Note: Descriptions are shown in the official language in which they were submitted.
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SIALIC ACID-BINDING IG-LIKE LECTIN (SIGLEC) GENE; OB-BINDING PROTEIN LIKE (OB-
BPL)
FIELD OF THE INVENTION
The invention relates to nucleic acid molecules, proteins encoded by such
nucleic acid molecules; and
use of the proteins and nucleic acid molecules
BACKGROUND OF THE INVENTION
The immunoglobulinsuperfamily (IgSF) encompasses a large number of cell
surface molecules which
play a vital role not only in immunity, but also in controlling the behaviour
of cells in various tissues, through
their ability to mediate cell surface recognition events. These molecules are
characterized by the presence of
at least one immunoglobulin (Ig) domain, a sandwich of two (3-sheets
stabilized by a conserved disulfide bond.
The core of this domain is composed of (3-strands A,B,E in one sheet and G,F,C
in the other, and arise from
the ends of the domain sequence (Williams and Barclay 1988). In between,
however, there is a great deal of
sequence length variation. Such Ig domains occur in two types, the V-set and
the C-set, and can be
distinguished based on patterns of conserved amino acid residues responsible
for forming the characteristic (3-
sheet sandwich. V-set domains consist of about 65-75 amino acid residues
between conserved cysteines,
whereas C-set domains have about 55-60 residues (reviewed in (Williams and
Barclay 1988)). The C-set
domains can be further divided into C I- and C2-sets, and are distinguished by
the fact that, although showing
signs of a C-set domain, the latter half of C2-set domains exhibit sequence
patterns more homologous to V-set
rather than C 1-set domains (Williams et al., 1989).
Recently, a novel family of structurally related IgSF molecules have been
identified, which mediate
2 0 protein-carbohydrate interactions through specific interactions with
sialic acid-containing glycoproteins and
glycolipids (Crocker et al., I 996). This family was originally referred to as
the sialoadhesins, but has recently
been designated the sialic acid-binding Ig-like lectin (Siglec) family
(Crocker et al., 1998). These molecules
are characterized by the presence of one N-terminal V-set domain, and a
variable number of downstream C2-set
domains, ranging from 16 in sialoadhesin to 1 in CD33 (Crocker et al., 1996).
Furthermore, these Ig-like
2 5 domains possess some unique features. In the V-set domain, the conserved
cysteine in (3-strand F of classic
V-set domains is absent, while a highly conserved cysteine is present in (3-
strand E in all siglecs identified so
far. This results in the cysteines in (3-strands B and E being next to each
other in one ~3-sheet, which likely
results in an intrasheet disulfide bond (Crocker et al., I 996; Williams et
al., 1989). There is also an additional
highly conserved cysteine residue in both the V-set and first C2-set domains
of all siglecs. In the V-set domain
3 0 it is located at the beginning of (3-strand B, while in the C2-set domain
it is found between ~i-strands B and C.
These two additional cysteines have been found to form an interdomain
disulfide bond, a feature unique to
siglecs (Crocker et al., 1996; Pedraza et al., 1990).
Currently, the siglec family consists of sialoadhesin (Siglec-I), CD22 (Siglec-
2), CD33 (Siglec-3),
myelin-associated glycoprotein (MAG) (Siglec-4a), Schwann cell myelin protein
(SMP) (Siglec-4b), OB-
3 5 binding protein 2 (Siglec-5), OB-binding protein 1 (Siglec-6), and
p75/AIRM 1 (Siglec-7) (Cornish et al., 1998;
Crocker et al., 1998; Falco et al., 1999; Nicoll et al., 1999; Patel et al.,
1999). Each member of the
SUBSTITUTE SHEET (RULE 26)
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Siglec family is expressed by specific cell types and exhibits a distinct
function. Sialoadhesin is a
macrophage-restricted adhesion molecule (Crocker et al., 1994), CD22 is B
lymphocyte-specific and
regulates its activation (Stamenkovic and Seed 1990), CD33 is a myeloid-
specific inhibitory receptor
(Ulyanova et al., 1999), and MAG functions in the formation and maintenance of
axonal myelin structure
(Li et al., 1998). Siglec-5 and -6 (OB-BP2 and -BP1, respectively) are
expressed in several tissues
including placenta and peripheral blood leukocytes, and have shown an in vitro
ability to bind leptin
(Cornish et al., 1998; Patel et al., 1999), while OB-BPL (p75/AIRM1) is an
inhibitory receptor expressed
predominantly on human natural killer cells (Falco et al., 1999; Nicoll et
al., 1999).
SUMMARY OF THE INVENTION
The present inventors have identified and characterized a gene encoding a
novel member of the
siglec family (OB-binding protein like or OB-BPL). The putative protein
product displays a high degree
of homology with siglec-7, as well as with siglec-5 and siglec-6. Further, it
possesses all the structural
features found in other siglecs. The gene was localized to 19q 13.4, 43.19 Kb
more telomeric than KLK-L6
(a member of the kallikrein gene family) through genomic sequencing data and
restriction mapping with
EcoRI. The novel siglec is encoded by 7 exons, with six intervening introns.
In addition, it is highly
expressed in bone marrow, placenta, spleen, and fetal liver, as well as other
tissues at lower levels.
The OB-BPL protein described herein is referred to as "OB-BPL Protein". The
gene encoding
the protein is referred to as " ob-bpl ".
Broadly stated the present invention relates to an isolated nucleic acid
molecule which comprises:
2 0 (i) a nucleic acid sequence encoding a protein having substantial sequence
identity with an
amino acid sequence of OB-BPL as shown in Table 5 or SEQ.ID.NO. 2 or 3;
(ii) a nucleic acid sequence encoding a protein comprising an amino acid
sequence of OB-
BPL as shown in Table S or SEQ.ID.NO. 2 or 3;
(iii) nucleic acid sequences complementary to (i);
2 5 (iv) a degenerate form of a nucleic acid sequence of (i);
(v) a nucleic acid sequence capable of hybridizing under stringent conditions
to a nucleic
acid sequence in (i), (ii) or (iii);
(vi) a nucleic acid sequence encoding a truncation, an analog, an allelic or
species variation
of a protein comprising an amino acid sequence of OB-BPL as shown in Table 5
or
3 0 SEQ.ID.NO. 2 or 3; or
(vii) a fragment, or allelic or species variation of (i), (ii) or (iii).
Preferably, a purified and isolated nucleic acid molecule of the invention
comprises:
(i) a nucleic acid sequence comprising the sequence of SEQ.ID.NO. 1 wherein T
can also
be U;
3 5 (ii) nucleic acid sequences complementary to (i), preferably complementary
to the full
nucleic acid sequence of SEQ.ID.NO. 1;
(iii) a nucleic acid capable of hybridizing under stringent conditions to a
nucleic acid of (i)
or (ii) and preferably having at least 18 nucleotides; or
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(iv) a nucleic acid molecule differing from any of the nucleic acids of (i) to
(iii) in codon
sequences due to the degeneracy of the genetic code.
The invention also contemplates a nucleic acid molecule comprising a sequence
encoding a
truncation of an OB-BPL Protein, an analog, or a homolog of an OB-BPL Protein
or a truncation thereof.
(OB-BPL Protein and truncations, analogs and homologs of OB-BPL Protein are
also collectively referred
to herein as " OB-BPL Related Proteins").
The nucleic acid molecules of the invention may be inserted into an
appropriate expression vector,
i.e. a vector that contains the necessary elements for the transcription and
translation of the inserted coding
sequence. Accordingly, recombinant expression vectors adapted for
transformation of a host cell may be
constructed which comprise a nucleic acid molecule of the invention and one or
more transcription and
translation elements linked to the nucleic acid molecule.
The recombinant expression vector can be used to prepare transformed host
cells expressing OB-
BPL Related Proteins. Therefore, the invention further provides host cells
containing a recombinant
molecule of the invention. The invention also contemplates transgenic non-
human mammals whose germ
cells and somatic cells contain a recombinant molecule comprising a nucleic
acid molecule of the invention,
in particular one which encodes an analog of the OB-BPL Protein, or a
truncation of the OB-BPL Protein.
The invention further provides a method for preparing OB-BPL Related Proteins
utilizing the
purified and isolated nucleic acid molecules of the invention. In an
embodiment a method for preparing an
OB-BPL Related Protein is provided comprising (a) transferring a recombinant
expression vector of the
2 0 invention into a host cell; (b) selecting transformed host cells from
untransformed host cells; (c) culturing
a selected transformed host cell under conditions which allow expression of
the OB-BPL Related Protein;
and (d) isolating the OB-BPL Related Protein.
The invention further broadly contemplates an isolated OB-BPL Protein
comprising an amino acid
sequence as shown in SEQ.ID.NO. 2 or 3
2 5 The OB-BPL Related Proteins of the invention may be conjugated with other
molecules, such as
proteins, to prepare fusion proteins. This may be accomplished, for example,
by the synthesis of N-terminal
or C-terminal fusion proteins.
The invention further contemplates antibodies having specificity against an
epitope of an OB-BPL
Related Protein of the invention. Antibodies may be labeled with a detectable
substance and used to detect
3 0 proteins of the invention in tissues and cells.
The invention also permits the construction of nucleotide probes which are
unique to the nucleic
acid molecules of the invention and/or to proteins of the invention.
Therefore, the invention also relates to
a probe comprising a nucleic acid sequence of the invention, or a nucleic acid
sequence encoding a protein
of the invention, or a part thereof. The probe may be labeled, for example,
with a detectable substance and
3 5 it may be used to select from a mixture of nucleotide sequences a nucleic
acid molecule of the invention
including nucleic acid molecules coding for a protein which displays one or
more of the properties of a
protein of the invention.
The invention still further provides a method for identifying a substance
which binds to a protein
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of the invention comprising reacting the protein with at least one substance
which potentially can bind with
the protein, under conditions which permit the formation of complexes between
the substance and protein
and detecting binding. Binding may be detected by assaying for complexes, for
free substance, or for non-
complexed protein. The invention also contemplates methods for identifying
substances that bind to other
intracellular proteins that interact with an OB-BPL Related Protein. Methods
can also be utilized which
identify compounds which bind to OB-BPL gene regulatory sequences (e.g.
promoter sequences).
Still further the invention provides a method for evaluating a compound for
its ability to modulate
the biological activity of an OB-BPL Related Protein of the invention. For
example a substance which
inhibits or enhances the interaction of the protein and a substance which
binds to the protein may be
evaluated. In an embodiment, the method comprises providing a known
concentration of an OB-BPL
Related Protein, with a substance which binds to the protein and a test
compound under conditions which
permit the formation of complexes between the substance and protein, and
removing andlor detecting
complexes.
Compounds which modulate the biological activity of a protein of the invention
may also be
identified using the methods of the invention by comparing the pattern and
level of expression of the protein
of the invention in tissues and cells, in the presence, and in the absence of
the compounds.
The proteins of the invention and substances and compounds identified using
the methods of the
invention, and peptides of the invention may be used to modulate the
biological activity of an OB-BPL
Related Protein of the invention, and they may be used in the treatment of
conditions such a disorders of
2 0 the hematopoietic system and in particular leukemias. Accordingly, the
substances and compounds may be
formulated into compositions for administration to individuals suffering from
a disorders of the
hematopoietic system.
Therefore, the present invention also relates to a composition comprising one
or more of a protein
of the invention, a peptide of the invention, or a substance or compound
identified using the methods of the
2 5 invention, and a pharmaceutically acceptable carrier, excipient or
diluent. A method for treating or
preventing cancer or a disorder of the hematopoietic system is also provided
comprising administering to
a patient in need thereof, an OB-BPL Related Protein of the invention, or a
composition of the invention.
Other objects, features and advantages of the present invention will become
apparent from the
following detailed description. It should be understood, however, that the
detailed description and the
3 0 specific examples while indicating preferred embodiments of the invention
are given by way of illustration
only, since various changes and modifications within the spirit and scope of
the invention will become
apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
3 5 Figure 1: Genomic Structure of a Novel Siglec. Shown are the exon/intron
boundaries, as well
as the predicted protein sequence. The single underlined region is the 5'
untranslated region, and the
double underlined region is the 3' untranslated region. In the shaded box is
the putative polyadenylation
signal.
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Figure 2: Hydophobicity Plot of the Novel Siglec. This shows the regions of
the putative novel
siglec protein which contain stretches of hydrophobic amino acid residues. As
is evident, there are two
such regions, the first corresponding to the signal peptide, and the second,
at around residues 350-370, the
putative transmembrane region.
Figure 3: Localization of the Novel Siglec Gene. The physical map of the
genomic area around
chromosome 19q 13.3-q 13.4 where the kallikrein gene family resides. Seven
additional kallikreins map in
the 132.1 Kb region (data not shown; see (Diamandis et al., 1999)). Gene
lengths are presented above each
arrow, and distances between genes are shown below. Arrows denote the
direction of transcription. The
novel siglec gene resides 43.2 Kb telomeric to the KLK-L6 gene. KLK,
kallikrein; PSA, prostate specific
antigen; KLK-L, kallikrein-like; NES1, normal epithelial cell-specific 1 gene;
TLSP, trypsin-like serine
protease.
Figure 4: Siglec Family Multiple Alignment. The novel siglec was aligned with
siglec-5 to -7 and
CD33, using ClustalX (Jeanmougin et al., 1998) (SEQ. ID. NOs. 10-13). The
signal peptide was
determined through computer prediction, and the Ig domain boundaries were
assigned based on exon
boundaries. The transmembrane domain was also predicted, while taking into
consideration exon
boundaries as well. The IT'IM-like and SLAM-like motifs are indicated, as are
the conserved cysteines (*)
which form the disulfide bonds of the Ig-like domains in siglecs, and the
conserved arginine and aromatic
residues (lx) which are responsible for sialic acid binding and specificity.
