1991) Human CYtokines
Blackwell Pub.
Assays for vascular biological activities are well known
in the art. They will cover angiogenic and angiostatic
activities in tumor, or other tissues, e.g., arterial smooth
muscle proliferation (see, e.g., Koyoma, et al. (1996) Cell
87:1069-1078), monocyte adhesion to vascular epithelium (see
McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc. See
also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995)
Am. J. Pathol. 147:668-677; Thyberg, et al. (1990)
Atherosclerosis 10:966-990; and Gumbiner (1996) Cell 84:345-
357.
Assays for neural cell biological activities are
described, e.g., in Wouterlood (ed. 1995) Neuroscience
Protocols modules 10, Elsevier; Methods in Neurosciences
Academic Press; and Neuromethods Humana Press, Totowa, NJ.
Methodology of developmental systems is described, e.g., in
Meisami (ed.) Handbook of Human Growth and Developmental
Bioloav CRC Press; and Chrispeels (ed.) Molecular Techniaues
and Approaches in Developmental Bioloay Interscience.
FACS analyses are described in Melamed, et al. (1990) Flow
_Cytometrv and Sorting Wiley-Liss, Inc., New York, NY; Shapiro
(1988) Practical Flow Cytometry Liss, New York, NY; and
Robinson, et al. (1993) Handbook of Flow Cvtometry Methods
Wiley-Liss, New York, NY.
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II. Cloning of Human DC-STAMP and DSP-1
The sequence of the primate, e.g., human, DC-STAMP gene is
provided in Table 1. The sequence is derived from sequence of
a cDNA clone isolated from dendritic cells. This sequence
allows preparation of PCR primers, or probes, to determine
cellular distribution of the gene. The sequence allows
isolation of genomic DNA which encode the message.
The ORF of DC-STAMP predicts a protein of 470 amino acids,
with a predicted molecular weight of around 53 kD and an
isoelectric point of 9.41. The amino terminus of the protein
starts with a short stretch of hydrophobic amino acids, which
predicts an uncleavable signal sequence (pSORT, Osaka
University, Japan). Hydrophobicity analysis of the sequence
revealed 5 strong and 2 weak hydrophobic stretches of 18-20
amino acids, suggesting that the DC-STAMP molecule is spanning
the membrane multiple times. The TM Predict program from BCM
Search Launcher (K. Hofman and W. Hofman) suggests a topology
model in which the DC-STAMP protein contains 7 transmembrane
spanning regions, with the N-terminus located outside and the
C-terminus on the luminal side of the membrane.
Interestingly, the DC-STAMP protein contains two pairs of
cysteine residues, one at the start of the first transmembrane
domain (TM1), the other at the end of the second transmembrane
domain (TM2). These cysteines might form a disulphide bridge
near the outer side of the membrane, and stabilize the protein
structure. Prosite analysis of the protein revealed 3
potential glycosylation sites, two on the second and one on the
third putative extracellular loop. In addition, there is a
consensus sequence for phosphorylation by protein kinase C
between the fifth and sixth transmembrane region, which is the
second intracellular loop according to the proposed topology.
The 72 amino acid cytoplasmic tail of DC-STAMP contains several
serine residues, two of which might serve as a target for
phosphorylation . Interestingly, the C-terminus of the DC-
STAMP protein is surprisingly rich in positively charged
residues, comprising 25% of the tail and conferring an overall
positive charge (+14).
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The DSP-1 gene was isolated from a cDNA library made from
human HEL cells.
Using the probe or PCR primers, various tissues or cell
types are probed to determine cellular distribution. PCR
products are cloned using, e.g., a TA cloning kit (Invitrogen).
The resulting cDNA plasmids are sequenced from both termini on
an automated sequencer (Applied Biosystems).
Leukocyte preparations
LO PBMC were obtained by leukophoresis of blood from healthy
donors, and adherence for 2 hours resulted in a non-adherent
PBL fraction. Monocytes were elutriated from PBMC by
counterflow centrifugation, resulting in a population of cells
that were greater than 85% CD14+. Fractions were cultured in
Iscove's medium supplemented with 5% FCS and 1%
antibiotics/antimyotics (Life Technologies Inc., Grand Island,
NY). Both the non-adherent PBL and total PBMC were stimulated
with phytohemagglutinin (PHA; 1 ~g/ml; Murex Diagnostics Ltd,
Dartford, England) and rIL-2 (200 U/ml; Cetus Corp.,
Emeryville, CA) for 16 h. Elutriated monocytes were stimulated
with 2 ~,g/ml LPS for 16 h.
DC were generated in vitro from monocytes using a
modification of described methods. See Ridge, et al. (1998)
Nature 393:474-478; and Bennett, et al. (1998) Nature 393:478-
480. Monocytes were cultured in AIM-V medium (Life
Technologies Ltd, Paisley, Scotland) supplemented with 5% fetal
calf serum and in the presence of 800 U/ml GM-CSF and 500 U/ml
IL-4 (both from Schering-Plough, The Netherlands) for 5-7 days.
Resulting DC were collected directly or after activation with
either LPS for 16 h (2 ~,g/ml), or after the sequential addition
of TNFa (10 ng/ml, 24 h) and the activating anti-CD40 antibody
MAB89, generously provided by DNAX, Palo Alto, CA (1.5 ~g/ml,
24 h). Purified tonsil B lymphocytes were isolated according
to the method described by Falkoff, et al. (1982). J. Immunol.
Methods 50:39-49.
cDNA library preparation
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Complementary DNA libraries were prepared. See Marland,
et al. (1997) in Ricciardi-Castognoli (ed.) Dendritic Cells in
Fundamental and Clinical Immunoloav, Vol 3, Plenum Publ.
Corporation; and Adema, et al. (1997) Nature 387:713-717.
5 Nucleotide sequences were analyzed against the non-redundant
GenBank and EMBL databases using the BLAST program. See
Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
Northern blot analysis for DC-STAMP
10 Total RNA was isolated from DC cultures, e.g., monocyte-
derived DC cultured for 7 days in IL-4 and GM-CSF, freshly
isolated leukocytes and cell lines using the guanidine
thiocyanate/cesium chloride procedure. Poly(A)+ RNA was
isolated from the DC fraction by affinity chromatography
15 (Oligotex, Qiagen). Per sample 20 ~g total RNA or 2 ~g
poly(A)+ RNA was resolved overnight on a formaldehyde gel and
transferred to a nylon membrane by capillary blotting.
Hybridization was performed overnight at 65° C in Church
solution (0.5 M NaHP04, pH 7.2; 7 °s SDS; 0,5 M EDTA). Multiple
20 tissue Northern blot #7780-1 (Clontech, Palo Alto, CA) was
probed and washed under stringent conditions according to the
manufacturer's recommendations. Both Northern blots were
probed with the 444 by SalI-RcaI fragment, comprising part of
the 3' UTR of DC-STAMP, randomly labeled with 32P (T7
25 QuickPrime Kit, Pharmacia).
RT-PCR
Total RNA was isolated using Trizol Reagent (Gibco BRL)
and treated with RNAse free DNAse (Boehringer Mannheim). 1 ~g
RNA was transcribed into cDNA using an oligodT primer and
30 Superscript II reverse transcriptase (RT, Gibco BRL). Half of
the cDNA was used to amplify the DC-STAMP message, according to
a standard PCR protocol (24 cycles). The primers were located
in the most 3' part of the DC-STAMP ORF, yielding a specific
product of 334 bp. As a control for RNA quality, the other
35 half of the cDNA was used to amplify a (3 actin product of 328
by (18 cycles). Southern blot analysis of the PCR products was
performed using an 32P-end labeled internal oligonucleotide
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66
(Klenow polymerase, Boehringer Mannheim) from either DC-STAMP
or ~i actin. Samples without RT were always completely
negative.
