Note: Descriptions are shown in the official language in which they were submitted.
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MURINE a(1,3)FUCOSYLTRANSFERASE Fuc-TVII, DNA ENCODING THE
SAME AND USES THEREOF
BACFGnOUND OF THE INVENTION
Field of the _Tnvention:
The present invention relates to murine
a(1,3)fucosyltransferases, Fuc--77II, DNA encoding such a
murine a(1,31 fucosvl t_ ansierase ='uc-=I, olasmids containing
such DNA, cells transformed with such a plasmid, a met:od for
producing a murine a(1,2)fucosyltransferase Fuc-TVII by
culturing such cells, monoclonal antibodies which specifically
bind to a murine a(1, 3) fucosyltransferase Fuc-TVII, and
immunoassays for detecting a murine a(1,3)fucosyitransferase
Fuc-'?'VII usin g suc h monoclonal antibodies.
Discussion ai the ?ackcround:
Cell adhesicn events between leukccvtes and endotheiial
calls operate to faci'_itate the exit of blood leukocrtes from
the vascular =ree. The select'-n family of cell adhesion
molecules, and their counter-receptors, function early in this
process, mediating transient adhesive contacts between
leukocytes and the endothelial cell monolayer. These
selec=in-cievendent adhesive contacts, =ovether with shear
forces impinging upon the leukocvte, cause the leukoc_:te to
"roll" alono =::e endotheiial monolayer. Leukccvte rolling, in
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turn, facilitates subsequent events that include leukocyte
activation, firm leukocyte-endothelial cell attachment, and
transendothelial migration (T.A. Springer, Cell, vol. 76, pp.
301-314 (1994); R.P. McEver et al, J. Biol. Chem., vol. 270,
pp. 11025-11028 (1995)).
E- and P-selectin, expressed by activated vascular
endothelial cells, recognize glycoprotein counter-receptors
displayed by leukocytes. Each of these selectins can operate
to mediate leukocyte rolling in the context of inflammation.
L-selectin has also been implicated in mediating leukocyte
adhesion to activated vascular endothelium, through
interactions with an as yet poorly understood endothelial cell
ligand (M.L. Arbones et al, Immunity, vol. 1, pp. 247-260
(1994) ; K. Ley et al, J. Exp. Med., vol. 181, pp. 669-675
(1995)). By contrast, lymphocyte.L-selectin recognizes
glycoprotein counter-receptors displayed by specialized
cuboidal endothelial cells that line high endothelial venules
(HEV) within lymph nodes and Peyer's patches. L-selectin-
dependent adhesive interactions in this context operate to
facilitate trafficking of lymphocytes (lymphocyte "homing") to
such lymphoid aggregates.
The NH.-terminal C-type mammalian lectin domain common to
each of the three selectin family members mediates cell
adhesion through calcium dependent interactions with specific
oligosaccharide ligands, displayed by leukocytes (E- and
P-selectin ligands) (R.P. McEver et al, J. Biol. Chem., vol.
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270, pp. 11025-11028 (1995); A. Varki, Proc. Natl. Acad. Sci.
U.S.A., vol. 91, pp. 7390-7397 (1994)), or by HEV (L-selectin)
(S.D. Rosen et al, Curr. Opin. Cell Biol., vol. 6, pp. 663-673
(1994)). Physiological ligand activity for E- and P-selectins
is critically dependent on the expression of a non-reducing
terminal tetrasaccharide termed sialyl Lewis x(sLe")
[NeuNAcc2,3Ga181,4(Fucacl,3)G1cNAc-R] (A. Varki, Proc. Natl.
Acad. Sci. U.S.A., vol. 91, pp. 7390-7397 (1994)), and/or its
difucosylated variant (T.P. Patel et al, Biochemistry,
vol. 33, pp. 14815-14824 (1994)). Howevler, E- and P-selectins
recognize this oligosaccharide in different contexts.
P-selectin-dependent cell adhesion is optimal when sLe" is
displayed by serine and threonine-linked oligosaccharides
residing on a specific protein termed P-selectin glycoprotein
ligand 1 (PSGL-1) (K.L. Moore et al, J. Cell. Biol., vol. 188,
pp. 445-456 (1992); D. Sako et al, Cell, vol. 75, pp. 1179-
1186 (1993)). sLe"-modified PSGL-1 also appears to represent a
high affinity counter-receptor for E-selectin (D. Asa et al,
J. Biol. Chem., vol. 270, pp. 11662-11670 (1995); K.D. Patel
et al, J. Clin. Invest., vol. 96, pp. 1887-1896 (1995)). A
distinct leukocyte glycoprotein termed E-selectin ligand i
(ESL-1) (M. Steegmaler et al, Nature, vol. 373, pp. 615-620
(1995)), and its c(1,3)fucosylated, asparagine-linked
oligosaccharides, may also function as an E-selectin counter-
receptor.