Figure 5: Phylogenetic Analysis of the Siglec Family. The phylogenetic tree
was created using
2 0 ClustalX (Jeanmougin et al., 1998) and TreeView (Page 1996). As is
evident, siglec-7 and the novel siglec
are very closely related, and they are both related to CD33, in addition to a
more distant relation to the other
siglecs.
Figure 6: Tissue Expression Profile of the Novel Siglec. RT-PCR was performed
on 28 tissue
total RNAs, for this novel siglec and actin (control gene). The novel siglec
is highly expressed in bone
2 5 marrow, placenta, spleen, and fetal liver. There is also a lower degree of
expression in many of the other
tissues, while it is absent in ovary, pancreas, skeletal muscle, and heart.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention there may be employed conventional
molecular biology,
microbiology, and recombinant DNA techniques within the skill of the art. Such
techniques are explained
3 0 fully in the literature. See for example, Sambrook, Fritsch, & Maniatis,
Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y); DNA
Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985);
Oligonucleotide Synthesis (M..J.
Gait ed. 1984); Nucleic Acid Hybridization B.D. Hames & S.J. Higgins eds.
(1985); Transcription and
Translation B.D. Hames & S.J. Higgins eds (1984); Animal Cell Culture R.I.
Freshney, ed. (1986);
3 5 Immobilized Cells and enzymes IRL Press, (1986); and B. Perbal, A
Practical Guide to Molecular Cloning
( 1984).
1. Nucleic Acid Molecules of the Invention
As hereinbefore mentioned, the invention provides an isolated nucleic acid
molecule having a
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sequence encoding an OB-BPL Protein. The term "isolated" refers to a nucleic
acid substantially free of
cellular material or culture medium when produced by recombinant DNA
techniques, or chemical reactants,
or other chemicals when chemically synthesized. An "isolated" nucleic acid may
also be free of sequences
which naturally flank the nucleic acid (i.e., sequences located at the 5' and
3' ends of the nucleic acid
molecule) from which the nucleic acid is derived. The term "nucleic acid" is
intended to include DNA and
RNA and can be either double stranded or single stranded. In an embodiment, a
nucleic acid molecule
encodes an OB-BPL Protein comprising an amino acid sequence as shown in
SEQ.ID.NO. 2 or 3,
preferably a nucleic acid molecule comprising a nucleic acid sequence as shown
in SEQ.ID.NO. 1.
The invention includes nucleic acid sequences complementary to a nucleic acid
encoding an OB-
BPL Protein comprising an amino acid sequence as shown in SEQ.)D.NO. 2 or 3,
preferably the nucleic
acid sequences complementary to a full nucleic acid sequence shown in
SEQ.ID.NO. 1.
The invention includes nucleic acid molecules having substantial sequence
identity or homology
to nucleic acid sequences of the invention or encoding proteins having
substantial identity or similarity to
the amino acid sequence shown in in SEQ.ID.NO. 2 or 3. Preferably, the nucleic
acids have substantial
sequence identity for example at least 65%, 70%, 75%, 80%, or 85% nucleic acid
identity; more preferably
90% nucleic acid identity; and most preferably at least 95%, 96%, 97%, 98%, or
99% sequence identity.
"Identity" as known in the art and used herein, is a relationship between two
or more amino acid sequences
or two or more nucleic acid sequences, as determined by comparing the
sequences. It also refers to the
degree of sequence relatedness between amino acid or nucleic acid sequences,
as the case may be, as
2 0 determined by the match between strings of such sequences. Identity and
similarity are well known terms
to skilled artisans and they can be calculated by conventional methods (for
example see Computational
Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics
and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer
Analysis of Sequence
Data, Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey,
1994; Sequence Analysis
2 5 in Molecular Biology, von Heinje, G. Acadmeic Press, 1987; and Sequence
Analysis Primer, Gribskov, M.
and Devereux, J. eds. M. Stockton Press, New York, 1991, Carillo, H. and
Lipman, D., SIAM J. Applied
Math. 48:1073, 1988). Methods which are designed to give the largest match
between the sequences are
generally preferred. Methods to determine identity and similarity are codified
in publicly available
computer programs including the GCG program package (Devereux J. et al.,
Nucleic Acids Research 12(1):
3 0 387, 1984); BLASTP, BLASTN, and FASTA (Atschul, S.F. et al. J. Molec.
Biol. 215: 403-410, 1990). The
BLAST X program is publicly available from NCBI and other sources (BLAST
Manual, Altschul, S. et al.
NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J. Mol. Biol. 215: 403-
410, 1990).
Isolated nucleic acid molecules encoding an OB-BPL Protein, and having a
sequence which differs
from a nucleic acid sequence of the invention due to degeneracy in the genetic
code are also within the
3 5 scope of the invention. Such nucleic acids encode functionally equivalent
proteins (e.g., an OB-BPL
Protein) but differ in sequence from the sequence of an OB-BPL Protein due to
degeneracy in the genetic
code. As one example, DNA sequence polymorphisms within the nucleotide
sequence of an OB-BPL
Protein may result in silent mutations which do not affect the amino acid
sequence. Variations in one or
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more nucleotides may exist among individuals within a population due to
natural allelic variation. Any and
all such nucleic acid variations are within the scope of the invention. DNA
sequence polymorphisms may
also occur which lead to changes in the amino acid sequence of an OB-BPL
Protein. These amino acid
polymorphisms are also within the scope of the present invention.
Another aspect of the invention provides a nucleic acid molecule which
hybridizes under stringent
conditions, preferably high stringency conditions to a nucleic acid molecule
which comprises a sequence
which encodes an OB-BPL Protein having an amino acid sequence shown in
SEQ.ID.NO. 2 or 3.
Appropriate stringency conditions which promote DNA hybridization are known to
those skilled in the art,
or can be found in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6.
For example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C,
followed by a wash of 2.0 x SSC
at 50°C may be employed. The stringency may be selected based on the
conditions used in the wash step.
By way of example, the salt concentration in the wash step can be selected
from a high stringency of about
0.2 x SSC at 50°C. In addition, the temperature in the wash step can be
at high stringency conditions, at
about65°C.
It will be appreciated that the invention includes nucleic acid molecules
encoding an OB-BPL
Related Protein including truncations of an OB-BPL Protein, and analogs of an
OB-BPL Protein as
described herein. The truncated nucleic acids or nucleic acid fragments may
correspond to the
transmembrane domain, cytoplasmic domain, IG domains,or ITIM-like or SLAM-like
motifs as described
in Table 4 and in Figure 4. It will further be appreciated that variant forms
of the nucleic acid molecules
2 0 of the invention which arise by alternative splicing of an mRNA
corresponding to a cDNA of the invention
are encompassed by the invention.
An isolated nucleic acid molecule of the invention which comprises DNA can be
isolated by
preparing a labelled nucleic acid probe based on all or part of a nucleic acid
sequence of the invention. The
labeled nucleic acid probe is used to screen an appropriate DNA library (e.g.
a cDNA or genomic DNA
2 5 library). For example, a cDNA library can be used to isolate a cDNA
encoding an OB-BPL Related Protein
by screening the library with the labeled probe using standard techniques.
Alternatively, a genomic DNA
library can be similarly screened to isolate a genomic clone encompassing a
gene encoding an OB-BPL
Related Protein. Nucleic acids isolated by screening of a cDNA or genomic DNA
library can be sequenced
by standard techniques.
3 0 An isolated nucleic acid molecule of the invention which is DNA can also
be isolated by
selectively amplifying a nucleic acid encoding an OB-BPL Related Protein using
the polymerise chain
reaction (PCR) methods and cDNA or genomic DNA. It is possible to design
synthetic oligonucleotide
primers from the nucleotide sequence of the invention for use in PCR. A
nucleic acid can be amplified from
cDNA or genomic DNA using these oligonucleotide primers and standard PCR
amplification techniques.
3 5 The nucleic acid so amplified can be cloned into an appropriate vector and
characterized by DNA sequence
analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by
a variety of techniques,
for example, by using the guanidinium-thiocyanate extraction procedure of
Chirgwin et al., Biochemistry,
18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse
transcriptase (for example,
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Moloney MLV reverse transcriptase available from GibcoBRL, Bethesda, MD, or
AMV reverse
transcriptase available from Seikagaku America, Inc., St. Petersburg, FL).
An isolated nucleic acid molecule of the invention which is RNA can be
isolated by cloning a
cDNA encoding an OB-BPL Related Protein into an appropriate vector which
allows for transcription of
the cDNA to produce an RNA molecule which encodes an OB-BPL Related Protein.
For example, a
cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7
promoter) in a vector, cDNA can
be transcribed in vitro with T7 polymerase, and the resultant RNA can be
isolated by conventional
techniques.
Nucleic acid molecules of the invention may be chemically synthesized using
standard techniques.
Methods of chemically synthesizing polydeoxynucleotides are known, including
but not limited to solid-
phase synthesis which, like peptide synthesis, has been fully automated in
commercially available DNA
synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et
al. U.S. Patent No. 4,458,066;
and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
Determination of whether a particular nucleic acid molecule encodes an OB-BPL
Related Protein
can be accomplished by expressing the cDNA in an appropriate host cell by
standard techniques, and
testing the expressed protein in the methods described herein. A cDNA encoding
an OB-BPL Related
Protein can be sequenced by standard techniques, such as dideoxynucleotide
chain termination or Maxam-
Gilbert chemical sequencing, to determine the nucleic acid sequence and the
predicted amino acid sequence
of the encoded protein.
2 0 The initiation codon and untranslated sequences of an OB-BPL Related
Protein may be determined
using computer software designed for the purpose, such as PC/Gene
(IntelliGenetics Inc., Calif.). The
intron-exon structure and the transcription regulatory sequences of a gene
encoding an OB-BPL Related
Protein may be confirmed by using a nucleic acid molecule of the invention
encoding an OB-BPL Related
Protein to probe a genomic DNA clone library. Regulatory elements can be
identified using standard
2 5 techniques. The function of the elements can be confirmed by using these
elements to express a reporter
gene such as the lacZ gene which is operatively linked to the elements. These
constructs may be introduced
into cultured cells using conventional procedures or into non-human transgenic
animal models. In addition
to identifying regulatory elements in DNA, such constructs may also be used to
identify nuclear proteins
interacting with the elements, using techniques known in the art.
3 0 In a particular embodiment of the invention, the nucleic acid molecules
isolated using the methods
described herein are mutant OB-BPL gene alleles. The mutant alleles may be
isolated from individuals
either known or proposed to have a genotype which contributes to the symptoms
of a disorder of the
hematopoietic system (e.g. leukemias). Mutant alleles and mutant allele
products may be used in
therapeutic and diagnostic methods described herein. For example, a cDNA of a
mutant OB-BPL gene may
3 5 be isolated using PCR as described herein, and the DNA sequence of the
mutant allele may be compared
to the normal allele to ascertain the mutations) responsible for the loss or
alteration of function of the
mutant gene product. A genomic library can also be constructed using DNA from
an individual suspected
of or known to carry a mutant allele, or a cDNA library can be constructed
using RNA from tissue known,
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or suspected to express the mutant allele. A nucleic acid encoding a normal OB-
BPL gene or any suitable
fragment thereof, may then be labeled and used as a probe to identify the
corresponding mutant allele in
such libraries. Clones containing mutant sequences can be purified and
subjected to sequence analysis. In
addition, an expression library can be constructed using cDNA from RNA
isolated from a tissue of an
individual known or suspected to express a mutant OB-BPL allele. Gene products
made by the putatively
mutant tissue may be expressed and screened, for example using antibodies
specific for an OB-BPL Related
Protein as described herein. Library clones identified using the antibodies
can be purified and subjected
to sequence analysis.
The sequence of a nucleic acid molecule of the invention, or a fragment of the
molecule, may be
inverted relative to its normal presentation for transcription to produce an
antisense nucleic acid molecule.
An antisense nucleic acid molecule may be constructed using chemical synthesis
and enzymatic ligation
reactions using procedures known in the art.
2. Proteins of the Invention
An amino acid sequence of an OB-BPL Protein comprises a sequence as shown in
SEQ.ID.NO.
2 or 3. The protein is highly expressed in bone marrow, placenta, spleen, and
fetal liver.
In addition to proteins comprising an amino acid sequence as shown in
SEQ.ID.NO. 2 or 3, the
proteins of the present invention include truncations of an OB-BPL Protein,
analogs of an OB-BPL
Protein, and proteins having sequence identity or similarity to an OB-BPL
Protein, and truncations thereof
as described herein (i.e. OB-BPL Related Proteins). Truncated proteins may
comprise peptides of between
2 0 3 and 70 amino acid residues, ranging in size from a tripeptide to a 70
mer polypeptide.
The truncated proteins may have an amino group (-NH2), a hydrophobic group
(for example,
carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-
fluorenylmethoxy-carbonyl (PMOC)
group, or a macromolecule including but not limited to lipid-fatty acid
conjugates, polyethylene glycol, or
carbohydrates at the amino terminal end. The truncated proteins may have a
carboxyl group, an amido
2 5 group, a T-butyloxycarbonyl group, or a macromolecule including but not
limited to lipid-fatty acid
conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.
The proteins of the invention may also include analogs of an OB-BPL Protein,
and/or truncations
thereof as described herein, which may include, but are not limited to an OB-
BPL Protein, containing one
or more amino acid substitutions, insertions, and/or deletions. Amino acid
substitutions may be of a
3 0 conserved or non-conserved nature. Conserved amino acid substitutions
involve replacing one or more
amino acids of an OB-BPL Protein amino acid sequence with amino acids of
similar charge, size, and/or
hydrophobicity characteristics. When only conserved substitutions are made the
resulting analog is
preferably functionally equivalent to an OB-BPL Protein. Non-conserved
substitutions involve replacing
one or more amino acids of the OB-BPL Protein amino acid sequence with one or
more amino acids which
3 5 possess dissimilar charge, size, and/or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into an OB-BPL Protein.