Library screening and 5' RACE PCR
100,000 colonies from a cDNA library derived from non-
stimulated DC were screened using the randomly labeled 444 by
RcaI/SalI fragment from the original 936 by cDNA clone as a
probe. The most 5' end of the DC-STAMP cDNA was isolated by 5'
RACE PCR. Briefly, 1 ~g of total DC RNA was transcribed into
cDNA (Superscript II Reverse Transcriptase, Gibco BRL), using a
DC-STAMP specific 5' RACE-1 primer. The cDNA was purified with
a QIAQuick PCR purification kit (Qiagen) and subsequently
tailed using 50 U of Terminal Transferase (Boehringer Mannheim)
in the presence of dCTP (5 ~M) and 0.75 mM CoCl2 (15 minutes
37° C). The tailed cDNA was extracted once with
phenol/chloroform and precipitated using glycogen (50 fig). 5%
of the tailed cDNA was used in a hemi-nested PCR reaction,
using nested DC-STAMP specific primer 5'RACE-2 and a 5' primer
annealing to the C-tail of the cDNA. 30 PCR cycles were
performed using a standard program (1 min 94° C, 1 min 58° C, 1
min 72° C, 10 min extension at 72° C). The resulting PCR
product was gel-purified and cloned into the TA-cloning vector
pGEM-T (Promega). The overlapping cDNA fragments were
sequenced by the dideoxy chain reaction (AutoRead, Sequencing
kit, Pharmacia Biotech) on the ALF Express automated sequencer
(Pharmacia Biotech). The complete ORF of DC-STAMP was
amplified from oligo dT transcribed cDNA (Superscript II, Gibco
BRL) using the Expand Long Template PCR System (30 cycles,
Boehringer Mannheim) and cloned into pGEM-T Easy. Sequence
analysis of several clones confirmed the sequence obtained by
5'RACE PCR.
DC-STAMP cDNA encodes a 470 amino acid multimembrane spanning
molecule
Since expression of DC-STAMP was specifically detected in
DC, a DC cDNA library was screened with a specific probe for
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DC-STAMP in order to obtain the full-length transcript.
Several DC-STAMP cDNA clones were isolated, of which the
longest clone contained an insert of 1.4 kb, identical to the
original clone at its 3' end. 5' RACE PCR resulted in the
cloning of the most 5' region of the DC-STAMP messenger.
Northern blot analysis using this 5' DC-STAMP fragment as a
probe resulted in the same 2.3 kb message as described,
indicating that both fragments belong to a single cDNA. The
cDNA encoding DC-STAMP has a total length of 1954 bp, which
nicely fits with the 2.3 kb messenger on Northern blot,
suggesting a poly A tail of around 350 bp. It contains a
single long ORF of 1410 nucleotides starting with the first ATG
codon at nucleotide 52, which is in the appropriate sequence
context for translation initiation (Kozak (1987) Nucleic Acid.
Res. 15:8125-8148), and is followed by a 490-nucleotide 3' UTR.
The poly A-tail is preceded by the polyadenylation signal
sequence ATTAAA. See Table 1.
Comparison of the DC-STAMP amino acid and nucleotide
sequence with known sequences in the GenBank/EMBL databases
revealed no homology, except two nucleotide matches with
unpublished EST fragments in the dbEST, derived from human skin
tumor and human neuroendocrine lung carcinoid (accession
numbers AA380009 and AI268407, respectively).
The sequence of the primate, e.g., human, DSP-1 gene is
provided in Table 2. The sequence is derived from sequence of
a cDNA clone isolated human HEL cells.
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III. Cellular Expression of Antigen
An appropriate probe or primers specific for cDNA encoding
primate antigen are prepared. Typically, the probe is labeled,
e.g., by random priming.
Southern Analysis: DNA (5 fig) from a primary amplified cDNA
library was digested with appropriate restriction enzymes to
release the inserts, run on a 1% agarose gel and transferred to a
nylon membrane (Schleicher and Schuell, Keene, NH).
Samples for human mRNA isolation may include: peripheral
LO blood mononuclear cells (monocytes, T cells, NK cells,
granulocytes, B cells), resting (T100); peripheral blood
mononuclear cells, activated with anti-CD3 for 2, 6, 12 h
pooled (T101); T cell, THO clone Mot 72, resting (T102); T
cell, THO clone Mot 72, activated with anti-CD28 and anti-CD3
for 3, 6, 12 h pooled (T103); T cell, THO clone Mot 72, anergic
treated with specific peptide for 2, 7, 12 h pooled (T104); T
cell, TH1 clone HY06, resting (T107); T cell, TH1 clone HY06,
activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled
(T108); T cell, TH1 clone HY06, anergic treated with specific
peptide for 2, 6, 12 h pooled (T109); T cell, TH2 clone HY935,
resting (T110); T cell, TH2 clone HY935, activated with anti-
CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); T cell tumor
lines Jurkat and Hut78, resting (T117); T cell clones, pooled
AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting
(T118); T cell random y8 T cell clones, resting (T119); CD28- T
cell clone; Splenocytes, resting (B100); Splenocytes, activated
with anti-CD40 and IL-4 (B101); B cell EBV lines pooled GIT49,
RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102); B cell line
JY, activated with PMA and ionomycin for 1, 6 h pooled (B103);
NK 20 clones pooled, resting (K100); NK 20 clones pooled,
activated with PMA and ionomycin for 6 h (K101); NKL clone,
derived from peripheral blood of LGL leukemia patient, IL-2
treated (K106); hematopoietic precursor line TFl, activated
with PMA and ionomycin for 1, 6 h pooled (C100); U937
premonocytic line, resting (M100); U937 premonocytic line,
activated with PMA and ionomycin for 1, 6 h pooled (M101);
elutriated monocytes, activated with LPS, IFNy, anti-IL-10 for
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1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes,
activated with LPS, IFNy, IL-10 for 1, 2, 6, 12, 24 h pooled
(M103); elutriated monocytes, activated with LPS, IFNy, anti-
IL-10 for 4, 16 h pooled (M106); elutriated monocytes,
activated with LPS, IFNy, IL-10 for 4, 16 h pooled (M107);
elutriated monocytes, activated LPS for 1 h (M108); elutriated
monocytes, activated LPS for 6 h (M109); DC 70% CDla+, from
CD34+ GM-CSF, TNFa 12 days, resting (D101); DC 70% CDla+, from
CD34+ GM-CSF, TNFa 12 days, activated with PMA and ionomycin
for 1 hr (D102); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days,
activated with PMA and ionomycin for 6 hr (D103); DC 95% CDla+,
from CD34+ GM-CSF, TNFa 12 days FAGS sorted, activated with PMA
and ionomycin for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+
GM-CSF, TNFa 12 days FACS sorted, activated with PMA and
ionomycin 1, 6 hr pooled (D105); DC CDla+ CD86+, from CD34+ GM-
CSF, TNFa 12 days FACS sorted, activated with PMA and ionomycin
for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5
days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days,
resting (D108); DC from monocytes GM-CSF, IL-4 5 days,
activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF,
IL-4 5 days, activated TNFa, monocyte supe for 4, 16 h pooled
(D110); epithelial cells, unstimulated; epithelial cells, IL-1(3
activated; lung fibroblast sarcoma line MRC5, activated with
PMA and ionomycin for 1, 6 h pooled (C101); kidney epithelial
carcinoma cell line CHA, activated with PMA and ionomycin for
1, 6 h pooled (C102).
Rodent counterparts, e.g., mouse, should be identified,
and their distributions will be similarly evaluated. Samples
for mouse mRNA isolation can include: resting mouse
fibroblastic L cell line (C200); Braf:ER (Braf fusion to
estrogen receptor) transfected cells, control (C201); Me114+
naive T cells from spleen, resting (T209); Me114+ naive T cells
from spleen, stimulated with IFNy, IL-12, and anti IL-4 to
polarize to TH1 cells, exposed to IFNy and IL-4 for 6, 12, 24
h, pooled (T210); Me114+ naive T cells from spleen, stimulated
with IL-4 and anti IFNy to polarize to Th2 cells, exposed to
IL-4 and anti IFNy for 6, 13, 24 h, pooled (T211); T cells, TH1
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polarized (Me114 bright, CD4+ cells from spleen, polarized for
7 days with IFN-y and anti IL-4; T200); T cells, TH2 polarized
(Me114 bright, CD4+ cells from spleen, polarized for 7 days
with IL-4 and anti-IFN-y; T201); T cells, highly TH1 polarized
5 3x from transgenic Balb/C (see Openshaw, et al. (1995) J. Exp.
Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 24 h
pooled; T202); T cells, highly TH2 polarized 3x from transgenic
Balb/C (activated with anti-CD3 for 2, 6, 24 h pooled (T203); T
cells, highly TH1 polarized 3x from transgenic C57 b1/6
10 (activated with anti-CD3 for 2, 6, 24 h pooled; T212); T cells,
highly TH2 polarized 3x from transgenic C57 b1/6 (activated
with anti-CD3 for 2, 6, 24 h pooled; T213); T cells, highly TH1
polarized (naive CD4+ T cells from transgenic Balb/C, polarized
3x with IFNy, IL-12, and anti-IL-4; stimulated with IGIF, IL-
15 12, and anti IL-4 for 6, 12, 24 h, pooled); CD44- CD25+ pre T
cells, sorted from thymus (T204); TH1 T cell clone D1.1,
resting for 3 weeks after last stimulation with antigen (T205);
TH1 T cell clone D1.1, 10 ~g/ml ConA stimulated 15 h (T206);
TH2 T cell clone CDC35, resting for 3 weeks after last
20 stimulation with antigen (T207); TH2 T cell clone CDC35, 10
~g/ml ConA stimulated 15 h (T208); unstimulated B cell line
CH12 (B201); unstimulated mature B cell leukemia cell line A20
(B200); unstimulated large B cells from spleen (B202); B cells
from total spleen, LPS activated (B203); metrizamide enriched
25 dendritic cells from spleen, resting (D200); dendritic cells
from bone marrow, resting (D201); unstimulated bone marrow
derived dendritic cells depleted with anti B220, anti CD3, and
anti Class II, cultured in GM-CSF and IL-4 (D202); bone marrow
derived dendritic cells depleted with anti B220, anti CD3, and
30 anti Class II, cultured in GM-CSF and IL-4, stimulated with
anti CD40 for 1, 5 d, pooled (D203); monocyte cell line RAW
264.7 activated with LPS 4 h (M200); bone-marrow macrophages
derived with GM and M-CSF (M201); bone-marrow macrophages
derived with GM-CSF, stimulated with LPS, IFNy, and IL-10 for
35 24 h (M205); bone-marrow macrophages derived with GM-CSF,
stimulated with LPS, IFNy, and anti IL-10 for 24 h (M206);
peritoneal macrophages (M207); macrophage cell line J774,
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resting (M202); macrophage cell line J774 + LPS + anti-IL-10 at
0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774 +
LPS + IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204); unstimulated
mast cell lines MC-9 and MCP-12 (M208); immortalized
endothelial cell line derived from brain microvascular
endothelial cells, unstimulated (E200); immortalized
endothelial cell line derived from brain microvascular
endothelial cells, stimulated overnight with TNFa (E201);
immortalized endothelial cell line derived from brain
microvascular endothelial cells, stimulated overnight with TNFa
(E202); immortalized endothelial cell line derived from brain
microvascular endothelial cells, stimulated overnight with TNFa
and IL-10 (E203); total aorta from wt C57 b1/6 mouse; total
aorta from 5 month ApoE KO mouse (X207); total aorta from 12
month ApoE KO mouse (X207); wt thymus (0214); total thymus,
rag-1 (0208); total kidney, rag-1 (0209); total kidney, NZ B/W
mouse; and total heart, rag-1 (0202).
To further analyze the expression pattern of DC-STAMP, RT-
PCR was performed on RNA from a panel of freshly isolated
resting or activated leukocyte populations and several cell
lines of haematopoietic as well as non-haematopoietic origin.
The PCR products were Southern blotted and hybridized with a
specific DC-STAMP oligonucleotide. A distinct band of the
expected size was detected in immature as well as in mature DC,
stimulated with either LPS or a combination of TNFa and an
activating anti-CD40 antibody. In contrast, freshly isolated
monocytes did not express the DC-STAMP RNA, even after
overnight stimulation with LPS. A low expression was detected
in total PBMC, which could be explained by the presence of
contaminating DC, and in the pre-monocytic cell line U937. The
f3-actin mRNA control was similar in all samples, indicating
that equal amounts of RNA were used for the RT-PCR.
Hybridization of a Northern blot containing mRNA from 12
human tissues (Clontech MTN # 7780-1) with a DC-STAMP specific
probe did not result in any detectable signal, not even after
exposure of the blot for several days. The absence of
detectable expression of DC-STAMP in the 12 different human
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tissues tested is consistent with a relatively low expression
in DC.
Initial distribution studies of the DSP-1 suggest that the
antigen is primarily expressed on monocytes, mast cells, T
cells, and NK cells. Thus, it is likely that the receptor has
a negative regulatory role for those cell types.
IV. Fusion Protein Constructs
The DC-STAMP ORF was amplified with Pwo DNA polymerase
(Boehringer Mannheim) using appropriate primers, containing a
EcoRI or BglII site, respectively, deleting the DC-STAMP STOP
codon. This PCR product was cloned as an EcoRI/BglII fragment
into the pN3-EGFP expression vector (Clontech, Palo Alto, CA),
digested with EcoRI and BamHI, inserting the DC-STAMP cDNA N-
terminal of the transcript encoding the enhanced green
fluorescent protein (EGFP). The CCR1 molecule was amplified by
RT-PCR using total RNA from monocytes and primers based on the
published sequence (see Adema, et al. (1997) Nature 387:713-
717; and Falkoff, et al. (1982). J. Immunol. Methods 50:39-49)
and cloned as a GFP fusion protein using a similar approach.
Primers contained a NotI or BamHI restriction site. The
digested PCR product was cloned into the NotI-BamHI digested
pBluescript SK -vector (Stratagene, La Jolla, CA) and
subsequently cloned as a Sacl-BamHI fragment into the
expression vector pN3-EGFP.
Similar constructs can be made using the DSP-1 sequences.
V. Cellular Localization of DC-STAMP
To determine the cellular localization of DC-STAMP, the
DC-STAMP-EGFP (Enhanced Green Fluorescent Protein) fusion
protein was subjected to analysis. This construct possessed
the EGFP sequence fused to the C-terminal of the DC-STAMP ORF.
293 cells were transfected with this construct, and analyzed by
Confocal Laser Scan Microscopy (CLSM). Since multimembrane
spanning proteins are very hydrophobic and complex proteins,
the CCR1 molecule, a 7 TM chemokine receptor expressed at the
cell membrane, was compared as a control. Transfection of
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CCR1-EGFP into 293 cells resulted in a bright membrane
fluorescence, often accompanied by an additional highly
fluorescent spot in the cytoplasm, possibly representing the
Golgi. Analysis of transient as well as stable transfectants
of the DC-STAMP-EGFP construct showed a similar fluorescence
staining pattern as seen for CCR1-EGFP, indicating that DC-
STAMP can also be expressed at the cell surface. Transfectants
expressing the EGFP protein alone showed a bright cytoplasmic
fluorescence, not localized to a particular cell structure.
The localization of the C-terminus of the DC-STAMP-EGFP
protein was determined by staining the DC-STAMP-EGFP transient
transfectants with polyclonal anti-GFP serum either before or
after permeabilization. Cytospin stainings showed that EGFP
could only be detected after permeabilization, indicating that
DC-STAMP has an intracellular C-terminus. The amount of
positive cells was consistent with the percentage of GFP
positive cells in the transient transfected bulk population as
observed by FACS analysis (30%). The few cells that stained
positive after pre-incubation with the anti-GFP serum were due
to leakage of the antibody into dead cells.
VI. Chromosome mapping of DC-STAMP and DSP-1
An isolated cDNA encoding the antigen is used. Chromosome
mapping is a standard technique. See, e.g., BIOS Laboratories
(New Haven, CT) and methods for using a mouse somatic cell
hybrid panel with PCR.
VII. Purification of DC-STAMP or DSP-1 Protein
Multiple transfected cell lines are screened for one which
expresses the desired antigen at a high level compared with
other cells. Various cell lines are screened and selected for
their favorable properties in handling. Natural antigen can be
isolated from natural sources, or by expression from a
transformed cell using an appropriate expression vector.
Purification of the expressed protein is achieved by standard
procedures, or may be combined with engineered means for
effective purification at high efficiency from cell lysates or
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supernatants. FLAG or His6 segments can be used for such
purification features. Alternatively, affinity chromatography
may be used with specific antibodies, see below. Protein is
produced in coli, insect cell, or mammalian expression systems,
as desired.
Human embryonic kidney (HEK) 293 cells were transfected
with 3 ~g DC-STAMP DNA using LipofectAMINE (Gibco BRL). See
Lamer, et al. (1994) J. Immunol. 153:2417-2428. 2 days after
transfection, cells were harvested and used for Confocal Laser
Scanning Microscopy (CLSM). Expression was checked by FACScan
analysis in the FITC channel (Becton Dickinson & Co., Oxnard,
CA) and usually 30-60% of the cells were positive for
expression. In order to obtain a stable bulk population, 6418
(1 mg/ml; Life Technologies Ltd, Paisley, Scotland) was added
to the culture medium at day 2 after transfection. After 1 to
2 weeks, cells were sorted for GFP expression on the Coulter
Epics Elite (Coulter, Hialeah, FL) and the resulting bulk
population was used for CLSM. Cells were stained with rabbit
polyclonal anti-GFP serum (kindly provided by E. Cuppen, Dept.
of Cell Biology and Histology, University of Nijmegen, The
Netherlands), either before or after cytospin preparations.