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Physiological L-selectin counter-receptors on HEV are
represented by the glycoproteins, G1yCAM-1 (L.A. Lasky et al,
Cell, vol. 69, pp. 927-938 (1992)), CD34 (S. Baumhueter et al,
Science, vol. 262, pp. 436-438 (1993)), and MadCAM-1 (E.L.
Berg et al, Nature, vol. 366, pp. 695-698 (1993)).
Biochemical studies indicate that L-selectin ligand activity
of these molecules is also critically dependent upon post-
translational modification by glycosylation. Early studies
documented a requirement for sialylation and sulfation
(Y. Imai et al, J. Cell Biol., vol. 113, pp. 1213-1221
(1991)), implied a requirement for a(1,3)fucosylation, and
indicated that these modifications are components of serine
and/or threonine-linked glycans. More recent oligosaccharide
structural analyses extend this work, and imply that high
affinity L-selectin ligand activity depends upon the sulfated
variant of the sLe" determinant,
NeuNAca2,3(S046)Ga1B1,4(Fuca1,3)G1cNAc-R (S. Hemmerich et al,
Biochemistry, vol. 33, pp. 4820-4829 (1994); S. Hemmerich et
al, Biochemistry, vol. 33, pp. 4830-4835 (1994); S. Hemmerich
et al, J. Biol. Chem., vol. 270, pp. 12035-12047 (1995)).
Expression of sLe' is determined by cell lineage-specific
expression of one or more a(1,3)fucosyltransferases
(S. Natsuka et al, Curr. Opin. Struc. Biol., vol. 4, pp. 683-
691 (1994)). These enzymes utilize the donor substrate GDP-
fucose, and catalyze a transglycosylation reaction involving
the addition of al,3-linked fucose to a common 3'-sialyl-N-
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acetyl-lactosamine precursor. It can be presumed that
expression of the sulfated variant of sLe" also depends upon
lineage-specific expression of c(1,3)fucosyltransf erase
activities operating on sulfate-modified 3'-sialyl-N-acetyl-
lactosamine precursors, or that create sLex moieties modified
subsequently by sulfation.
The identity of the ac(1,3)fucosyltransferase(s)
responsible for selectin ligand expression in leukocytes is
not well-defined, and HEV-specific a(1,3)fucosyltransferases
have not been described. To date, five different human
a(1,3)fucosyltransf erases have been cloned (J.F. Kukowska-
Latallo et al, Genes & Dev., vol. 4, pp. 1288-1303 (1990);
B.W. Weston et al, J. Biol. Chem., vol. 267, pp. 24575-24584
(1992); B.W. Weston et al, J. Biol. Chem., vol. 267, pp. 4152-
4160 (1992); J.B. Lowe et al, J. Biol. Chem., vol. 266,
pp. 17467-i7477 (1991); S.E. Goelz et al, Cell, vol. 63,
pp. 1349-1356 (1990); R. Kumar et al, J. Biol. Chem.,
vol. 266, pp. 21777-21783 (1991); S. Natsuka et al, J. Biol.
Chem., vol. 269, pp. 16789-16794 (1994); K. Sasaki et al, J.
Biol. Chem., vol. 269, pp. 14730-14737 (1994)). Northern blot
and molecular cloning analyses imply that two of these, termed
Fuc-TIV (J.B. Lowe et al, J. Biol. Chem., vol. 266, pp. 17467-
17477 (1991); S.E. Goelz et al, Cell, vol. 63, pp. 1349-1356
(1990); R. Kumar et al, J. Biol. Chem., vol. 266, pp. 21777-
21783 (1991)) and Fuc-TVII (S. Natsuka et al, J. Biol. Chem.,
vol. 269, pp. 16789-16794 (1994); K. Sasaki et al, J. Biol.
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Chem., vol. 269, pp. 14730-14737 (1994)), are expressed in
leukocyte cells, and represent candidates for critical
participation in selectin ligand expression. The role of Fuc-
TIV (also known as ELAM-1 Ligand Fucosyl Transferase, or ELFT)
in this process is not clear, however. While Fuc-TIV/ELFT is
able to efficiently utilize non-sialylated N-acetyl-
lactosamine precursors to direct expression of the Le" moiety
(J.B. Lowe et al, J. Biol. Chem., vol. 266, pp. 17467-17477
(1991); R. Kumar et al, J. Biol. Chem., vol. 266, pp. 21777-
21783 (1991)), this enzyme cannot determsne Le" expression in
all cellular contexts (S. Goelz et al, J. Biol. Chem.,
vol. 269, pp. 1033-1040 (1994)), and its ability to do so in
leukocytes, or in leukocyte progenitors, has not been
demonstrated. By contrast, Fuc-TVII is apparently able to
determine sLe" expression in all mammalian cellular contexts
examined, where sLe" synthesis is biochemically possible
(S. Natsuka et al, J. Biol. Chem., vol. 269, pp. 16789-16794
(1994); K. Sasaki et al, J. Biol. Chem., vol. 269, pp. 14730-
14737 (1994)). Neither enzyme has been tested for its ability
to participate in the synthesis of L-selectin ligands
represented by the sulfated sLe" determinant.