Amino acid
insertions may consist of single amino acid residues or sequential amino acids
ranging from 2 to 15 amino
acids in length.
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Deletions may consist of the removal of one or more amino acids, or discrete
portions from an OB-
BPL Protein sequence. The deleted amino acids may or may not be contiguous.
The lower limit length of
the resulting analog with a deletion mutation is about 10 amino acids,
preferably 20 to 40 amino acids.
The proteins of the invention include proteins with sequence identity or
similarity to an OB-BPL
Protein and/or truncations thereof as described herein. Such OB-BPL Proteins
include proteins whose
amino acid sequences are comprised of the amino acid sequences of OB-BPL
Protein regions from other
species that hybridize under selected hybridization conditions (see discussion
of stringent hybridization
conditions herein) with a probe used to obtain an OB-BPL Protein. These
proteins will generally have the
same regions which are characteristic of an OB-BPL Protein. Preferably a
protein will have substantial
sequence identity for example, about 65%, 70%, 75%, 80%, or 85% identity,
preferably 90% identity, more
preferably at least 95%, 96%, 97%, 98%, or 99% identity, and most preferably
98% identity with an amino
acid sequence shown in in SEQ.ID.NO. 2 or 3. A percent amino acid sequence
homology, similarity or
identity is calculated as the percentage of aligned amino acids that match the
reference sequence using
known methods as described herein.
The invention also contemplates isoforms of the proteins of the invention. An
isoform contains
the same number and kinds of amino acids as a protein of the invention, but
the isoform has a different
molecular structure. Isoforms contemplated by the present invention preferably
have the same properties
as a protein of the invention as described herein.
The present invention also includes OB-BPL Related Proteins conjugated with a
selected protein,
2 0 or a marker protein (see below) to produce fusion proteins. Additionally,
immunogenic portions of an OB-
BPL Protein and an OB-BPL Protein Related Protein are within the scope of the
invention.
AN OB-BPL Related Protein of the invention may be prepared using recombinant
DNA methods.
Accordingly, the nucleic acid molecules of the present invention having a
sequence which encodes an OB-
BPL Related Protein of the invention may be incorporated in a known manner
into an appropriate
2 5 expression vector which ensures good expression of the protein. Possible
expression vectors include but
are not limited to cosmids, plasmids, or modified viruses (e.g. replication
defective retroviruses,
adenoviruses and adeno-associated viruses), so long as the vector is
compatible with the host cell used.
The invention therefore contemplates a recombinant expression vector of the
invention containing
a nucleic acid molecule of the invention, and the necessary regulatory
sequences for the transcription and
3 0 translation of the inserted protein-sequence. Suitable regulatory
sequences may be derived from a variety
of sources, including bacterial, fungal, viral, mammalian, or insect genes
[For example, see the regulatory
sequences described in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic
Press, San Diego, CA (1990)]. Selection of appropriate regulatory sequences is
dependent on the host cell
chosen as discussed below, and may be readily accomplished by one of ordinary
skill in the art. The
3 5 necessary regulatory sequences may be supplied by the native OB-BPL
Protein and/or its flanking regions.
The invention further provides a recombinant expression vector comprising a
DNA nucleic acid
molecule of the invention cloned into the expression vector in an antisense
orientation. That is, the DNA
molecule is linked to a regulatory sequence in a manner which allows for
expression, by transcription of
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the DNA molecule, of an RNA molecule which is antisense to the nucleic acid
sequence of a protein of the
invention or a fragment thereof. Regulatory sequences linked to the antisense
nucleic acid can be chosen
which direct the continuous expression of the antisense RNA molecule in a
variety of cell types, for
instance a viral promoter and/or enhancer, or regulatory sequences can be
chosen which direct tissue or cell
type specific expression of antisense RNA.
The recombinant expression vectors of the invention may also contain a marker
gene which
facilitates the selection of host cells transformed or transfected with a
recombinant molecule of the
invention. Examples of marker genes are genes encoding a protein such as 6418
and hygromycin which
confer resistance to certain drugs, (3-galactosidase, chloramphenicol
acetyltransferase, firefly luciferase,
or an immunoglobulin or portion thereof such as the Fc portion of an
immunoglobulin preferably IgG. The
markers can be introduced on a separate vector from the nucleic acid of
interest.
The recombinant expression vectors may also contain genes which encode a
fusion moiety which
provides increased expression of the recombinant protein; increased solubility
of the recombinant protein;
and aid in the purification of the target recombinant protein by acting as a
ligand in affinity purification.
For example, a proteolytic cleavage site may be added to the target
recombinant protein to allow separation
of the recombinant protein from the fusion moiety subsequent to purification
of the fusion protein. Typical
fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia),
pMAL (New England
Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse
glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the
recombinant protein.
2 0 The recombinant expression vectors may be introduced into host cells to
produce a transformant
host cell. "Transformant host cells" include host cells which have been
transformed or transfected with a
recombinant expression vector of the invention. The terms "transformed with",
"transfected with",
"transformation" and "transfection" encompass the introduction of a nucleic
acid (e.g. a vector) into a cell
by one of many standard techniques. Prokaryotic cells can be transformed with
a nucleic acid by, for
2 5 example, electroporation or calcium-chloride mediated transformation. A
nucleic acid can be introduced
into mammalian cells via conventional techniques such as calcium phosphate or
calcium chloride co-
precipitation, DEAF-dextran-mediated transfection, lipofectin, electroporation
or microinjection. Suitable
methods for transforming and transfecting host cells can be found in Sambrook
et al. (Molecular Cloning:
A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)),
and other laboratory
3 0 textbooks.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host
cells. For example,
the proteins of the invention may be expressed in bacterial cells such as E.
coli, insect cells (using
baculovirus), yeast cells, or mammalian cells. Other suitable host cells can
be found in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
CA (1991)
3 5 A host cell may also be chosen which modulates the expression of an
inserted nucleic acid
sequence, or modifies (e.g. glycosylation or phosphorylation) and processes
(e.g. cleaves) the protein in
a desired fashion. Host systems or cell lines may be selected which have
specific and characteristic
mechanisms for post-translational processing and modification of proteins. For
example, eukaryotic host
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cells including CHO, VERO, BHK, HeLA, COS, MDCK, 293, 3T3, and WI38 may be
used. For long-term
high-yield stable expression of the protein, cell lines and host systems which
stably express the gene
product may be engineered.
Host cells and in particular cell lines produced using the methods described
herein may be
particularly useful in screening and evaluating compounds that modulate the
activity of an OB-BPL
Related Protein.
The proteins of the invention may also be expressed in non-human transgenic
animals including
but not limited to mice, rats, rabbits, guinea pigs, micro-pigs, goats, sheep,
pigs, non-human primates (e.g.
baboons, monkeys, and chimpanzees) [see Hammer et al. (Nature 315:680-683,
1985), Palmiter et al.
(Science 222:809-814, 1983), Brinster et al. (Proc Natl. Acad. Sci USA
82:44384442, 1985), Palmiter and
Brinster (Cell. 41:343-345, 1985) and U.S. Patent No. 4,736,866)]. Procedures
known in the art may be
used to introduce a nucleic acid molecule of the invention encoding an OB-BPL
Related Protein into
animals to produce the founder lines of transgenic animals. Such procedures
include pronuclear
microinjection, retrovirus mediated gene transfer into germ lines, gene
targeting in embryonic stem cells,
electroporation of embryos, and sperm-mediated gene transfer.
The present invention contemplates a transgenic animal that carries the OB-BPL
gene in all their
cells, and animals which carry the transgene in some but not all their cells.
The transgene may be integrated
as a single transgene or in concatamers. The transgene may be selectively
introduced into and activated in
specific cell types (See for example, Lasko et al, 1992 Proc. Natl. Acad. Sci.
USA 89: 6236). The transgene
2 0 may be integrated into the chromosomal site of the endogenous gene by gene
targeting. The transgene may
be selectively introduced into a particular cell type inactivating the
endogenous gene in that cell type (See
Gu et al Science 265: 103-106).
The expression of a recombinant OB-BPL Related Protein in a transgenic animal
may be assayed
using standard techniques. Initial screening may be conducted by Southern Blot
analysis, or PCR methods
2 5 to analyze whether the transgene has been integrated. The level of mRNA
expression in the tissues of
transgenic animals may also be assessed using techniques including Northern
blot analysis of tissue
samples, in situ hybridization, and RT-PCR. Tissue may also be evaluated
immunocytochemically using
antibodies against OB-BPL Protein.
Proteins of the invention may also be prepared by chemical synthesis using
techniques well known
3 0 in the chemistry of proteins such as solid phase synthesis (Merrifield,
1964, J. Am. Chem. Assoc. 85:2149-
2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of
Organic Chemistry, ed. E.
Wansch, Vol. 15 I and II, Thieme, Stuttgart).
N-terminal or C-terminal fusion proteins comprising an OB-BPL Related Protein
of the invention
conjugated with other molecules, such as proteins, may be prepared by fusing,
through recombinant
3 5 techniques, the N-terminal or C-terminal of an OB-BPL Related Protein, and
the sequence of a selected
protein or marker protein with a desired biological function. The resultant
fusion proteins contain OB-BPL
Protein fused to the selected protein or marker protein as described herein.
Examples of proteins which
may be used to prepare fusion proteins include immunoglobulins, glutathione-S-
transferase (GST),
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hemagglutinin (HA), and truncated myc.
3. Antibodies
OB-BPL Related Proteins of the invention can be used to prepare antibodies
specific for the
proteins. Antibodies can be prepared which bind a distinct epitope in an
unconserved region of the protein.
An unconserved region of the protein is one that does not have substantial
sequence homology to other
proteins. A region from a conserved region such as a well-characterized domain
can also be used to prepare
an antibody to a conserved region of an OB-BPL Related Protein. Antibodies
having specificity for an
OB-BPL Related Protein may also be raised from fusion proteins created by
expressing fusion proteins
in bacteria as described herein.
The invention can employ intact monoclonal or polyclonal antibodies, and
immunologically active
fragments (e.g. a Fab, (Fab)2 fragment, or Fab expression library fragments
and epitope-binding fragments
thereof), an antibody heavy chain, and antibody light chain, a genetically
engineered single chain Fv
molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for
example, an antibody which
contains the binding specificity of a murine antibody, but in which the
remaining portions are of human
origin. Antibodies including monoclonal and polyclonal antibodies, fragments
and chimeras, may be
prepared using methods known to those skilled in the art.
4. Applications of the Nucleic Acid Molecules OB-BPL Related Proteins and
Antibodies of
the Invention
The nucleic acid molecules, OB-BPL Related Proteins, and antibodies of the
invention may be
2 0 used in the prognostic and diagnostic evaluation of cancer or disorders of
the hematopoietic system, and
the identification of subjects with a predisposition to cancer or
hematopoietic disorders (Section 4.1.1 and
4.1.2). Methods for detecting nucleic acid molecules and OB-BPL Related
Proteins of the invention, can
be used to monitor cancer or hematopoietic disorders by detecting OB-BPL
Related Proteins and nucleic
acid molecules encoding OB-BPL Related Proteins. It would also be apparent to
one skilled in the art that
2 5 the methods described herein may be used to study the developmental
expression of OB-BPL Related
Proteins and, accordingly, will provide further insight into the role of OB-
BPL Related Proteins. The
applications of the present invention also include methods for the
identification of compounds that
modulate the biological activity of OB-BPL or OB-BPL Related Proteins (Section
4.2). The compounds,
antibodies etc. may be used for the treatment of cancer or hematopoietic
disorders (Section 4.3).
3 0 4.1 Diaenostic Methods
A variety of methods can be employed for the diagnostic and prognostic
evaluation of cancer or
disorders of the hematopoietic system (e.g. leukemias), and the identification
of subjects with a
predisposition to cancer or hematopoietic disorders. Such methods may, for
example, utilize nucleic acid
molecules of the invention, and fragments thereof, and antibodies directed
against OB-BPL Related
3 5 Proteins, including peptide fragments. In particular, the nucleic acids
and antibodies may be used, for
example, for: (1) the detection of the presence of OB-BPL mutations, or the
detection of either over- or
under-expression of OB-BPL mRNA relative to a non-disorder state or the
qualitative or quantitative
detection of alternatively spliced forms of OB-BPL transcripts which may
correlate with certain conditions
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or susceptibility toward such conditions; and (2) the detection of either an
over- or an under-abundance of
OB-BPL Related Proteins relative to a non- disorder state or the presence of a
modified (e.g., less than full
length) OB-BPL Protein which correlates with a disorder state, or a
progression toward a disorder state.
The methods described herein may be performed by utilizing pre-packaged
diagnostic kits
comprising at least one specific OB-BPL nucleic acid or antibody described
herein, which may be
conveniently used, e.g., in clinical settings, to screen and diagnose patients
and to screen and identify those
individuals exhibiting a predisposition to developing a disorder.
Nucleic acid-based detection techniques are described, below, in Section
4.1.1. Peptide detection
techniques are described, below, in Section 4.1.2. The samples that may be
analyzed using the methods of
the invention include those which are known or suspected to express OB-BPL or
contain OB-BPL Related
Proteins. The samples may be derived from a patient or a cell culture, and
include but are not limited to
biological fluids, tissue extracts, freshly harvested cells, and lysates of
cells which have been incubated in
cell cultures.