Cytospins were fixed with acetone for 10 minutes, incubated
with a horse anti-mouse biotinylated antibody and positive
cells visualized by immunoperoxidase staining (Vectastain Elite
ABC kit, Vector Laboratories, Burlingame, USA; AEC Substrate
Kit, Zymed Laboratories, CA).
Confocal laser scanning microscopy
Cells were attached to poly-1-lysine coated glass slides,
after which GFP-fusion protein distribution was determined by
Confocal Laser Scanning Microscopy (CLSM) at 488 nm with a
krypton/argon Laser (Biorad 1000, Hercules, CA). The CSLM
settings were: lens, 60x; gain, 1100-1350; pinhole, 1.5 Vim; and
magnification, 60x.
VIII. Isolation of Homologous Genes
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The DC-STAMP or DSP-1 cDNA, or other species counterpart
sequence, can be used as a hybridization probe to screen a
library from a desired source, e.g., a primate cell cDNA
library. Many different species can be screened both for
5 stringency necessary for easy hybridization, and for presence
using a probe. Appropriate hybridization conditions will be
used to select for clones exhibiting specificity of cross
hybridization.
Screening by hybridization using degenerate probes based
10 upon the peptide sequences will also allow isolation of
appropriate clones. Alternatively, use of appropriate primers
for PCR screening will yield enrichment of appropriate nucleic
acid clones.
Similar methods are applicable to isolate either species,
15 polymorphic, or allelic variants. Species variants are
isolated using cross-species hybridization techniques based
upon isolation of a full length isolate or fragment from one
species as a probe.
Alternatively, antibodies raised against human antigen
20 will be used to screen for cells which express cross-reactive
proteins from an appropriate, e.g., cDNA library. The purified
protein or defined peptides are useful for generating
antibodies by standard methods, as described above. Synthetic
peptides or purified protein are presented to an immune system
25 to generate monoclonal or polyclonal antibodies. See, e.g.,
Coligan (1991) Current Protocols in Immunoloay Wiley/Greene;
and Harlow and Lane (1989) Antibodies' A Laboratory Manual Cold
Spring Harbor Press. The resulting antibodies are used for
screening, purification, or diagnosis, as described.
IX. Preparation of Antibodies Specific for Antigen
Synthetic peptides or purified protein are presented to an
immune system to generate monoclonal or polyclonal antibodies.
See, e.g., Coligan (1991) Current Protocols in Immunoloay
Wiley/Greene; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press. Polyclonal serum,
or hybridomas may be prepared. In appropriate situations, the
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binding reagent is either labeled as described above, e.9.,
fluorescence or otherwise, or immobilized to a substrate for
panning methods. Immunoselection, absorptions, and related
techniques are available to prepare selective reagents, e.g.,
exhibiting the desired spectrum of selectivity for binding.
X. Evaluation of Breadth of Biological Functions
DC-STAMP could possibly serve as a receptor for growth
factors or hormones, which upon activation drive the
differentiation into DC, or modulate DC function by directing T
cell responses. Another possibility is a putative role for DC-
STAMP as a receptor connecting the neuro-endocrine system to
the immune system. Analysis of the effects of differentiation,
maturation by various stimuli, and co-culture with T cells on
the expression levels of DC-STAMP, will provide more insight
into the specific function of this novel multimembrane surface
receptor on DC.
Recently, DC pulsed with tumor antigens have been
successfully used in vivo for the induction of anti-tumor T
cell reactivity in melanoma patients. Nestle, et al. (1998)
Nat. Med. 4:328-332. Thus, DC treated with these reagents may
be useful in cell based therapies, e.g., cell transfer or in
vitro cell treatments.
Biological activities of antibody to antigen are tested,
based, in part, on the sequence and structural homology between
the DC-STAMP and other membrane proteins. Initially, assays
that show biological activities of 7TM receptors are examined.
For the DSP-1, biological activities related to the function of
monocyte, T, NK, and/or mast cells will be tested. Thus, assay
for effects of polyclonal antibodies likely to contain
antagonist antibodies affecting cells possessing the antigens
will be tested. Primary assays include chemotaxis assays for
the various cell types, e.g., monocytes, T, NK, and/or mast
cells. Mast cell specific assays include, e.g., IgE mediated
degranulation assays, mast cell chemotaxis assays, and effects
on SCF/.IL-6 induced mast cell proliferation assays.
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Similarly, assays for effects on T cell, NK cells, or monocytes
will be tested.
A. Effects on proliferation/differentiation of progenitor
cells
The effect on proliferation or differentiation of various
cell types are evaluated with various concentrations of
antibody. A dose response analysis is performed.
In particular, antibodies will be tested on cord blood
cells to see if they have effects on proliferation or
differentiation of early progenitor cells derived therefrom.
Preferably, the cells are early precursor cells, e.g., stem
cells, originating from, e.g., cord blood, bone marrow, thymus,
spleen, or CD34+ progenitor cells. The antibodies will be
tested for effects on myeloid and/or erythroid precursors,
including B cell precursors.
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B. Effects of antibodies on proliferation
Total PBMC are isolated from buffy coats of normal healthy
donors by centrifugation through ficoll-hypaque as described
(Boyum, et al.). PBMC are cultured in 200 ~1 Yssel's medium
(Gemini Bioproducts, Calabasas, CA) containing 1°s human AB
serum in 96 well plates (Falcon, Becton-Dickinson, NJ) in the
absence or presence of antibodies. Cells are cultured in
medium alone or in combination with 100 U/ml IL-2 (R&D Systems)
for 120 hours. 3H-Thymidine (0.1 mCi) is added during the last
six hours of culture and 3H-Thymidine incorporation determined
by liquid scintillation counting.
The antibodies would be tested for blocking signaling
activity in many other biological assay systems, e.g., on T-
cells, B-cells, NK, macrophages, dendritic cells, mast cells,
hematopoietic progenitors, etc.
Antibodies are evaluated for effects in
macrophage/dendritic cell activation and antigen presentation
assays, T cell cytokine production or proliferation in response
to antigen or allogeneic stimulus. See, e.g., de Waal Malefyt
et al. (1991) J. Exp. Med. 174:1209-1220; de Waal Malefyt et
al. (1991) J. Exp,. Med. 174:915-924; Fiorentino, et al. (1991)
J. Immunol: 147, 3815-3822; Fiorentino, et al. (1991) J.
Immunol. 146:3444-3451; and Groux, et al. (1996) J. Ex~. Med.
184:19-29. Antibodies will be tested for ability to affect
mast cell degranulation, chemotaxis, etc.
Antibodies will also be evaluated for effects on NK cell
stimulation. Assays may be based, e.g., on Hsu, et al. (1992)
Internat: Immunol. 4:563-569; and Schwarz, et al. (1994) J.
Immunother. 16:95-104. Other assays are applied to evaluate
effects on cytotoxic T cells and LAK cells. See, e.g., Namien
and Mire-Sluis (1998).
B cell growth and differentiation effects will be
analyzed, e.g., by the methodology described, e.g., in
Defrance, et al. (1992). J. Exp. Med. 175:671-682; Rousset, et
al. (1992) Proc. Nat'1 Acad. Sci. USA 89:1890-1893; including
IgG2 and IgA2 switch factor assays. Note that, unlike COS7
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supernatants, NIH3T3 and COP supernatants apparently do not
interfere with human B cell assays.
C. Effects on the expression of cell surface molecules on
human monocytes
Monocytes are purified by negative selection from
peripheral blood mononuclear cells of normal healthy donors.
Briefly, 3 x 108 ficoll banded mononuclear cells are incubated
on ice with a cocktail of monoclonal antibodies (Becton-
Dickinson; Mountain View, CA). consisting, e.g., of 200 ~,1 of
aCD2 (Leu-5A), 200 ~1 of aCD3 (Leu-4), 100 ~1 of aCD8 (Leu 2a),
100 ~1 of aCDl9 (Leu-12 ), 100 ~,1 of aCD20 (Leu-16), 100 ~l of
aCD56 (Leu-19), 100 ~,1 of aCD67 (IOM 67; Immunotech, Westbrook,
ME), and anti-glycophorin antibody (lOF7MN, ATCC, Rockville,
MD). Antibody bound cells are washed and then incubated with
sheep anti-mouse IgG coupled magnetic beads (Dynal, Oslo,
Norway) at a bead to cell ratio of 20:1. Antibody bound cells
are separated from monocytes by application of a magnetic
field. Subsequently, human monocytes are cultured in Yssel's
medium (Gemini Bioproducts, Calabasas, CA) containing to human
AB serum in the absence or presence of antibodies.
Analyses of the expression of cell surface molecules can
be performed by direct immunofluorescence. For example, 2 x
105 purified human monocytes are incubated in phosphate
buffered saline (PBS) containing 1% human serum on ice for 20
minutes. Cells are pelleted at 200 x g. Cells are resuspended
in 20 ml PE or FITC labeled mAb. Following an additional 20
minute incubation on ice, cells are washed in PBS containing 1%
human serum followed by two washes in PBS alone. Cells are
fixed in PBS containing 1% paraformaldehyde and analyzed on
FACScan flow cytometer (Becton Dickinson; Mountain View, CA).
Exemplary mAbs are used, e.g.. CDllb (anti-mac h , CDllc (a
gp150/95), CD14 (Leu-M3), CD54 (Leu 54), CD80 (anti-BB1/B7),
HLA-DR (L243) from Becton-Dickinson and CD86 (FUN 1;
Pharmingen), CD64 (32.2; Medarex), CD40 (mAb89; Schering-Plough
France) .
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D. Effects of antibodies on cytokine production by human
monocytes
Human monocytes are isolated as described and cultured in
Yssel's medium (Gemini Bioproducts, Calabasas, CA) containing
5 1% human AB serum in the absence or presence of antibodies. In
addition, monocytes are stimulated with LPS (E. coli 0127: B8
Difco) in the absence or presence of antibodies and the
concentration of cytokines (IL-1(3, IL-6, TNFa, GM-CSF, and IL-
10) in the cell culture supernatant determined by ELISA.
10 Additional assays will be tested in the areas of bone
remodeling, chondriocytes, neurons, adipocytes,
gastrointestinal epithelium, or bronchial epithelium.
XI. Generation and Analysis of Genetically Altered Animals
15 Transgenic mice can be generated by standard methods.
Such animals are useful to determine the effects of deletion of
the gene, in specific tissues, or completely throughout the
organism. Such may provide interesting insight into
development of the animal or particular tissues in various
20 stages. Moreover, the effect on various responses to
biological stress can be evaluated. See, e.g., Hogan, et al.
(1995) Manipulating the Mouse Embryo: A Laboratory Manual (2d
ed.) Cold Spring Harbor Laboratory Press.
25 All references cited herein are incorporated herein by
reference to the same extent as if each individual publication
or patent application was specifically and individually
indicated to be incorporated by reference in its entirety for
all purposes.
30 Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
35 claims, along with the full scope of equivalents to which such
claims are entitled.
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1
SEQUENCE SUBMISSION
SEQ ID N0: 1 is primate, e.g., human, DC-STAMP nucleic acid sequence.
SEQ ID N0: 2 is primate DC-STAMP polypeptide sequence.
SEQ ID NO: 3 is reverse translation of primate DC-STAMP.
SEQ ID NO: 4 is primate, e.g., human, DSP-1L nucleic acid sequence.
SEQ ID N0: 5 is primate DSP-1L polypeptide sequence.
SEQ ID NO: 6 is primate, e.g., human, DSP-1S nucleic acid sequence.
SEQ ID NO: 7 is primate DSP-1S polypeptide sequence.
SEQ ID N0: 8 is reverse translation of primate DSP-1L.
SEQ ID NO: 9 is reverse translation of primate DSP-1S.
<110> Zlot, Constance F.
Adema, Gosse J.
Figdor, Carl
Phillips, Joseph H.
<120> Mammalian Genes; Related Reagents and Methods
<130> DX1051Q
<140>
<141>
<160> 9
<170> PatentIn Ver.
2.0
<210> 1
<211> 1960
<212> DNA
<213> primate
<220>
<221> CDS
<222> (58)..(1467)
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4 0 ggggggtggc atttctgcat tcgaagaaga atctgagaga aacctgacgc agggagc 57
atg ggt atc tgg acc tca ggc act gat atc ttc cta agt ctt tgg gag 105
Met Gly Ile Trp Thr Ser Gly Thr Asp Ile Phe Leu Ser Leu Trp Glu
1 5 10 15
att tac gtg tct cca aga agc ccc gga tgg atg gac ttt atc cag cat 153
Ile Tyr Val Ser Pro Arg Ser Pro Gly Trp Met Asp Phe Ile Gln His
20 25 30
5 0 ttg gga gtt tgc tgt ttg gtt get ctt att tca gtg ggc ctc ctg tct 201
Leu Gly Val Cys Cys Leu Val Ala Leu Ile Ser Val Gly Leu Leu Ser
35 40 45
gtg gcc gcc tgc tgg ttt ctg cca tca atc ata gcg gcc get gcc tcc 249
5 5 Val Ala Ala Cys Trp Phe Leu Pro Ser Ile Ile Ala Ala Ala Ala Ser
55 60
tgg att atc acg tgt gtt ctg ctg tgt tgc tcc aag cat gca cga tgt 297
Trp Ile Ile Thr Cys Val Leu Leu Cys Cys Ser Lys His Ala Arg Cys
65 70 75 80
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2
ttt att ctt ctt gtc ttt ctc tct tgt ggc ctg cgt gaa ggc agg aat 345
Phe Ile Leu Leu Val Phe Leu Ser Cys Gly Leu Arg Glu Gly Arg Asn
85 90 95
get ttg att gca get ggc aca ggg atc gtc atc ttg gga cac gta gaa 393
Ala Leu Ile Ala Ala Gly Thr Gly Ile Val Ile Leu Gly His Val Glu
100 105 110
aat att ttt cac aac ttt aaa ggt ctc cta gat ggt atg act tgc aac 441
Asn Ile Phe His Asn Phe Lys Gly Leu Leu Asp Gly Met Thr Cys Asn
115 120 125
cta agg gca aag agc ttt tcc ata cat ttt cca ctt ttg aaa aaa tat 489
Leu Arg Ala Lys Ser Phe Ser Ile His Phe Pro Leu Leu Lys Lys Tyr
130 135 140
att gag gca att cag tgg att tat ggc ctt gcc act cca cta agt gta 537
Ile Glu Ala Ile Gln Trp Ile Tyr Gly Leu Ala Thr Pro Leu Ser Val
2 0 145 150 155 160
ttt gat gac ctt gtt tct tgg aac cag acc ctg gca gtc tct ctt ttc 585
Phe Asp Asp Leu Val Ser Trp Asn Gln Thr Leu Ala Val Ser Leu Phe
165 170 175