Oligonucleotides or longer fragments derived from any of the nucleic acid
molecules of the
invention may be used as targets in a microarray. The microarray can be used
to simultaneously monitor
the expression levels of large numbers of genes and to identify genetic
variants, mutations, and
polymorphisms. The information from the microarray may be used to determine
gene function, to
understand the genetic basis of a disorder, to diagnose a disorder, and to
develop and monitor the activities
of therapeutic agents.
2 0 The preparation, use, and analysis of microarrays are well known to a
person skilled in the art.
(See, for example, Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796;
Schena, et al. (1996) Proc. Natl.
Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995), PCT Application
W095/251116; Shalom D. et
al. (I 995) PCT application W095/35505; Heller, R. A. et al. (1997) Proc.
Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
2 5 4.1.1 Methods for Detecting Nucleic Acid Molecules of the Invention
The nucleic acid molecules of the invention allow those skilled in the art to
construct nucleotide
probes for use in the detection of nucleic acid sequences of the invention in
samples. Suitable probes
include nucleic acid molecules based on nucleic acid sequences encoding at
least 5 sequential amino acids
from regions of the OB-BPL Protein, preferably they comprise 15 to 30
nucleotides. A nucleotide probe
3 0 may be labeled with a detectable substance such as a radioactive label
which provides for an adequate
signal and has sufficient half-life such as 3zP, 3H, ~4C or the like. Other
detectable substances which may
be used include antigens that are recognized by a specific labeled antibody,
fluorescent compounds,
enzymes, antibodies specific for a labeled antigen, and luminescent compounds.
An appropriate label may
be selected having regard to the rate of hybridization and binding of the
probe to the nucleotide to be
3 5 detected and the amount of nucleotide available for hybridization. Labeled
probes may be hybridized to
nucleic acids on solid supports such as nitrocellulose filters or nylon
membranes as generally described in
Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The
nucleic acid probes may
be used to detect genes, preferably in human cells, that encode OB-BPL Related
Proteins. The nucleotide
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probes may also be useful in the diagnosis of disorders of the hematopoietic
system or cancer; in
monitoring the progression of such disorders; or monitoring a therapeutic
treatment.
The probe may be used in hybridization techniques to detect genes that encode
OB-BPL Related
Proteins. The technique generally involves contacting and incubating nucleic
acids (e.g. recombinant DNA
molecules, cloned genes) obtained from a sample from a patient or other
cellular source with a probe of the
present invention under conditions favorable for the specific annealing of the
probes to complementary
sequences in the nucleic acids. After incubation, the non-annealed nucleic
acids are removed, and the
presence of nucleic acids that have hybridized to the probe if any are
detected.
The detection of nucleic acid molecules of the invention may involve the
amplification of specific
gene sequences using an amplification method such as PCR, followed by the
analysis of the amplified
molecules using techniques known to those skilled in the art. Suitable primers
can be routinely designed
by one of skill in the art.
Genomic DNA may be used in hybridization or amplification assays of biological
samples to
detect abnormalities involving ob-bpl structure, including point mutations,
insertions, deletions, and
chromosomal rearrangements. For example, direct sequencing, single stranded
conformational
polymorphism analyses, heteroduplex analysis, denaturing gradient gel
electrophoresis, chemical mismatch
cleavage, and oligonucleotide hybridization may be utilized.
Genotyping techniques known to one skilled in the art can be used to type
polymorphisms that are
in close proximity to the mutations an OB-BPL gene. The polymorphisms may be
used to identify
2 0 individuals in families that are likely to carry mutations. If a
polymorphism exhibits linkage disequalibrium
with mutations in an OB-BPL gene, it can also be used to screen for
individuals in the general population
likely to carry mutations. Polymorphisms which may be used include restriction
fragment length
polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat
polymorphisms (SSLPs).
A probe of the invention may be used to directly identify RFLPs. A probe or
primer of the
2 5 invention can additionally be used to isolate genomic clones such as YACs,
BACs, PACs, cosmids, phage
or plasmids. The DNA in the clones can be screened for SSLPs using
hybridization or sequencing
procedures.
Hybridization and amplification techniques described herein may be used to
assay qualitative and
quantitative aspects of OB-BPL expression. For example, RNA may be isolated
from a cell type or tissue
3 0 known to express OB-BPL and tested utilizing the hybridization (e.g.
standard Northern analyses) or PCR
techniques referred to herein. The techniques may be used to detect
differences in transcript size which may
be due to normal or abnormal alternative splicing. The techniques may be used
to detect quantitative
differences between levels of full length and/or alternatively splice
transcripts detected in normal
individuals relative to those individuals exhibiting symptoms of a
hematopoietic disorder or other disease
3 5 conditions.
The primers and probes may be used in the above described methods in situ i.e
directly on tissue
sections (fixed and/or frozen) of patient tissue obtained from biopsies or
resections.
4.1.2 Methods for Detectins OB-BPL Related Proteins
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Antibodies specifically reactive with an OB-BPL Related Protein, or
derivatives, such as enzyme
conjugates or labeled derivatives, may be used to detect OB-BPL Related
Proteins in various samples (e.g.
biological materials). They may be used as diagnostic or prognostic reagents
and they may be used to detect
abnormalities in the level of OB-BPL Related Proteins expression, or
abnormalities in the structure, and/or
temporal, tissue, cellular, or subcellular location of an OB-BPL Related
Protein. Antibodies may also be
used to screen potentially therapeutic compounds in vitro to determine their
effects on disorders of the
hematopoietic system, and other conditions. In vitro immunoassays may also be
used to assess or monitor
the efficacy of particular therapies. The antibodies of the invention may also
be used in vitro to determine
the level of OB-BPL expression in cells genetically engineered to produce an
OB-BPL Related Protein.
The antibodies may be used in any known immunoassays which rely on the binding
interaction
between an antigenic determinant of an OB-BPL Related Protein and the
antibodies. Examples of such
assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA),
immunofluorescence,
immunoprecipitation, latex agglutination, hemagglutination, and histochemical
tests. The antibodies may
be used to detect and quantify OB-BPL Related Proteins in a sample in order to
determine its role in
particular cellular events or pathological states, and to diagnose and treat
such pathological states.
In particular, the antibodies of the invention may be used in immuno-
histochemical analyses, for
example, at the cellular and sub-subcellular level, to detect an OB-BPL
Related Protein, to localize it to
particular cells and tissues, and to specific subcellular locations, and to
quantitate the level of expression.
Cytochemical techniques known in the art for localizing antigens using light
and electron
2 0 microscopy may be used to detect an OB-BPL Related Protein. Generally, an
antibody of the invention
may be labeled with a detectable substance and an OB-BPL Related Protein may
be localised in tissues and
cells based upon the presence of the detectable substance. Examples of
detectable substances include, but
are not limited to, the following: radioisotopes (e.g., 3 H, 14C, 3sS izsl
isil) fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors), luminescent labels such as luminol;
enzymatic labels (e.g., horseradish
2 5 peroxidase, beta-galactosidase, luciferase, alkaline phosphatase,
acetylcholinesterase), biotinyl groups
(which can be detected by marked avidin e.g., streptavidin containing a
fluorescent marker or enzymatic
activity that can be detected by optical or calorimetric methods),
predetermined polypeptide epitopes
recognized by a secondary reporter (e.g., leucine zipper pair sequences,
binding sites for secondary
antibodies, metal binding domains, epitope tags). In some embodiments, labels
are attached via spacer arms
3 0 of various lengths to reduce potential steric hindrance. Antibodies may
also be coupled to electron dense
substances, such as ferritin or colloidal gold, which are readily visualised
by electron microscopy.
The antibody or sample may be immobilized on a carrier or solid support which
is capable of
immobilizing cells, antibodies etc. For example, the carrier or support may be
nitrocellulose, or glass,
polyacrylamides, gabbros, and magnetite. The support material may have any
possible configuration
3 5 including spherical (e.g. bead), cylindrical (e.g. inside surface of a
test tube or well, or the external surface
of a rod), or flat (e.g. sheet, test strip). Indirect methods may also be
employed in which the primary
antigen-antibody reaction is amplified by the introduction of a second
antibody, having specificity for the
antibody reactive against OB-BPL Related Protein. By way of example, if the
antibody having specificity
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against an OB-BPL Related Protein is a rabbit IgG antibody, the second
antibody may be goat anti-rabbit
gamma-globulin labeled with a detectable substance as described herein.
Where a radioactive label is used as a detectable substance, an OB-BPL Related
Protein may be
localized by radioautography. The results of radioautography may be
quantitated by determining the density
of particles in the radioautographs by various optical methods, or by counting
the grains.
4.2 Methods for Identifyine or Evaluating Substances/Compounds
The methods described herein are designed to identify substances that modulate
the biological
activity of an OB-BPL Related Protein including substances that bind to OB-BPL
Related Proteins, or bind
to other proteins that interact with an OB-BPL Related Protein, to compounds
that interfere with, or
enhance the interaction of an OB-BPL Related Protein and substances that bind
to the OB-BPL Related
Protein or other proteins that interact with an OB-BPL Related Protein.
Methods are also utilized that
identify compounds that bind to OB-BPL regulatory sequences.
The substances and compounds identified using the methods of the invention
include but are not
limited to peptides such as soluble peptides including Ig-tailed fusion
peptides, members of random peptide
libraries and combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino
acids, phosphopeptides (including members of random or partially degenerate,
directed phosphopeptide
libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-
idiotypic, chimeric, single chain
antibodies, fragments, (e.g. Fab, F(ab)2, and Fab expression library
fragments, and epitope-binding
fragments thereof)], and small organic or inorganic molecules. The substance
or compound may be an
2 0 endogenous physiological compound or it may be a natural or synthetic
compound.
Substances which modulate an OB-BPL Related Protein can be identified based on
their ability
to bind to an OB-BPL Related Protein. Therefore, the invention also provides
methods for identifying
substances which bind to an OB-BPL Related Protein. Substances identified
using the methods of the
invention may be isolated, cloned and sequenced using conventional techniques.
A substance that
2 5 associates with a polypeptide of the invention may be an agonist or
antagonist of the biological or
immunological activity of a polypeptide of the invention.
The term "agonist", refers to a molecule that increases the amount of, or
prolongs the duration of,
the activity of the polypeptide. The term "antagonist" refers to a molecule
which decreases the biological
or immunological activity of the polypeptide. Agonists and antagonists may
include proteins, nucleic acids,
3 0 carbohydrates, or any other molecules that associate with a polypeptide of
the invention.
Substances which can bind with an OB-BPL Related Protein may be identified by
reacting an OB-
BPL Related Protein with a test substance which potentially binds to an OB-BPL
Related Protein, under
conditions which permit the formation of substance-OB-BPL Related Protein
complexes and removing
and/or detecting the complexes. The complexes can be detected by assaying for
substance-OB-BPL Related
3 5 Protein complexes, for free substance, or for non-complexed OB-BPL Related
Protein. Conditions which
permit the formation of substance-OB-BPL Related Protein complexes may be
selected having regard to
factors such as the nature and amounts of the substance and the protein.
The substance-protein complex, free substance or non-complexed proteins may be
isolated by
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conventional isolation techniques, for example, salting out, chromatography,
electrophoresis, gel filtration,
fractionation, absorption, polyacrylamide gel electrophoresis, agglutination,
or combinations thereof. To
facilitate the assay of the components, antibody against OB-BPL Related
Protein or the substance, or
labeled OB-BPL Related Protein, or a labeled substance may be utilized. The
antibodies, proteins, or
substances may be labeled with a detectable substance as described above.
AN OB-BPL Related Protein, or the substance used in the method of the
invention may be
insolubilized. For example, an OB-BPL Related Protein, or substance may be
bound to a suitable carrier
such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl
cellulose polystyrene, filter paper,
ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl
vinyl-ether-malefic acid
copolymer, amino acid copolymer, ethylene-malefic acid copolymer, nylon, silk,
etc. The carrier may be in
the shape of, for example, a tube, test plate, beads, disc, sphere etc. The
insolubilized protein or substance
may be prepared by reacting the material with a suitable insoluble carrier
using known chemical or physical
methods, for example, cyanogen bromide coupling.
The invention also contemplates a method for evaluating a compound for its
ability to modulate
the biological activity of an OB-BPL Related Protein of the invention, by
assaying for an agonist or
antagonist (i.e. enhancer or inhibitor) of the binding of an OB-BPL Related
Protein with a substance which
binds with an OB-BPL Related Protein. The basic method for evaluating if a
compound is an agonist or
antagonist of the binding of an OB-BPL Related Protein and a substance that
binds to the protein, is to
prepare a reaction mixture containing the OB-BPL Related Protein and the
substance under conditions
2 0 which permit the formation of substance-OB-BPL Related Protein complexes,
in the presence of a test
compound. The test compound may be initially added to the mixture, or may be
added subsequent to the
addition of the OB-BPL Related Protein and substance. Control reaction
mixtures without the test
compound or with a placebo are also prepared. The formation of complexes is
detected and the formation
of complexes in the control reaction but not in the reaction mixture indicates
that the test compound
2 5 interferes with the interaction of the OB-BPL Related Protein and
substance. The reactions may be carried
out in the liquid phase or the OB-BPL Related Protein, substance, or test
compound may be immobilized
as described herein. The ability of a compound to modulate the biological
activity of an OB-BPL Related
Protein of the invention may be tested by determining the biological effects
on cells.
It will be understood that the agonists and antagonists i.e. inhibitors and
enhancers that can be
3 0 assayed using the methods of the invention may act on one or more of the
binding sites on the protein or
substance including agonist binding sites, competitive antagonist binding
sites, non-competitive antagonist
binding sites or allosteric sites.
The invention also makes it possible to screen for antagonists that inhibit
the effects of an agonist
of the interaction of OB-BPL Related Protein with a substance which is capable
of binding to the OB-BPL
3 5 Related Protein. Thus, the invention may be used to assay for a compound
that competes for the same
binding site of an OB-BPL Related Protein.
The invention also contemplates methods for identifying compounds that bind to
proteins that
interact with an OB-BPL Related Protein. Protein-protein interactions may be
identified using conventional
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methods such as co-immunoprecipitation, crosslinking and co-purification
through gradients or
chromatographic columns. Methods may also be employed that result in the
simultaneous identification of
genes which encode proteins interacting with an OB-BPL Related Protein. These
methods include probing
expression libraries with labeled OB-BPL Related Protein.
Two-hybrid systems may also be used to detect protein interactions in vivo.
Generally, plasmids
are constructed that encode two hybrid proteins. A first hybrid protein
consists of the DNA-binding domain
of a transcription activator protein fused to an OB-BPL Related Protein, and
the second hybrid protein
consists of the transcription activator protein's activator domain fused to an
unknown protein encoded by
a cDNA which has been recombined into the plasmid as part of a cDNA library.
The plasmids are
transformed into a strain of yeast (e.g. S. cerevisiae) that contains a
reporter gene (e.g. lacZ, luciferase,
alkaline phosphatase, horseradish peroxidase) whose regulatory region contains
the transcription activator's
binding site. The hybrid proteins alone cannot activate the transcription of
the reporter gene. However,
interaction of the two hybrid proteins reconstitutes the functional activator
protein and results in expression
of the reporter gene, which is detected by an assay for the reporter gene
product.
It will be appreciated that fusion proteins may be used in the above-described
methods. In
particular, OB-BPL Related Proteins fused to a glutathione-S-transferase may
be used in the methods.
The reagents suitable for applying the methods of the invention to evaluate
compounds that
modulate an OB-BPL Related Protein may be packaged into convenient kits
providing the necessary
materials packaged into suitable containers. The kits may also include
suitable supports useful in
2 0 performing the methods of the invention.
4.3 Compositions and Treatments
The proteins of the invention, substances or compounds identified by the
methods described
herein, antibodies, and antisense nucleic acid molecules of the invention may
be used for modulating the
biological activity of an OB-BPL Related Protein, and they may be used in the
treatment of conditions such
2 5 as cancer and disorders of the hematopoietic system, in particular
leukemias.
Hematopoietic disorders include but are not limited to myeloproliferative or
other proliferative
disorders of blood forming organs such as thromocythemias, polycythemias, and
leukemias (acute
myelogenous leukemia, chronic myelogenous leukemia). The proteins, substances,
compounds, antibodies,
and antisense nucleic acid molecules of the invention may be used in
conjunction with bone marrow
3 0 transplant, or in the treatment of aplasia or myelosuppression caused by
radiation, chemical treatment, or
chemotherapy. They may also be used to treat hematopoietic disorders
associated with viral or bacterial
infections.
Accordingly, the substances, antibodies, peptides, and compounds may be
formulated into
pharmaceutical compositions for administration to subjects in a biologically
compatible form suitable for
3 5 administration in vivo. By "biologically compatible form suitable for
administration in vivo" is meant a
form of the active substance to be administered in which any toxic effects are
outweighed by the therapeutic
effects. The active substances may be administered to living organisms
including humans, and animals.
Administration of a therapeutically active amount of a pharmaceutical
composition of the present invention
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is defined as an amount effective, at dosages and for periods of time
necessary to achieve the desired result.
For example, a therapeutically active amount of a substance may vary according
to factors such as the
disease state, age, sex, and weight of the individual, and the ability of
antibody to elicit a desired response
in the individual. Dosage regima may be adjusted to provide the optimum
therapeutic response. For
example, several divided doses may be administered daily or the dose may be
proportionally reduced as
indicated by the exigencies of the therapeutic situation.
The active substance may be administered in a convenient manner such as by
injection
(subcutaneous, intravenous, etc.), oral administration, inhalation,
transdermal application, or rectal
administration. Depending on the route of administration, the active substance
may be coated in a material
to protect the substance from the action of enzymes, acids and other natural
conditions that may inactivate
the substance.
The compositions described herein can be prepared by eR r se known methods for
the preparation
of pharmaceutically acceptable compositions which can be administered to
subjects, such that an effective
quantity of the active substance is combined in a mixture with a
pharmaceutically acceptable vehicle.
Suitable vehicles are described, for example, in Remington's Pharmaceutical
Sciences (Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On
this basis, the
compositions include, albeit not exclusively, solutions of the active
substances in association with one or
more pharmaceutically acceptable vehicles or diluents, and contained in
buffered solutions with a suitable
pH and iso-osmotic with the physiological fluids.
2 0 Vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses,
or from various
bacterial plasmids, may be used to deliver nucleic acid molecules to a
targeted organ, tissue, or cell
population. Methods well known to those skilled in the art may be used to
construct recombinant vectors
which will express antisense nucleic acid molecules of the invention. (See,
for example, the techniques
described in Sambrook et al (supra) and Ausubel et al (supra)).
2 5 The nucleic acid molecules comprising full length cDNA sequences and/or
their regulatory
elements enable a skilled artisan to use sequences encoding a protein of the
invention as an investigative
tool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol 13:98-104) or
antisense (Eguchi et al
(1991) Annu Rev Biochem 60:631-652) regulation of gene function. Such
technology is well known in the
art, and sense or antisense oligomers, or larger fragments, can be designed
from various locations along the
3 0 coding or control regions.
Genes encoding a protein of the invention can be turned off by transfecting a
cell or tissue with
vectors which express high levels of a desired OB-BPL-encoding fragment. Such
constructs can inundate
cells with untranslatable sense or antisense sequences. Even in the absence of
integration into the DNA,
such vectors may continue to transcribe RNA molecules until all copies are
disabled by endogenous
3 5 nucleases.
Modifications of gene expression can be obtained by designing antisense
molecules, DNA, RNA
or PNA, to the regulatory regions of a gene encoding a protein of the
invention, ie, the promoters,
enhancers, and introns. Preferably, oligonucleotides are derived from the
transcription initiation site, eg,
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between -10 and +10 regions of the leader sequence. The antisense molecules
may also be designed so that
they block translation of mRNA by preventing the transcript from binding to
ribosomes. Inhibition may also
be achieved using "triple helix" base-pairing methodology. Triple helix
pairing compromises the ability of
the double helix to open sufficiently for the binding of polymerases,
transcription factors, or regulatory
molecules. Therapeutic advances using triplex DNA were reviewed by Gee J E et
al (In: Huber B E and
B I Carr (1994) Molecular and Immunologic Approaches, Futura Publishing Co, Mt
Kisco N.Y.).
Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of
RNA. Ribozymes
act by sequence-specific hybridization of the ribozyme molecule to
complementary target RNA, followed
by endonucleolytic cleavage. The invention therefore contemplates engineered
hammerhead motif ribozyme
molecules that can specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding
a protein of the invention.
Specific ribozyme cleavage sites within any potential RNA target may initially
be identified by
scanning the target molecule for ribozyme cleavage sites which include the
following sequences, GUA,
GUU and GUC. Once the sites are identified, short RNA sequences of between 15
and 20 ribonucleotides
corresponding to the region of the target gene containing the cleavage site
may be evaluated for secondary
structural features which may render the oligonucleotide inoperable. The
suitability of candidate targets
may also be determined by testing accessibility to hybridization with
complementary oligonucleotides using
ribonuclease protection assays.
Methods for introducing vectors into cells or tissues include those methods
discussed herein and
2 0 which are suitable for in vivo, in vitro and ex vivo therapy. For ex vivo
therapy, vectors may be introduced
into stem cells obtained from a patient and clonally propagated for autologous
transplant into the same
patient (See U.S. Pat. Nos. 5,399,493 and 5,437,994). Delivery by transfection
and by liposome are well
known in the art.
The nucleic acid molecules disclosed herein may also be used in molecular
biology techniques that
2 5 have not yet been developed, provided the new techniques rely on
properties of nucleotide sequences that
are currently known, including but not limited to such properties as the
triplet genetic code and specific
base pair interactions.
The invention also provides methods for studying the function of a polypeptide
of the invention.
Cells, tissues, and non-human animals lacking in expression or partially
lacking in expression of a nucleic
3 0 acid molecule or gene of the invention may be developed using recombinant
expression vectors of the
invention having specific deletion or insertion mutations in the gene. A
recombinant expression vector
may be used to inactivate or alter the endogenous gene by homologous
recombination, and thereby create
a deficient cell, tissue, or animal.
Null alleles may be generated in cells, such as embryonic stem cells by
deletion mutation. A
3 5 recombinant gene may also be engineered to contain an insertion mutation
that inactivates the gene. Such
a construct may then be introduced into a cell, such as an embryonic stem
cell, by a technique such as
transfection, electroporation, injection etc. Cells lacking an intact gene may
then be identified, for example
by Southern blotting, Northern Blotting, or by assaying for expression of the
encoded polypeptide using
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the methods described herein. Such cells may then be fused to embryonic stem
cells to generate transgenic
non-human animals deficient in a polypeptide of the invention. Germline
transmission of the mutation may
be achieved, for example, by aggregating the embryonic stem cells with early
stage embryos, such as 8 cell
embryos, in vitro; transferring the resulting blastocysts into recipient
females and; generating germline
transmission of the resulting aggregation chimeras. Such a mutant animal may
be used to define specific
cell populations, developmental patterns and in vivo processes, normally
dependent on gene expression.
The invention thus provides a transgenic non-human mammal all of whose germ
cells and somatic
cells contain a recombinant expression vector that inactivates or alters a
gene encoding a OB-BPL Related
Protein. In an embodiment the invention provides a transgenic non-human mammal
all of whose germ cells
and somatic cells contain a recombinant expression vector that inactivates or
alters a gene encoding an OB-
BPL Related Protein resulting in an OB-BPL Related Protein associated
pathology. Further the invention
provides a transgenic non-human mammal which doe not express an OB-BPL Related
Protein of the
invention. In an embodiment, the invention provides a transgenic non-human
mammal which doe not
express an OB-BPL Related Protein of the invention resulting in an OB-BPL
Related Protein associated
pathology. AN OB-BPL Related Protein pathology refers to a phenotype observed
for an OB-BPL Related
Protein homozygous mutant.
A transgenic non-human animal includes but is not limited to mouse, rat,
rabbit, sheep, hamster,
dog, cat, goat, and monkey, preferably mouse.
The invention also provides a transgenic non-human animal assay system which
provides a model
2 0 system for testing for an agent that reduces or inhibits a pathology
associated with an OB-BPL Related
Protein, preferably an OB-BPL Related Protein associated pathology,
comprising:
(a) administering the agent to a transgenic non-human animal of the invention;
and
(b) determining whether said agent reduces or inhibits the pathology (e.g. OB-
BPL Related
Protein associated pathology) in the transgenic non-human animal relative to a
transgenic
2 5 non-human animal of step (a) which has not been administered the agent.
The agent may be useful in the treatment and prophylaxis of conditions such as
cancer or
hematopoietic disorders as discussed herein. The agents may also be
incorporated in a pharmaceutical
composition as described herein.
The activity of the proteins, substances, compounds, antibodies, nucleic acid
molecules, agents,
3 0 and compositions of the invention may be confirmed in animal experimental
model systems. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical procedures
in cell cultures or with
experimental animals, such as by calculating the EDso ( the dose
therapeutically effective in 50% of the
population) or LDso (the dose lethal to 50% of the population) statistics. The
therapeutic index is the dose
ratio of therapeutic to toxic effects and it can be expressed as the EDSO/LDso
ratio. Pharmaceutical
3 5 compositions which exhibit large therapeutic indices are preferred.
The following non-limiting examples are illustrative of the present invention:
Example
MATERIALS AND METHODS
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New Gene Identification
Nucleotide sequencing data of approximately 130 Kb on chromosome 19q13.4 was
obtained from
the Lawrence Livermore National Laboratory (LLNL) web site (http://www-
bio.llnl. o~v/~enome/~enome.html), in the form of one contig. This genomic
sequence was subjected to a
number of computer algorithms (gene prediction programs) designed to predict
the presence of putative
new genes. All programs used were previously thoroughly evaluated using a
large number of known genes
(Yousef et al., 1999a). Based on these results, the most reliable algorithms -
GeneBuilder (gene prediction)
(httv://125.itba.mi.cnr.it/~web ene/~enebuilder html) and GeneBuilder (exon
prediction)
(http://125.itba.mi.cnr.it/~web~e/genebuilder html); Grail 2
(http://compbio.ornl.gov); and GENE1D-3
(http://apolo.imim.es/geneid.html) - were selected for further use.
Expressed sequence tag (EST) identification
The genomic sequence of the putative new gene was subjected to a homology
search against the
human EST database using the BLASTN algorithm
(htto://www.ncbi.nlm.nih.gov/BLAST) (Altschul et al.,
1997). Clones showing >95% homology were obtained from the LM.A.G.E.
consortium through Research
Genetics Inc. (Huntsville, AL). The clone obtained was then propagated
according to the suppliers
instructions, purified, and sequenced from both directions with an automated
sequencer, using the insert-
flanking vector primers T3 and T7.