agt ccc agc cat gtc ctg gag gca cag cta aat gac agc aaa ggg gaa 633
Ser Pro Ser His Val Leu Glu Ala Gln Leu Asn Asp Ser Lys Gly Glu
180 185 190
3 0 gtc ctg agc gtc ttg tac cag atg gca aca acc aca gag gtg ttg tcc 681
Val Leu Ser Val Leu Tyr Gln Met Ala Thr Thr Thr Glu Val Leu Ser
195 200 205
tcc ctg ggt cag aag cta ctt gcc ttt gca ggg ctt tcg ctc gtc ctg 729
Ser Leu Gly Gln Lys Leu Leu Ala Phe Ala Gly Leu Ser Leu Val Leu
210 215 220
ctt ggc act ggc ctc ttc atg aag cga ttt ttg ggc cct tgt ggt tgg 777
Leu Gly Thr Gly Leu Phe Met Lys Arg Phe Leu Gly Pro Cys Gly Trp
4 0 225 230 235 240
aag tat gaa aac atc tac atc acc aga caa ttt gtt cag ttt gat gaa 825
Lys Tyr Glu Asn Ile Tyr Ile Thr Arg Gln Phe Val Gln Phe Asp Glu
245 250 255
agg gag aga cat caa cag agg ccc tgt gtg ctc ccg ctg aat aag gag 873
Arg Glu Arg His Gln Gln Arg Pro Cys Val Leu Pro Leu Asn Lys Glu
260 265 270
5 0 gaa agg agg aag tat gtc atc atc ccg act ttc tgg ccg act cct aaa 921
Glu Arg Arg Lys Tyr Val Ile Ile Pro Thr Phe Trp Pro Thr Pro Lys
275 280 285
gaa agg aaa aac ctg ggg ctg ttt ttc ctc ccc ata ctt atc cat ctc 969
Glu Arg Lys Asn Leu Gly Leu Phe Phe Leu Pro Ile Leu Ile His Leu
290 295 300
tgc atc tgg gtg ctg ttt gca get gta gat tat ctg ctg tat cgg ctc 1017
Cys Ile Trp Val Leu Phe Ala Ala Val Asp Tyr Leu Leu Tyr Arg Leu
305 310 315 320
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
3
att ttc tca gtg agc aag cag ttt caa agc ttg cca ggg ttt gag gtt 1065
Ile Phe Ser Val Ser Lys Gln Phe Gln Ser Leu Pro Gly Phe Glu Val
325 330 335
cac ttg aaa ctg cac gga gag aaa caa gga act caa gat att atc cat 1113
His Leu Lys Leu His Gly Glu Lys Gln Gly Thr Gln Asp Ile Ile His
340 345 350
gat tct tcc ttt aat ata tct gtg ttt gaa ccc aac tgt atc cca aaa 1161
Asp Ser Ser Phe Asn Ile Ser Val Phe Glu Pro Asn Cys Ile Pro Lys
355 360 365
cca aaa ttc ctt cta tct gag acc tgg gtt cct ctc agt gtt att ctt 1209
Pro Lys Phe Leu Leu Ser Glu Thr Trp Val Pro Leu Ser Val Ile Leu
370 375 380
ttg ata tta gtg atg ctg gga ctg ttg tcc tct atc ctt atg caa ctt 1257
Leu Ile Leu Val Met Leu Gly Leu Leu Ser Ser Ile Leu Met Gln Leu
2 0 385 390 395 400
aaa atc ctg gtg tca gca tct ttc tac ccc agc gtg gag agg aag cgc 1305
Lys Ile Leu Val Ser Ala Ser Phe Tyr Pro Ser Val Glu Arg Lys Arg
405 410 415
atc caa tat ctg cat gca aag ctg ctt aaa aaa aga tca aag cag ccg 1353
Ile Gln Tyr Leu His Ala Lys Leu Leu Lys Lys Arg Ser Lys Gln Pro
420 425 430
3 0 ctg gga gaa gtc aaa aga cgg ctg agt ctc tat ctt aca aag att cat 1401
Leu Gly Glu Val Lys Arg Arg Leu Ser Leu Tyr Leu Thr Lys Ile His
435 440 445
ttc tgg ctt cca gtc ctg aaa atg att agg aag aag caa atg gac atg 1449
Phe Trp Leu Pro Val Leu Lys Met Ile Arg Lys Lys Gln Met Asp Met
450 455 460
gca agt gca gac aag tca tgagagaccc cgactactcc tcagccacat 1497
Ala Ser Ala Asp Lys Ser
465 470
cgcaccaaca attctcttca ggtctaggat ggcagtcact attcatgccg gataatagag 1557
aactatgtga cgcagtcctc tcaggagtct gagtttacag agccaacttg cagcacctgg 1617
ttatgcctcc tttcatctca aagccaaaga gctgccaggt aaatggttat gtggtctatg 1677
ttccaaacaa accacatgat cttgcctgtg tcacaatgta acaagactct agctgggtcc 1737
cctggtgatg agtttcagca tagaataatg ttcaaggaaa agaaaacgaa aacagtttaa 1797
atctctacca cagcctcaca agcaaatgct aaggggaaca tacatgtaaa aagccagcaa 1857
actatcttca aactcttccg tccttaatgt cttccatggc tattgccccc acaatggtct 1917
cttttctccc tgctccctta ttaaagaact ctttctgaaa ccc 1960
<210> 2
<211> 470
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
4
<212>
PRT
<213> imate
pr
<400>
2
Met GlyIle TrpThrSer GlyThrAsp IlePheLeu SerLeuTrp Glu
1 5 10 15
Ile TyrVal SerProArg SerProGly TrpMetAsp PheIleGln His
20 25 30
Leu GlyVal CysCysLeu ValAlaLeu IleSerVal GlyLeuLeu Ser
35 40 45
Val AlaAla CysTrpPhe LeuProSer IleIleAla AlaAlaAla Ser
50 55 60
Trp IleIle ThrCysVal LeuLeuCys CysSerLys HisAlaArg Cys
65 70 75 80
2 Phe IleLeu LeuValPhe LeuSerCys GlyLeuArg GluGlyArg Asn
0
85 90 95
Ala LeuIle AlaAlaGly ThrGlyIle ValIleLeu GlyHisVal Glu
100 105 110
Asn IlePhe HisAsnPhe LysGlyLeu LeuAspGly MetThrCys Asn
115 120 125
Leu ArgAla LysSerPhe SerIleHis PheProLeu LeuLysLys Tyr
130 135 140
Ile GluAla IleGlnTrp IleTyrGly LeuAlaThr ProLeuSer Val
145 150 155 160
3 Phe AspAsp LeuValSer TrpAsnGln ThrLeuAla ValSerLeu Phe
5
165 170 175
Ser ProSer HisValLeu GluAlaGln LeuAsnAsp SerLysGly Glu
180 185 190
Val LeuSer ValLeuTyr GlnMetAla ThrThrThr GluValLeu Ser
195 200 205
Ser LeuGly GlnLysLeu LeuAlaPhe AlaGlyLeu SerLeuVal Leu
210 215 220
Leu GlyThr GlyLeuPhe MetLysArg PheLeuGly ProCysGly Trp
225 230 235 240
5 Lys TyrGlu AsnIleTyr IleThrArg GlnPheVal GlnPheAsp Glu
0
245 250 255
Arg GluArg HisGlnGln ArgProCys ValLeuPro LeuAsnLys Glu
260 265 270
Glu ArgArg LysTyrVal IleIlePro ThrPheTrp ProThrPro Lys
275 280 285
Glu ArgLys AsnLeuGly LeuPhePhe LeuProIle LeuIleHis Leu
290 295 300
CA 02391669 2002-05-14
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Cys Ile Trp Val Leu Phe Ala Ala Val Asp Tyr Leu Leu Tyr Arg Leu
305 310 315 320
5 Ile Phe Ser Val Ser Lys Gln Phe Gln Ser Leu Pro Gly Phe Glu Val
325 330 335
His Leu Lys Leu His Gly Glu Lys Gln Gly Thr Gln Asp Ile Ile His
340 345 350
Asp Ser Ser Phe Asn Ile Ser Val Phe Glu Pro Asn Cys Ile Pro Lys
355 360 365
Pro Lys Phe Leu Leu Ser Glu Thr Trp Val Pro Leu Ser Val Ile Leu
370 375 380
Leu Ile Leu Val Met Leu Gly Leu Leu Ser Ser Ile Leu Met Gln Leu
385 390 395 400
2 0 Lys Ile Leu Val Ser Ala Ser Phe Tyr Pro Ser Val Glu Arg Lys Arg
405 410 415
Ile Gln Tyr Leu His Ala Lys Leu Leu Lys Lys Arg Ser Lys Gln Pro
420 425 430
Leu Gly Glu Val Lys Arg Arg Leu Ser Leu Tyr Leu Thr Lys Ile His
435 440 445
Phe Trp Leu Pro Val Leu Lys Met Ile Arg Lys Lys Gln Met Asp Met
450 455 460
Ala Ser Ala Asp Lys Ser
465 470
40
<210> 3
<211> 1410
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc_feature
<222> (1) . (1410)
<223> n may be a, c, g, or t
<400> 3
atgggnatht ggacnwsngg nacngayath ttyytnwsny tntgggarat htaygtnwsn 60
ccnmgnwsnc cnggntggat ggayttyath carcayytng gngtntgytg yytngtngcn 120
ytnathwsng tnggnytnyt nwsngtngcn gcntgytggt tyytnccnws nathathgcn 180
gcngcngcnw sntggathat hacntgygtn ytnytntgyt gywsnaarca ygcnmgntgy 240