Molecular Characterization of a Novel Siglec
The sequence derived from the computer predicted exons of the putative new
gene was also used
2 0 to search the non-redundant protein sequence database, using the BLASTP
algorithm (Altschul et al.,
1997). Several proteins showing a high degree of homology were selected, and
their nucleotide coding
sequences were aligned with predicted coding sequence using the ClustalX
multiple alignment program
(Jeanmougin et al., 1998). From this, regions on the putative gene were
selected which showed the least
amount of homology to the others and PCR primers were designed: Fl
(TCACCGGCTCTCTGTGAATG
2 5 - SEQ.)D.NO. 4 ) and R1 (GTCTTCTGCCCAAGGTTCAG - SEQ.m.NO. 5). Using these
primers, PCR
was performed on bone marrow cDNA, prepared as discussed below, and chosen
based on the tissue
expression results. The PCR conditions were as follows: 2.5 units HotStarTaq
polymerase (Qiagen,
Valencia, CA), 1X PCR buffer with 1.5 mM MgCl2 (Qiagen), 1 pl cDNA, 200 uM
dNTPs
(deoxynucleoside triphosphates), and 250 ng of primers, using the
Mastercycler~ gradient thermocycler
3 0 (Eppendorf Scientific, Inc., Westbury, NY). The temperature profile was:
denaturation at 95°C for 15 min.
followed by 94°C for 30 s., annealing at 58°C for 30 s., and
extension at 72°C for 1 min. for a total of 35
cycles, followed by a final extension at 72°C for 10 min. The PCR
product was subjected to electropheresis
on a 2% agarose gel and stained with ethidium bromide. Aliquots of the PCR
products were subsequently
extracted from the gel and the purified DNA was directly sequenced using an
automated sequencer.
3 5 In order to verify the sequence surrounding the proposed start codon,
another set of primers were
designed, again derived from regions showing low homology with other known
genes: F3
(TCCTCTAAGTCTTGAGCCCG - SEQ.ID.NO. 6) and R3 (CAGACGTTGAGATGGACGGT -
SEQ.ID.NO. 7). PCR was performed using bone marrow cDNA, prepared as described
below. The
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conditions used for the PCR reaction were identical to those discussed
previously, with electrophoresis of
the PCR product on a 2% agarose gel, gel extraction, and automated sequencing
as before.
Following final characterization of the genomic structure of this novel
siglec, the putative protein
product was aligned with the protein sequences of the other siglec family
members using the ClustalX
multiple sequence alignment tool. Further, phylogenetic analysis was performed
using ClustalX in
combination with TreeView (Page 1996).
Sequence analysis tools, available through the Internet, were also utilized to
detect the presence
of possible sites of post-translational modification on the putative protein.
The analysis programs PROSITE
motif search (http~//www easy ch/prosit~ (Bairoch et al., 1997), and NetOGlyc
2.0 (Hansen et al., 1995;
Hansen et al., 1998) were used to detect N- and O-glycosylation, as well as
the presence of kinase
phosphorylation motifs. Further, the putative protein was assessed for the
presence of a possible signal
peptide, using SignalP vl.l (http://www.cbs.dtu.uk/) (Nielsen et al., 1997).
For the prediction of
transmembrane domains, two independent algorithms were used, TMpred
(http://www.ch.embnet.or~Jsoftware/TMPRED form html) and DAS
(http://www.biokemi.su.se/~serve>~.
In addition, the hydropathic profile of this novel siglec was determined,
using the Kyte-Doolittle method
(httn://bioinformatics.weizmann.ac.il/hydroph/plot hydroph html).
Mapping and Chromosomal Localization of a Novel Siglec
As mentioned previously, the contig on which the novel siglec gene was
identified was obtained
from the LLNL. EcoRI restriction maps were obtained from LLNL, and also
generated using the Webcutter
2 0 restriction analysis tool (http://www.firstmarker.com/cutter/cutter2
html), for both this contig, as well as
the adjacent more centromeric contigs, containing the recently identified
kallikrein gene family (Diamandis
et al., 1999; Yousef et al., 1999a). Overlapping restriction fragments were
identified and used to order the
contigs and determine the distance between KLK-L6, the most telomeric member
of the kallikrein gene
family, and this novel siglec.
2 5 Tissue Expression
Total RNA from 28 normal human tissues was obtained (Clontech, Palo Alto, CA,
USA), and
reverse transcription was performed using Superscript IITM, according to the
manufacturer's instructions
(Gibco BRL, Gaithersburg, MD, USA). PCR was then performed using primers F2
(CGTGGGAGATACGGGCATAG - SEQ.ID.N0.8) and R2 (AAAAGGGAGGGCACAGTGTG -
3 0 SEQ.ID.NO. 9), using the same PCR conditions described previously. PCR for
actin was also performed
as describe elsewhere (Yousef et al., 1999b), as a control for cDNA quality.
RESULTS
Identification of a Novel Siglec on 19q13.4
Computer analysis of the approximately 130 Kb contig predicted a putative new
gene consisting
3 5 of six exons. Five of these were predicted by at least three programs,
with only one exon being predicted
by two of the four programs (Table 1). Homology search for the putative new
gene against the human EST
database revealed the presence of one unique EST (GenBank accession #
AA936059) which showed 98%
identity to the sixth predicted exon.
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The entire insert of this EST was sequenced, followed by alignment of this
nucleotide sequence
with the genomic sequence of the putative gene, using the "BLAST 2 sequences"
program. This revealed
the presence of an additional area, between predicted exons 5 and 6, with 98%
identity to the EST. This
suggested that there was an additional exon in this area which was not
detected by the prediction algorithms
used.
Characterization of the Genomic Structure of the Novel Siglec Gene and its
Protein Product
With the aid of unique primers, designed as discussed in the experimental
section, RT-PCR was
performed on bone marrow cDNA and two additional products were isolated, both
encompassing multiple
predicted exons. Upon sequencing of these PCR products, the presence of all
six predicted exons, as well
as the newly identified exon, found from the EST sequence were confirmed. With
both cDNA and genomic
sequence at hand, the genomic organization of this new gene was determined
(Figure 1). The gene encoding
this novel siglec encompasses a genomic area of 5,421 bp. It is composed of
seven exons, with six
intervening introns. The lengths of the exons are 509, 279, 48, 267, 91, 97,
and 417 bp, respectively. All
the intron/exon splice sites and their flanking sequences are closely related
to the consensus splice sites (-
mGTAAGT...CAGm-, where m is any base) (Iida 1990).
The proposed protein coding region of the novel siglec gene consists of 1,392
nucleotides,
producing a 463 amino acid protein, with a predicted molecular mass of 50.1
kDa, excluding any post-
translational modifications. The translation initiation codon (ATG) at
position 1171 of the first exon
(according to the numbering of SEQ. ID. NO. 1 and GenBank Accession No.
AF135027), was chosen
2 0 because: 1) the flanking region surrounding that codon closely matches the
Kozak consensus sequence for
translational initiation, particularly at position -3 (a purine), which
appears to be the most highly conserved
(Kozak 1991); 2) using this initiation codon, the proposed protein contains an
N-terminal signal sequence
which shows a high degree of homology to other similar proteins (see below).
The 3' terminus of the novel
siglec gene was verified by the presence of a poly dA tail present in the EST
sequence. Further, it is
2 5 evident from Figure 1 that this gene possesses a 5' untranslated region of
at least 88 nucleotides, as well
as a 3' untranslated region of 228 nucleotides.
Examination of the hydrophobicity profile of the novel siglec protein revealed
two regions with
long stretches of hydrophobic residues. The first of these occurs at the N-
terminus, suggesting the presence
of a signal peptide (Figure 2). This is consistent with findings from a signal
sequence prediction program
3 0 (Nielsen et al., 1997), which predicts a 17 amino acid residue signal
sequence. The second region occurs
between residues 349 and 370, suggestive of a transmembrane domain, and is
consistent with results from
transmembrane region prediction programs. Based on this information, the
protein product of this novel
gene is likely a type I transmembrane protein, after cleavage of the 17
residue signal sequence.
Through the use of sequence analysis tools, the various putative post-
translational modification
3 5 sites were identified (Table 2). There are numerous potential sites in
this novel siglec where there could
be either O- or N-glycosylation. Furthermore, several possible sites of
phosphorylation have been
identified for CAMP-dependent protein kinase, protein kinase C, and casein
kinase 2.
Mapping and Chromosomal Localization of a Novel Siglec
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The contig in which the gene encoding a novel siglec was identified is located
at 19q13.4,
telomeric to the kallikrein gene KLK3 (PSA). Previous studies have identified
and mapped the kallikrein
gene family locus on this region of chromosome 19 (Diamandis et al., 1999;
Yousef et al., 1999a). The
contig containing the novel siglec gene was found, through EcoRI restriction
mapping, to be located
adjacent to this kallikrein gene family. The novel siglec gene is located
43.19 Kb more telomeric than
KLK-L6, at 19q13.4. A detailed physical map of the area which contains some
known genes and the newly
identified siglec gene is shown in Figure 3. By computer analysis, no other
genes were predicted between
KLK-L6 and this novel siglec.
Homology with other Siglec Family Members
Using the predicted protein sequence, a homology search was performed against
the GenBank
database using the BLASTP program. The novel siglec showed a high degree of
homology to other known
members of the Siglec family (Table 3). A multiple alignment of this novel
siglec with the other family
members was also perfomed using the ClustalX alignment program. As is evident
in Figure 4, the N-
terminal signal sequence is highly conserved within this family of proteins.
Furthermore, the protein
contains Ig domains typically found in Siglec family members: an N-terminal V-
set domain, followed by
multiple C2-set domains (Crocker et al., 1996). This novel siglec contains a
total of 3 Ig domains, a V-set
and two C2-set domains, based on homology with known Ig domains. As shown in
Table 4, the V-set and
first C2-set domains are highly similar to Siglec-7 and CD33, with the second
C2-set showing highest
homologies with Siglec-7 and Siglec-6. The novel siglec exhibits conservation
of the cysteine residues in
2 0 the V-set and first C2-set domains, which form the two characteristic
disulfide bridges in other Siglec
family members. In the V-set domain, Cys 41 and Cys 102 form an intrasheet
disulfide bond, whereas Cys
36 and Cys 170 of the first C2-set domain are likely to form the interdomain
disulfide bond, based on
findings for other siglecs (Crocker et al., 1996; Williams et al., 1989). The
V-set domain also possesses
a conserved arginine which has been found to be essential for sialic-acid
binding (van der Merwe et al.,
2 5 1996), as well as two conserved aromatic residues in (3-strands A and G
which have been found to make
hydrophobic contacts with the N-acetyl and glycerol side groups of N-acetyl
neuraminic acid (May et al.,
1998). As is evident from Figure 4, this novel siglec also possesses the
critical arginine, at position 120,
as well as the aromatic residue in (3-strand G; however it lacks the aromatic
residue in the A (3-strand. The
domain boundaries were determined based on the one domain: one exon rule
(Williams and Barclay 1988),
3 0 while taking into consideration the domain assignments of others (Cornish
et al., 1998; Crocker et aL.,
1998; Falco et al., 1999; Nicoll et al., 1999; Patel et al., 1999).
Examination of the transmembrane and intracellular domains of Siglec family
members reveals
that it is more variable than the extracellular domain. However, there are
regions that show a high level
of conservation. As shown in Figure 4, all the Siglecs possess a single
transmembrane domain, consisting
3 5 of approximately 25 residues. In addition, within the cytoplasmic domain,
there are two highly conserved
motifs. The first of these, L(HQ)YA(SV)L, exhibits similarity to an
immunoreceptor tyrosine kinase
inhibitory motif (ITIM), which has a 6 amino acid consensus sequence
(ILV)xYxx(LV) (Burshtyn et al.,
1997; Vivier and Daeron 1997). The second motif, TEYSE(IV), is homologous to a
sequence (TxYxx(IV))
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recently found in the signaling lymphocyte activation molecule (SLAM) which is
responsible for the
binding of the SLAM-associated protein (SAP) (Coffey et al., 1998; Sayos et
al., 1998).
Phylogenetic analysis of the entire siglec family was performed using ClustalX
and TreeView.
This revealed that the novel siglec is very closely related to Siglec-7,
followed by CD33 (Figure 5). It is
evident that this novel gene, which encodes a putative siglec protein is the
newest member of the siglec
family. It possesses all the necessary features, including the Ig-like
domains, the type I transmembrane
topology, as well as the conserved cytoplasmic motifs, and shows a close
phylogenetic relationship to the
other siglec family members.
Tissue Expression Profile of a Novel Siglec
RT-PCR was performed on a panel of tissue-specific total RNA preparations
(Figure 6). High
levels of expression of the novel siglec were found in bone marrow, placenta,
spleen, and fetal liver. Lower
levels of expression were also evident in fetal brain, stomach, lung, thymus,
prostate, brain, mammary,
adrenal gland, colon, trachea, cerebellum, testis, small intestine, and spinal
cord. Expression of this novel
siglec was absent in heart, skeletal muscle, pancreas, and ovary. All PCR
products obtained were of equal
length, and corresponded to the length of the product obtained from
overlapping EST (accession #
AA936059). Sequencing of the PCR products ensured specificity.
DISCUSSION
Using the positional candidate gene approach a novel gene belonging to the
siglec family was
identified. This gene is comprised of 7 exons, with 6 intervening introns. The
coding region of this gene
2 0 is composed of 1,392 nucleotides, producing a 463 amino acid protein, with
a predicted molecular mass
of 50.1 kDa. This gene is located at 19q13.4, 43.19 Kb telomeric to the newly
identified kallikrein KLK-
L6. The high degree of homology between this novel siglec and other siglecs
provides strong evidence that
this protein also plays a role in sialic acid-dependent protein-glycoprotein
or -glycolipid interactions. It
possesses the unique pattern of conserved cysteine residues in its Ig-like
domains, which are found only
2 5 in members of the siglec family. Further, this novel siglec possesses the
conserved arginine residue, which
has been found to be essential for sialic acid binding (van der Merwe et al.,
1996). Of note, however, is
that it only possesses one of the two conserved aromatic residues in the V-set
domain, which may be
suggestive of a unique sialic acid specificity, differing from that of
previously identified siglecs.
The tissue expression profile of the novel siglec was examined and it was
found to be highly
3 0 expressed in bone marrow, placenta, spleen, and fetal liver. The high
level of expression of this novel
siglec in bone marrow is consistent with findings from groups investigating
the other siglec family
members. All currently known siglecs have been found to be expressed in some
type of bone marrow stem
cell-derived cell, ranging from myeloid progenitor cells for CD33 to natural
killer cells for Siglec-7 and
B lymphocytes for CD22. It is likely that this novel siglec is predominantly
expressed on a distinct subset
3 5 of immune cells, where it plays an intercellular signaling role. This is
supported by the presence of ITIM-
like and SLAM-like motifs in the cytoplasmic domain of this novel siglec, with
similar domains in other
siglecs. ITIM motifs are consensus binding sites for the SH2 (src homology 2)
domains of the phosphatases
SHP-1 and SHP-2 (Borges et al., 1997; Le Drean et al., 1998). It has been
reported that the
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
-28-
phosphorylation of the ITIM-like motif in CD22, the phosphatase SHP-1 is
recruited, suggesting a possible
function of this siglec as a B cell receptor-associated negative co-receptor
(Vivier and Daeron 1997). The
second cytoplasmic motif has been identified in SLAM and several SLAM-like
proteins, a family of
immunoregulatory molecules of the IgSF, and is responsible for the binding of
a new SH2-containing
molecule, SAP (Coffey et al., 1998; Sayos et al., 1998). The binding of SAP
was shown to inhibit the
binding of SHP-2 to its respective binding site on these SLAM proteins. The
presence of such a motif in
the novel siglec, and other siglecs, suggests that there may be a similar
regulatory mechanism present in
the cytoplasmic domains of siglecs, with SAP inhibiting the binding of SHP-1
and SHP-2 to the TTIM-like
motif.
The regulation of SHP-1 and SHP-2 binding to ITIM motifs, and thus their
activation, very likely
affects downstream tyrosine-kinase dependent pathways by regulating the
phosphorylation state of
components in these pathways. Thus, the siglec family of ITIM and SLAM-bearing
receptors probably play
a role in controlling the activation of a number of cell types. By extension,
it is possible that these siglecs
may be involved in the regulation of tumour growth. CD33 has already been
identified as an important
marker for the diagnosis of acute myelogenous leukemia (AML), particularly for
the undifferentiated form,
and serves to distinguish AML from lymphoid leukemias (Bernstein et al., 1992;
Dinndorf et al., 1986;
Griffin et al., 1984). Recently, Kossman et. al. and Sievers et. al. have
reported the use of anti-CD33
monoclonal antibodies in phase I studies for the treatment of AML, and have
shown selective ablation of
malignant hematopoiesis (Kossman et al., 1999; Sievers et al., 1999). The
newly identified member of the
2 0 siglec family may have utility as a target for immunological
antineoplastic therapy.
Having illustrated and described the principles of the invention in a
preferred embodiment, it
should be appreciated to those skilled in the art that the invention can be
modified in arrangement and detail
2 5 without departure from such principles. All modifications coming within
the scope of the following claims
are claimed.
All publications, patents and patent applications referred to herein are
incorporated by reference
in their entirety to the same extent as if each individual publication, patent
or patent application was
specifically and individually indicated to be incorporated by reference in its
entirety.
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
-29-
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2 5 May, A. P., Robinson, R. C., Vinson, M., Crocker, P. R. and Jones, E. Y. (
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Nicoll, G., Ni, J., Liu, D., Klenerman, P., Munday, J., Dubock, S., Mattei, M.
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Pedraza, L., Owens, G. C., Green, L. A. and Salzer, J. L. ( 1990). The myelin-
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Table 1: Genomic organization of a novel siglec.
Exon Exon Predicted3
No.
Coding
Regions
No.
of
EST
Intron
base pairs Match2 Phase
From (bp) To (bp)
1 1083 1591 509 - I B,C
2 1793 2071 279 - I A,B,C,D
3 2277 2324 48 - I A,B,D
4 3226 3492 267 - I A,B,C
4145 4235 91 - 0 A,B,C,D
6 4610 4706 97 + 0 -
7 6087 6503 417 + - A,B,C
1. The coding region shown includes the 5' untranslated3' untranslated
region in exon 1, and the
region in exon 7. Numbers refer to GenBank
accession no. AF135027.
5 EST; GenBank accession no. AA936059
2.
3. The exon prediction programs are as follows: ); B) GeneBuilder
A) GeneBuilder (gene prediciton
(exon prediction); C) Grail 2; D) GENEID-3.
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Table 2: Putative post-translational modification sites in the novel siglec.
Modifications Residue Position2
O-glycosylation Thr 76, 192, 193
Ser 184, 186, 195
N-glycosylation Asn 101, 138, 161, 225,
231,
238, 256, 334
cAMP-dependent Protein Kinase phosphorylation374
Ser/Thr
Protein Kinase C phosphorylation Ser/T'hr 372, 377, 421
Casein Kinase 2 phosphorylation Ser/Thr 387 412 425 452
1. The proposed O-glycosylation sites wereetOGlyc 2.0 (Hansen
determined through N
et al., 1998). The remainder of the post-translational
modifications were predicted by
PROSITE (Hansen et al., 1995).
2. The residue numbering is according to el siglec, as shown
the numbering of the nov in
Figure 4.
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Table 3: Overall homology of this novel siglec with other known siglecs.
Siglec Family Members Homology to the Novel
Siglec2
% identit % similarity
Siglec-7 (p75/AIRM1) (AF170485) 75 80
Siglec-5 (OB-BP2) (U71383) 52 65
CD33 (M23197) 52 64
Siglec-6 (OB-BP1) (U71382) 49 60
Sialoadhesin (Z36293) 27 43
CD22 (X52785) 26 42
Myelin associated glycoprotein (MAG) 25 42
(M29273)
1. GenBank accession numbers for each
of the siglec family members is also
shown, in
brackets.
2. Homology was determined using ithm (Altschul et al.,
the BLASTP algor 1997).
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Table 4: Ig-like domain homology between the novel siglec and other siglec
family membersl
Homolo ous Protein Domain % identit% similarit
Siglec-7 (p75/A1RM1)1 75 78
CD33 1 61 71
Novel Siglec Siglec-5 (OB-BP2) 1 54 67
Ig 1
(V-set) Siglec-6 (OB-BP1) 1 54 62
MAG 1 32 48
Sialoadhesin 1 29 48
CD22 1 28 44
Siglec-7 (p75/AIRMI)2 89 93
CD33 2 63 75
Novel Siglec Siglec-6 (OB-BP1) 2 58 70
Ig 2
(C2-set) Siglec-5 (OB-BP2) 2 58 71
Sialoadhesin 2 30 46
12 31 44
MAG 2 25 46
CD22 2 27 43
Siglec-7 (p75/AIRM1)3 76 79
Siglec-6 (OB-BP1) 3 52 67
Novel Siglec Siglec-5 (OB-BP2) 3 48 62
Ig 3
(C2-set) Sialoadhesin 13 33 48
7 31 42
15 28 40
MAG 3 27 49
i. ~emsanx accession numbers for the listed siglecs are the same as those
shown in Table 3.
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
-36-
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CA 02366087 2001-09-10
FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets
publishing international applications under the PCT.
AL Albania ES Spain LS Lesotho SI Slovenia
AM Armenia FI Finland LT Lithuania SK Slovakia
AT Austria FR France LU Luxembourg SN Senegal
AU Australia GA Gabon LV Latvia SZ Swaziland
AZ Azerbaijan CB United KingdomMC Monaco TD Chad
BA Bosnia and GE Georgia MD Republic of TG Togo
Herzegovina Moldova
BB Barbados GH Ghana MG Madagascar TJ Tajikistan
BE Belgium GN Guinea MK The former TM Turkmenistan
Yugoslav
BF Burkina Faso GR Greece Republic of TR Turkey
Macedonia
BG Bulgaria HU Hungary ML Mali TT Trinidad
and Tobago
BJ Benin IE Ireland MN Mongolia UA Ukraine
BR Brazil IL Israel MR Mauritania UG Uganda
BY Belarus IS Iceland MW Malawi US United States
of America
CA Canada IT Italy MX Mexico UZ Uzbekistan
CF Central AfricanJP Japan NE Niger VN Viet Nam
Republic
CG Congo KE Kenya NL Netherlands YU Yugoslavia
CH Switzerland KG Kyrgyzstan NO Norway ZW Zimbabwe
CI CBte d'ivoireKP Democratic NZ New Zealand
People's
CM Cameroon Republic PL Poland
of Korea
CN China KR Republic PT Portugal
of Korea
CU Cuba KZ Kazakstan RO Romania
CZ Czech RepublicLC Saint Lucia RU Russian Federation
DE Germany LI LiechtensteinSD Sudan
DK Denmark LK Sri Lanka SE Sweden
EE Estonia LR Liberia SG Singapore
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
1
Sequence Listing
SEQ.ID.NO. 1
OB-BPL NA
1 ctgacatcgg tctaaggagg
ctccaccaca caggatactc
ccagttcctc ccagcccagc
61 cacacagtta caaaaaatcttctgctgaattcaggtgcatgtttcttctc
ccatgaaatc
1 121 ctgcccatcg cggagggtgcatgccccggggctggtgtcctcactccaca
0
tctcctgttc
181 accgggaccc ctgggagcagagcttgcagaagcaaagccccagggccagc
tgatccctca
241 tgttctggag taaccagggaagtgtggctgagcgagacatcggtggtgaa
gaaacccttc
301 gtggtgcagt gaggaaaggagaaatatcttcccttttgaaatctgcccct
tttcttccga
361 atttcctccc ttccaagccccacagtacaacagtcacagcctcagtttcc
cagacctcct
2 421 gcgagccagg ctcccctctgtgtccttggcgtgtatcaacacatagaatc
0
ctcacctcca
481 cagccccatg tccctctgctcagtcctcctgagattgaacccctgaccta
cgggagagtg
541 accccatcct gcctgtgccccccaactgaagctcctgccgtggacagctc
2 agacgtcagg
5
601 agcctccatg tctcccctccgcacagtgaccccttggggacagtgtccag
ctctgctgtg
661 ttgacctcgt tgtgcagaaagggggtgcccacctcgtgcttgcttggggg
ggatgagagg
3 721 ctgcctgctc accccacctgcacccccatctctcatggccccaagtccca
0
cctctgagct
781 acgcagatat ccacagcctctgactcaggggtctggccagatgggactca
ttttcaccct
841 gcagaaaatg tcacctaaggggaagggcattgagagggaggcaggaggtg
35 gggtgggccc
901 tgcatggggt gggaatctgggtgagtctgtctcccgctctggcctcaggg
acccaggagt
961 gaacatgggg tggtggacggtggatctcccagggctgacccgggcctgac
agtgtctggg
40 1021 tgtaaagttc ctcctctgaggaggtcactgttccgacctcgcccctgtct
tcctgtaggg