ttyathytny tngtnttyyt nwsntgyggn ytnmgngarg gnmgnaaygc nytnathgcn 300
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
6
gcnggnacng gnathgtnat hytnggncay gtngaraaya thttycayaa yttyaarggn 360
ytnytngayg gnatgacntg yaayytnmgn gcnaarwsnt tywsnathca yttyccnytn 420
ytnaaraart ayathgargc nathcartgg athtayggny tngcnacncc nytnwsngtn 480
ttygaygayy tngtnwsntg gaaycaracn ytngcngtnw snytnttyws nccnwsncay 540
gtnytngarg cncarytnaa ygaywsnaar ggngargtny tnwsngtnyt ntaycaratg 600
gcnacnacna cngargtnyt nwsnwsnytn ggncaraary tnytngcntt ygcnggnytn 660
wsnytngtny tnytnggnac nggnytntty atgaarmgnt tyytnggncc ntgyggntgg 720
aartaygara ayathtayat hacnmgncar ttygtncart tygaygarmg ngarmgncay 780
carcarmgnc cntgygtnyt nccnytnaay aargargarm gnmgnaarta ygtnathath 840
ccnacnttyt ggccnacncc naargarmgn aaraayytng gnytnttytt yytnccnath 900
ytnathcayy tntgyathtg ggtnytntty gcngcngtng aytayytnyt ntaymgnytn 960
athttywsng tnwsnaarca rttycarwsn ytnccnggnt tygargtnca yytnaarytn 1020
2 5 cayggngara arcarggnac ncargayath athcaygayw snwsnttyaa yathwsngtn 1080
ttygarccna aytgyathcc naarccnaar ttyytnytnw sngaracntg ggtnccnytn 1140
wsngtnathy tnytnathyt ngtnatgytn ggnytnytnw snwsnathyt natgcarytn 1200
aarathytng tnwsngcnws nttytayccn wsngtngarm gnaarmgnat hcartayytn 1260
caygcnaary tnytnaaraa rmgnwsnaar carccnytng gngargtnaa rmgnmgnytn 1320
wsnytntayy tnacnaarat hcayttytgg ytnccngtny tnaaratgat hmgnaaraar 1380
caratggaya tggcnwsngc ngayaarwsn 1410
<210> 4
<211> 942
<212> DNA
<213> primate
<220>
<221> CDS
<222> (1) . . (939)
<220>
<221> mat~eptide
<222> (64)..(939)
<400> 4
atg gcc tta cca acc gccttgctc ctgccgcta gccttgctg ctc
gtg 48
Met Ala Leu Pro Thr AlaLeuLeu LeuProLeu AlaLeuLeu Leu
Val
-20 -15 -10
cac gcc gcc agg gat tacaaggac gatgacgac aagatcgat ctg
ccg 96
His Ala Ala Arg Asp TyrLysAsp AspAspAsp LysIleAsp Leu
Pro
-5 -1 1 5 10
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
7
agc aaa tgc agg acc gtg gcg ggc ccc gtg ggg gga tcc ctg agt gtg 144
Ser Lys Cys Arg Thr Val Ala Gly Pro Val Gly Gly Ser Leu Ser Val
20 25
cag tgt ccc tat gag aag gaa cac agg acc ctc aac aaa tac tgg tgc 192
Gln Cys Pro Tyr Glu Lys Glu His Arg Thr Leu Asn Lys Tyr Trp Cys
30 35 40
10 aga cca cca cag att ttc cta tgt gac aag att gtg gag acc aaa ggg 240
Arg Pro Pro Gln Ile Phe Leu Cys Asp Lys Ile Val Glu Thr Lys Gly
45 50 55
tca gca gga aaa agg aac ggc cga gtg tcc atc agg gac agt cct gca 288
15 Ser Ala Gly Lys Arg Asn Gly Arg Val Ser Ile Arg Asp Ser Pro Ala
60 65 70 75
aac ctc agc ttc aca gtg acc ctg gag aat ctc aca gag gag gat gca 336
Asn Leu Ser Phe Thr Val Thr Leu Glu Asn Leu Thr Glu Glu Asp Ala
80 85 90
ggc acc tac tgg tgt ggg gtg gat aca ccg tgg ctc cga gac ttt cat 384
Gly Thr Tyr Trp Cys Gly Val Asp Thr Pro Trp Leu Arg Asp Phe His
95 100 105
gat ccc gtt gtc gag gtt gag gtg tcc gtg ttc ccg gca tca acg tca 432
Asp Pro Val Val Glu Val Glu Val Ser Val Phe Pro Ala Ser Thr Ser
110 115 120
3 0 atg aca cct gca agt atc act gcg gcc aag acc tca aca atc aca act 480
Met Thr Pro Ala Ser Ile Thr Ala Ala Lys Thr Ser Thr Ile Thr Thr
125 130 135
gca ttt cca cct gta tca tcc act acc ctg ttt gca gtg ggt gcc acc 528
Ala Phe Pro Pro Val Ser Ser Thr Thr Leu Phe Ala Val Gly Ala Thr
140 145 150 155
cac agt gcc agc atc cag gag gaa act gag gag gtg gtg aac tca cag 576
His Ser Ala Ser Ile Gln Glu Glu Thr Glu Glu Val Val Asn Ser Gln
160 165 170
ctc ccg ctg ctc ctc tcc ctg ctg gca ttg ttg ctg ctt ctg ttg gtg 624
Leu Pro Leu Leu Leu Ser Leu Leu Ala Leu Leu Leu Leu Leu Leu Val
175 180 185
ggg gcc tcc ctg cta gcc tgg agg atg ttt cag aaa tgg atc aaa get 672
Gly Ala Ser Leu Leu Ala Trp Arg Met Phe Gln Lys Trp Ile Lys Ala
190 195 200
5 0 ggt gac cat tca gag ctg tcc cag aac ccc aag cag get gcc acg cag 720
Gly Asp His Ser Glu Leu Ser Gln Asn Pro Lys Gln Ala Ala Thr Gln
205 210 215
agt gag ctg cac tac gca aat ctg gag ctg ctg atg tgg cct ctg cag 768
Ser Glu Leu His Tyr Ala Asn Leu Glu Leu Leu Met Trp Pro Leu Gln
220 225 230 235
gaa aag cca gca cca cca agg gag gtg gag gtg gaa tac agc act gtg 816
Glu Lys Pro Ala Pro Pro Arg Glu Val Glu Val Glu Tyr Ser Thr Val
240 245 250
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
8
gcc tcc ccc agg gaa gaa ctt cac tat gcc tcg gtg gtg ttt gat tct 864
Ala Ser Pro Arg Glu Glu Leu His Tyr Ala Ser Val Val Phe Asp Ser
255 260 265
aac acc aac agg ata get get cag agg cct cgg gag gag gaa cca gat 912
Asn Thr Asn Arg Ile Ala Ala Gln Arg Pro Arg Glu Glu Glu Pro Asp
270 275 280
tca gat tac agt gtg ata agg aag aca tag 942
Ser Asp Tyr Ser Val Ile Arg Lys Thr
285 290
<210> 5
<211> 313
<212> PRT
<213> primate
<400> 5
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
-20 -15 -10
His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Ile Asp Leu
-5 -1 1 5 10
Ser Lys Cys Arg Thr Val Ala Gly Pro Val Gly Gly Ser Leu Ser Val
15 20 25
3 0 Gln Cys Pro Tyr Glu Lys Glu His Arg Thr Leu Asn Lys Tyr Trp Cys
30 35 40
Arg ProProGln IlePheLeu CysAspLys IleVal GluThrLys Gly
45 50 55
Ser AlaGlyLys ArgAsnGly ArgValSer IleArg AspSerPro Ala
60 65 70 75
Asn LeuSerPhe ThrValThr LeuGluAsn LeuThr GluGluAsp Ala
80 85 90
Gly ThrTyrTrp CysGlyVal AspThrPro TrpLeu ArgAspPhe His
95 100 105
4 Asp ProValVal GluValGlu ValSerVal PhePro AlaSerThr Ser
5
110 115 120
Met ThrProAla SerIleThr AlaAlaLys ThrSer ThrIleThr Thr
125 130 135
Ala PheProPro ValSerSer ThrThrLeu PheAla ValGlyAla Thr
140 145 150 155
His SerAlaSer IleGlnGlu GluThrGlu GluVal ValAsnSer Gln
160 165 170
Leu ProLeuLeu LeuSerLeu LeuAlaLeu LeuLeu LeuLeuLeu Val
175 180 185
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
9
Gly SerLeu LeuAlaTrp ArgMetPhe GlnLysTrp IleLysAla
Ala
190 195 200
Gly AspHisSer GluLeuSer GlnAsnPro LysGlnAla AlaThrGln
205 210 215
Ser GluLeuHis TyrAlaAsn LeuGluLeu LeuMetTrp ProLeuGln
220 225 230 235
Glu LysProAla ProProArg GluValGlu ValGluTyr SerThrVal
240 245 250
Ala SerProArg GluGluLeu HisTyrAla SerValVal PheAspSer
255 260 265
Asn ThrAsnArg IleAlaAla GlnArgPro ArgGluGlu GluProAsp
270 275 280
Ser AspTyrSer ValIleArg LysThr
285 290
<210> 6
<211> 603
2 <212> DNA
5
<213> primate
<220>
<221> CDS
<222> (1) .