1081 cctcctctaa gtcttgagcccgcagttcctgagagaagaaccctgaggaa
cagacgttcc
1141 ctcgcggccc tggcacctctaaccccagacatgctgctgctgctgctgcc
45 cctgctctgg
1201 gggagggaga gggcggaaggacagacaagtaaactgctgacgatgcagag
ttccgtgacg
1261 gtgcaggaag gcctgtgtgtccatgtgccctgctccttctcctacccctc
gcatggctgg
5 1321 atttaccctg gcccagtagttcatggctactggttccgggaaggggccaa
0
tacagaccag
1381 gatgctccag tggccacaaacaacccagctcgggcagtgtgggaggagac
tcgggaccga
1441 ttccacctcc ttggggacccacataccaagaattgcaccctgagcatcag
55 agatgccaga
1501 agaagtgatg cggggagatacttctttcgtatggagaaaggaagtataaa
atggaattat
1561 aaacatcacc ggctctctgtgaatgtgacaggtaaggcacaggctccagg
aaaggccaca
60 1621 gggaaaggtc atgggggcggcagggaaaggctgggatggagcccctgccc
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
2
caggagaggg
1681 cttagggtga agcgagttggctcagggcaggagctggaccagagcctgag
ctccccccag
1741 ggctgcacca tggatcctctgacctgatcctgagtccccctctcttcacc
agccttgacc
1801 cacaggccca acatcctcatcccaggcaccctggagtccggctgccccca
gaatctgacc
1861 tgctctgtgc cctgggcctgtgagcaggggacaccccctatgatctcctg
gatagggacc
1921 tccgtgtccc ccctggacccctccaccacccgctcctcggtgctcaccct
catcccacag
1981 ccccaggacc atggcaccagcctcacctgtcaggtgaccttccctggggc
cagcgtgacc
2041 acgaacaaga ccgtccatctcaacgtgtcctgtgagtgctgggccgggac
gcctgggtcc
2101 ctgatggggt gagcgtcaagcctggacactgggtgctgggtcccggaatc
tgggctggtg
2161 gtggggtcag gaggacactggctctgccttccctgtttatgcggctcctg
gggacagaca
2 2221 gggccagtgt ccccagccctcacagtgatgcgggtctccatgtctttctg
0
tcccagaccc
2281 gcctcagaac ttgaccatgactgtcttccaaggagacggcacaggtagga
tggagctccc
2341 tccctggggc tggaggagcagggccttcaggtcaggatggggctggctta
2 ttcctcaacc
5
2401 tggactcact ttggcaaacagggatgtccttgtgggtgaactcagggccc
ctctgtatcc
2461 ttaggcccca aggccacttgttcccatcctcccatcacctcccttggact
cccccacaca
3 2521 cccccccctc agcctcaaacaagaagagggtggcattcacacagcaggac
0
caggctttga
2581 ggctccttct catgtatctcctgaatacatctccacccttatctgtttat
ttctgatagt
2641 tctgatctaa gtacttctggacaggtgataaatgtccatgggcaaaaatt
3 caaattgcag
5
2701 agcaaaggct ctcctccgatgcctgcccccctccccagaaccaaccactg
tccatccagg
2761 ctgccctgag tctcggtttgtacacctggaggatctcagaggtggtttga
cgtccgtagt
4 2821 gagactgtcc gcaccctcctctagggctgtgtgtgagtccactgcatgga
0
tggactctga
2881 ttttgtggca tctcctaatggaagatcacggcactaatttcatcctacgg
caggatagaa
2941 caatcttgta tctacttccacaggaatatctaagcctgtgggttaagttc
45 ctaaaagcaa
3001 aatgtagcta cattatatgttctttcttattttgaaagataagcccaaac
tgttctcgat
3061 gaagcgggga gaagtttacattcccagcagtgagtggtgaaagtgtgtgt
ttccagaact
5 3121 tcagtctatg tctgtgtgtcagttgctgtcatcagtctctttctgtatcc
0
ttcctttttc
3181 tccagatcta tgtatctctctgaccctctgtctctttttctacagtatcc
acagtcttgg
3241 gaaatggctc atctctgtcactcccagagggccagtctctgcgcctggtc
55 tgtgcagttg
3301 atgcagttga cagcaatccccctgccaggctgagcctgagctggagaggc
ctgaccctgt
3361 gcccctcaca gccctcaaacccgggggtgctggagctgccttgggtgcac
ctgagggatg
60 3421 cagctgaatt cacctgcagagctcagaaccctctcggctctcagcaggtc
tacctgaacg
3481 tctccctgca gagtgagtgcaccagtatgctggggaggggctggagagga
gaacacacct
3541 cctccaccct tagtaactgctgagcgtggaccttcagagaggagctccgc
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
3
tctggtctgt
3601 gctcagctgt gaggtctggaacttccctgggacccacagcaccactgtcc
tcttcctgcc
3661 agggaagggt tgtggggtggggagagggcaggagtggatctcagagggga
caggatgggg
3721 ccggacaggt gtgtttagggagacaagcgcctttctttgcagggctgaac
tggagtcaca
3781 caactgagat acttgctttgagcatcaaattaaaaaaaagaaaaagccca
gcaagtcagc
3841 aatcaaatga aatcatattgcaatgcaataatcttttaaaaaaagtaaaa
attgaatgca
3901 aaacaaattc attaatggataaaatattaaaattgtgaaaaaaaacccca
aaaggaatgg
3961 ctggcacttg cacgcctcactggcctcaggaagagtctctccatgtcctg
ctctctctca
4021 ttcctgttct ttgtgtctggaaaggggaagtggaaatagaagtctaggac
cctacaggaa
4081 gtgggaggag aagagacccaattctctatgatatatcacaaaaataactc
ccatctgtca
2 4141 acaggcaaag ccacatcaggagtgactcagggggtggtcgggggagctgg
0
agccacagcc
4201 ctggtcttcc tgtccttctgcgtcatcttcgttgtgtaagcatggaccct
agagagggag
4261 ggagggagag ccctgggggaggacaggctggaagctggatccctgaagcc
2 agagctggag
5
4321 ggacctggat gggtcaagagcttggggcaagaaggaggtcacaggtgcat
ggtgagaatt
4381 ccatgtgggc ctgtgtttgaggagctttgagtctgtggcaaaccttggta
cccactgtcc
3 4441 aggagaagag agcctctgttctcaaccttggggtctctaagactggacca
0
ctgctttccc
4501 acctcagtca cccctgcagtcccttaataggaaacacatgggggtacctg
gtctgcccac
4561 cgcaccccaa tctgaccacactgaaaggctctctggtctcttcactcaga
35 gtgaggtcct
4621 gcaggaagaa atcggcaaggccagcagcgggcgtgggagatacgggcata
gaggatgcaa
4681 acgctgtcag gggttcagcctctcaggtgagtgatgtggactctccacag
ccagcatgta
4 4741 gcctggacac ctcccacaggatgacccccaggactaatcagctgggcgta
0
gccaaagtta
4801 cctcctctct gttcttcctttcttctctgtagccccaaatcacaatgttt
ggttggtttc
4861 ctcccctaag aacagcttttattgtctctgctccctatcctgacccttca
45 ttgctgaggc
4921 ctgaggatct ctgtcttttgttccctcacctgtctgcctgtctcctctcc
tttcctgcct
4981 ggggggactg tccagaagacatcatcgtccagttcctctgcatttgaaca
gctgttcccc
5 5041 cacccctcaa taccgtttagagcagaagccagcaaatactatctgtcagg
0
gacagataga
5101 aactattttc ggcttcatgggccacacagtctcattgcagctcctcaaat
ctgctgttgt
5161 agcaagaaag aagccatataccctgtgtaaacaaatgaatatggctgtgt
55 gccaataaaa
5221 ctattcacaa acataaagagtgggctggatatgactcagatactgtagtt
tgacaacccc
5281 tgatctagag taaaaatcccaaactctatagcctgcagcagtgcacattc
tgactttttt
60 5341 tgtttttttt tttttttgttgttgttgtttttgagacagagtcttgctct
gtcgcccagg
5401 ctggagtgca gtggtgcgatctctgctcactgcaacttccaccttccggg
ttcaagccat
5461 tctcctgcct cagcctccggagtagctgggactacaggcgcctgccacca
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
4
cgcccagcta
5521 atttttttgt atttttagtagagacggggtttcactgtgttagccaggat
ggtctcagtc
5581 tcctgacctt gtgatctgcccaccttggcttcccgaagtgctgggattac
aggcgtgagc
5641 cactgtgacc ggccacattctgaccttttaagcacctacctctccactag
ggcaagaaca
5701 agggtgaagt gagtgaggctgttgcctcaagtgcattttttcgtttgttt
gtttttgttt
5761 tttgagatgg agtctcgctctgtcacccaggatgtagtgcagtggcacaa
tcttggctta
5821 ctgcaacctc tgcctcctaggttcaagcgattctcctgcctcagcctcct
gagtagctgg
5881 gattaaaggt gcacaccaccacacctggctaattttgtatttttagtaga
gacagggttt
5941 caccatgttg gccaggctggtctcaaactcctgacctcaggtgatccgcc
tacctcagcc
6001 tcctgaagag ctgggattacagatgtgagccaccgcgccccatcctcact
gtctgctctg
2 6061 actcacttct ctctcccatgtctcaggggcccctgactgaaccttgggca
0
gaagacagtc
6121 ccccagacca gcctcccccagcttctgcccgctcctcagtgggggaagga
gagctccagt
6181 atgcatccct cagcttccagatggtgaagccttgggactcgcggggacag
2 gaggccactg
5
6241 acaccgagta ctcggagatcaagatccacagatgagaaactgcagagact
caccctgatt
6301 gagggatcac agcccctccaggcaagggagaagtcagaggctgattcttg
tagaattaac
3 6361 agccctcaac gtgatgagctatgataacactatgaattatgtgcagagtg
0
aaaagcacac
6421 aggctttaga gtcaaagtatctcaaacctgaatccacactgtgccctccc
ttttattttt
6481 ttaactaaaa gacagacaaattcct
35
SEQ.ID.NO. 2
OB-BPL AA
PPLSLEPAVPERRTLRNRRSLAALAPLTPDMLLLLLPLLWGRERAEGQTSKLLTMQSSV
TVQEGLCVHVPCSFSYPSHGWIYPGPWHGWFREGANTDQDAPVATNNPARAWEETR
DRFHLLGDPHTKNCTLSIRDARRSDAGRYFFRMEKGSIKWNYKHHRLSVNVTALTHRPN
ILIPGTLESGCPQNLTCSVPWACEQGTPPMISWIGTSVSPLDPSTTRSSVLTLIPQPQD
HGTSLTCQVTFPGASVTTNKTVHLNVSYPPQNLTMTVFQGDGTGQSLRLVCAVDAVDSN
PPARLSLSWRGLTLCPSQPSNPGVLELPWVHLRDAAEFTCRAQNPLGSQQWLNVSLQK
ATSGVTQGWGGAGATALVFLSFCVIFVGPLTEPWAEDSPPDQPPPASARSSVGEGELQ
YASLSFQMVKPWDSRGQEATDTEYSEIKIHR
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
SEQ.ID.NO. 3
OB-BPL AA
MLLLLLPLLWGRERAEGQTSKLLTMQSSVTVQEGLCVHVPCSFSPSHGWIYPGPVVH
5 GYWFREGANTDQDAPVATNNPARAVWEETRDRFHLLGDPHTKNCLSIRDARRSDAGR
YFFRMEKGSIKWNYKHHRLSVNVTALTHRPNILIPGTLESGCPQNLTCSVPWACEQG
TPPMISWIGTSVSPLDPSTTRSSVLTLIPQPQDHGTSLTCQVTFPGASVTTNKTVHL
NVSYPPQNLTMTVFQGDGTVSTVLGNGSSLSLPEGQSLRLVCAVDAVDSNPPARLSL
SWRGLTLCPSQPSNPGVLELPWVHLRDAAEFTCRAQNPLGSQQWLNVSLQSKATSG
VTQGWGGAGATALVFLSFCVIFVWRSCRKKSARPAAGVGDTGIEDANAVRGSASQ
GPLTEPWAEDSPPDQPPPASARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSE
IKIHR
SEQ.ID.NO. 4
TCACCGGCTCTCTGTGAATG
SEQ.ID.NO. 5
GTCTTCTGCCCAAGGTTCAG
SEQ.ID.NO. 6
TCCTCTAAGTCTTGAGCCCG
SEQ.ID.NO. 7
CAGACGTTGAGATGGACGGT
3 0 SEQ.ID.N0.8
CGTGGGAGATACGGGCATAG
SEQ.ID.NO. 9
AAAAGGGAGGGCACAGTGTG
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
SEQ.ID.NO. 10
Siglec-7
MLLLLLLPLLWGRERVEGQKSNRKDYSLTMQSSVTVQEGMCVHVRCSFSYPVDSQTDSDPVHGY
WFRAGNDISWKAPVATNNPAWAVQEETRDRFHLLGDPQTKNCTLSIRDARMSDAGRYFFRMEKG
NIKWNYKYDQLS VNVTALTHRPNILIPGTLESGCFQNLTCS VPWACEQGTPPMIS WMGTS VSPLHP
STTRSSVLTLIPQPQHHGTSLTCQVTLPGAGVTTNRTIQLNVSYPPQNLTVTVFQGEGTASTALGNS
SSLSVLEGQSLRLVCAVDSNPPARLSWTWRSLTLYPSQPSNPLVLELQVHLGDEGEFTCRAQNSLG
SQHVSLNLSLQQEYTGKMRPVSGVLLGAVGGAGATALVFLSFCVIFIVVRSCRKKSARPAADVGDI
GMKDANTIRGSASQGNLTESWADDNPRHHGLAAHSSGEEREIQYAPLSFHKGEPQDLSGQEATNN
EYSEIKIPK
SEQ.ID.NO. 11
CD33
MPLLLLLPLLWAGALAMDPNFWLQVQES VT VQEGLC VLVPCTFFHPIPYYDKNSPVHGYWFREG
AIISGDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKS
2 0 PQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIIT
PRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGLVHGAIGGA
GVTALLALCLCLIFFIVKTHRRKAARTAVGSNDTHPTTGSASPKHQKNSKLHGPTETSSCSGAAPT
VEM-DEELHYASLNFHGMNPSKDTSTEYSEVRTQ
2 5 SEQ.ID.NO. 12
Siglec-6
MLP-LLLPLLWAGALAQERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEG
3 0 ADVPVATNDPDEEVQEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRL,KSKWMKYGYTSS
KIYVRVMALTHRPNISIPGPGV WPSSNLTCS VPW VCEQGTPPIFS WMSAAPHLLGPRTTQSS VLTIT
PAQDHSTNLTCQVTFPGAGVTMERTIQLNVSYAPQKVAISIFQGNSAAFKILQNTSSLPVLEGQALR
LLCDADGNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVH
WKPEGRAGGVLGAV WGASITTLVFLC VCFIFRVKTRRKKAAQPVQNTDDVNPVMVSGSRGHQHQ
3 5 FQTGIVSDHPA EAGPISEDEQELHYAVLHFHKVQPQEPKVTDTEYSEIKIHK
CA 02366087 2001-09-10
WO 00/53747 PCT/CA00/00259
SEQ.ID.NO. I 3
Siglec-5
MLPLLLLPLLWGGSLQEKPWELQVQKSVTVQEGLCVLVPCSFSYPWRSWYSSPPLYW
WFRDGEIPYYAEWATNNPDRRVKPETQGRFRLLGDVQKKNCSLSIGDARMEDTGSYF
RVERGRDVKYSYQQNKLNLEVTALIEKPDIHFLEPLESGRPTRLSCSLPGSCEAGPPLT
FSWTGNALSPLDPETTRSSELTLTPRPEDHGTNLTCQMKRQGAQVTTERTVQLNVSYAPQT
ITIFRNGIALEILQNTSYLPVLEGQALRLLCDAPSNPPAHLSWFQGSPALNATPISNTGILELRRVRSA
EEGGFTCRAQHPLGFLQIFLNLSVYSLPQLLGPSCSWEAEGLHCRCSFRARPAPSLCWRLEEKPLEG
NSSQGSFKVNSSSAGPWANSSLILHGGLSSDLKVSCKAWNIYGSQSGS VLLLQGRSNLGTGV VPAA
LGGAGVMALLCICLCLIFFLIVKARRKQAAGRPEKMDDEDPIMGTITSGSRKKPWPDSPGDQASPP
GDAPPLEE-QKELHYASLSFSEMKSREPKDQEAPSTTEYSEIKTSK