. (600)
<220>
<221> mat~eptide
<222> (64)..(600)
<400> 6
atg gcc tta gtgacc gccttgctc ctgccgcta gccttgctg ctc 48
cca
Met Ala Leu ValThr AlaLeuLeu LeuProLeu AlaLeuLeu Leu
Pro
-20 -15 -10
cac gcc gcc ccggat tacaaggac gatgacgac aagatcgat atg 96
agg
His Ala Ala ProAsp TyrLysAsp AspAspAsp LysIleAsp Met
Arg
-5 -1 1 5 10
4 aca cct gca atcact gcggccaag acctcaaca atcacaact gca 144
5 agt
Thr Pro Ala IleThr AlaAlaLys ThrSerThr IleThrThr Ala
Ser
15 20 25
ttt cca cct tcatcc actaccctg tttgcagtg ggtgccacc cac 192
gta
Phe Pro Pro SerSer ThrThrLeu PheAlaVal GlyAlaThr His
Val
30 35 40
agt gcc agc caggag gaaactgag gaggtggtg aactcacag ctc 240
atc
Ser Ala Ser GlnGlu GluThrGlu GluValVal AsnSerGln Leu
Ile
45 50 55
ccg ctg ctc tccctg ctggcattg ttgctgctt ctgttggtg ggg 288
ctc
Pro Leu Leu SerLeu LeuAlaLeu LeuLeuLeu LeuLeuVal Gly
Leu
65 70 75
60
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
gcc tcc ctg cta gcc tgg agg atg ttt cag aaa tgg atc aaa get ggt 336
Ala Ser Leu Leu Ala Trp Arg Met Phe Gln Lys Trp Ile Lys Ala Gly
80 85 90
5 gac cat tca gag ctg tcc cag aac ccc aag cag get gcc acg cag agt 384
Asp His Ser Glu Leu Ser Gln Asn Pro Lys Gln Ala Ala Thr Gln Ser
95 100 105
gag ctg cac tac gca aat ctg gag ctg ctg atg tgg cct ctg cag gaa 432
1 0 Glu Leu His Tyr Ala Asn Leu Glu Leu Leu Met Trp Pro Leu Gln Glu
110 115 120
aag cca gca cca cca agg gag gtg gag gtg gaa tac agc act gtg gcc 480
Lys Pro Ala Pro Pro Arg Glu Val Glu Val Glu Tyr Ser Thr Val Ala
125 130 135
tcc ccc agg gaa gaa ctt cac tat gcc tcg gtg gtg ttt gat tct aac 528
Ser Pro Arg Glu Glu Leu His Tyr Ala Ser Val Val Phe Asp Ser Asn
140 145 150 155
acc aac agg ata get get cag agg cct cgg gag gag gaa cca gat tca 576
Thr Asn Arg Ile Ala Ala Gln Arg Pro Arg Glu Glu Glu Pro Asp Ser
160 165 170
2 5 gat tac agt gtg ata agg aag aca tag 603
Asp Tyr Ser Val Ile Arg Lys Thr
175
<210> 7
<211>
200
<212>
PRT
<213>
primate
<400>
7
Met AlaLeu ProValThr AlaLeuLeu LeuProLeu AlaLeu LeuLeu
-20 -15 -10
His AlaAla ArgProAsp TyrLysAsp AspAspAsp LysIle AspMet
-5 -1 1 5 10
Thr ProAla SerIleThr AlaAlaLys ThrSerThr IleThr ThrAla
15 20 25
4 Phe ProPro ValSerSer ThrThrLeu PheAlaVal GlyAla ThrHis
5
30 35 40
Ser AlaSer IleGlnGlu GluThrGlu GluValVal AsnSer GlnLeu
50 55
Pro LeuLeu LeuSerLeu LeuAlaLeu LeuLeuLeu LeuLeu ValGly
65 70 75
Ala SerLeu LeuAlaTrp ArgMetPhe GlnLysTrp IleLys AlaGly
55 80 85 90
Asp HisSer GluLeuSer GlnAsnPro LysGlnAla AlaThr GlnSer
95 100 105
CA 02391669 2002-05-14
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11
Glu Leu His Tyr Ala Asn Leu Glu Leu Leu Met Trp Pro Leu Gln Glu
110 115 120
Lys Pro Ala Pro Pro Arg Glu Val Glu Val Glu Tyr Ser Thr Val Ala
125 130 135
Ser Pro Arg Glu Glu Leu His Tyr Ala Ser Val Val Phe Asp Ser Asn
140 145 150 155
Thr Asn Arg Ile Ala Ala Gln Arg Pro Arg Glu Glu Glu Pro Asp Ser
160 165 170
Asp Tyr Ser Val Ile Arg Lys Thr
175
<210> 8
<211> 939
<212> DNA
2 0 <213> Artificial Sequence
30
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc_feature
<222> (1) . (939)
<223> n may be a, c, g, or t
<400> 8
atggcnytnc cngtnacngc nytnytnytn ccnytngcny tnytnytnca ygcngcnmgn 60
ccngaytaya argaygayga ygayaarath gayytnwsna artgymgnac ngtngcnggn 120
ccngtnggng gnwsnytnws ngtncartgy ccntaygara argarcaymg nacnytnaay 180
aartaytggt gymgnccncc ncarathtty ytntgygaya arathgtnga racnaarggn 240
4 0 wsngcnggna armgnaaygg nmgngtnwsn athmgngayw snccngcnaa yytnwsntty 300
acngtnacny tngaraayyt nacngargar gaygcnggna cntaytggtg yggngtngay 360
acnccntggy tnmgngaytt ycaygayccn gtngtngarg tngargtnws ngtnttyccn 420
gcnwsnacnw snatgacncc ngcnwsnath acngcngcna aracnwsnac nathacnacn 480
gcnttyccnc cngtnwsnws nacnacnytn ttygcngtng gngcnacnca ywsngcnwsn 540
5 0 athcargarg aracngarga rgtngtnaay wsncarytnc cnytnytnyt nwsnytnytn 600
gcnytnytny tnytnytnyt ngtnggngcn wsnytnytng cntggmgnat gttycaraar 660
tggathaarg cnggngayca ywsngarytn wsncaraayc cnaarcargc ngcnacncar 720
wsngarytnc aytaygcnaa yytngarytn ytnatgtggc cnytncarga raarccngcn 780
ccnccnmgng argtngargt ngartaywsn acngtngcnw snccnmgnga rgarytncay 840
taygcnwsng tngtnttyga ywsnaayacn aaymgnathg cngcncarmg nccnmgngar 900
CA 02391669 2002-05-14
WO 01/36463 PCT/US00/31167
12
gargarccng aywsngayta ywsngtnath mgnaaracn 939
<210> 9
<211> 600
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: reverse
translation
<220>
<221> misc feature
<222> (1). (600)
<223> n
may be
a, c, g,
or t
<400> 9
2 atggcnytnc cngtnacngcnytnytnytnccnytngcnytnytnytncaygcngcnmgn60
0
ccngaytaya argaygaygaygayaarathgayatgacnccngcnwsnathacngcngcn120
aaracnwsna cnathacnacngcnttyccnccngtnwsnwsnacnacnytnttygcngtn180
ggngcnacnc aywsngcnwsnathcargargaracngargargtngtnaaywsncarytn240
ccnytnytny tnwsnytnytngcnytnytnytnytnytnytngtnggngcnwsnytnytn300
3 gcntggmgna tgttycaraartggathaargcnggngaycaywsngarytnwsncaraay360
0
ccnaarcarg cngcnacncarwsngarytncaytaygcnaayytngarytnytnatgtgg420
ccnytncarg araarccngcnccnccnmgngargtngargtngartaywsnacngtngcn480
wsnccnmgng argarytncaytaygcnwsngtngtnttygaywsnaayacnaaymgnath540
gcngcncarm gnccnmgngargargarccngaywsngaytaywsngtnathmgnaaracn600