Thus, there remains a need for additional
a(1,3)fucosyltransferases and methods, cells, plasmids, and
DNA useful for preparing the same. There also remains a need
for antibodies and immunoassays useful for detecting such
a(1,3)fucosyltransf erases.
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SUMMARY OF THE INVENTION
Accordingly, it is one object of the present invention to provide novel
a(1,3)fucosyltransferases.
The invention specifically relates to a polypeptide having an amino acid
sequence which comprises an amino acid subsequence, said amino acid
subsequence being selected from the group consisting of:
a) the amino acid sequence encoded by the DNA sequence
corresponding to from position 996 to 1149 and 2067 to 3079 of SEQ ID NO:1;
and
b) the amino acid sequence encoded by the DNA sequence
corresponding to from position 1947 to 1959 and 2067 to 3079 of SEQ ID NO:1.
It is another object of the present invention to provide
novel DNA encoding such a a(1,3)fucosyltransferase.
It is another object of the present invention to provide
novel plasmids containing such DNA.
It is another object of the present invention to provide
novel cells transfor:neci with such a plasmid.
It is another object of the present invention to provide
a novel method for preparing such an a(1,3)f;:cosyltransferase
by culturing such a transformed cell.
It is another object of the present invention to provide
novel monoclonal antibodies which bind specif"ically to such an
a(1,3)fucosyltransferase.
It is another object of the present Invention to provide
a novel immunoassay for deteczing such an
* (1,3)fucosyltransrerase using such a monoclonal antibody.
It is another object of the present invention to provide
a novel method to fucosylate moieties by means of such an
a(1,3)fucosyltransferase.
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These and other objects, which will become apparent
during the following detailed description, have been achieved
by the inventors' cloning of c(1,3)fucosyltransferase Fuc-
TVII.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the :nvention and many of
the attendant acivantages thereof will be readily obtained as
the same becomes better understood by reference to the
following detailed description when considered in connection
with the accompanying drawings, wherein:
Figure la shows the structure and functional activities
of the :nurine Fuc-'Z'VII gene and cDNAs. The multi-exon
structure of the murir.e Fuc-TVII gene is shown at top.
Numbering below the schematic corresponds-to the nucleotide
positions of intron-exon boundaries, and the first (1) and
last (3520) nucleotides of the known sequence of the locus.
Intron-exon boundaries are defined by comparison of the cDNA
sequences to the corresponding genomic DNA sequence (see
Fig. 2 and SEQ ID N0:1). Numbering above the schematic, immediately above
"ATG"s, corresnonds to the nucleotide oosition of the first
nucleoticie in each cf the three potential 4-nitiation codons,
as discussed in detail below. Numbering above the schematic,
immediately above "STOP"s, corresponds to the nucleotide
position of the translational termination codon (TGA; base
pairs 1900-1902) localized to exon 3b, that truncates the
potential open reading frame initiated by the start codon at
nucleotide 996-99B iz cDNA classes represented by cDNAs 6 and
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(see text for details). Representative members of the five
str=.:cturally different classes cf Fuc-'I'VI? cDNAs isolated from
the :~urine cvtotoxic T-lymphocyte cell line 14-7fd are
~
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schematically represented below the gene structure schematic.
The cDNAs shown are the representative member of each class
with the longest 5' extension. The number of cDNAs isolated
in each class is indicated in the column labeled "number of
cDNAs". Each cDNA was transiently expressed in COS-7 cells
(see the Experimental Procedures in the Examples). The
transfected COS-7 cells were then subjected to flow cytometry
analysis to characterize the cell surface glycosylation
phenotype determined by each cDNA. The fraction of sialyl
Lewis x-positive cells in the transfectea population (positive
staining with the monoclonal antibody CSLEX-1) normalized to
transfection efficiency as determined by chloramphenicol
acetyl transferase activity encoded by a co-transfected
plasmid vector encoding this enzyme (see the Experimental
Procedures in the Examples) is indicated in the column labeled
"% CSLEX-1 positive". These results represent the fraction of
antigen-positive cells observed above a background of 2%
staining obtained with the negative control vector pCDM8.
Extracts were also prepared from the transfected cells, and
were subjected to in vitro a(1,3)fucosyltransferase assays
using 5 mM sialyl-N-acetyllactosamine as an acceptor (see the
Experimental Procedures in the Examples). The specific
activity of the a(1,3)fucosyltransferase activity encoded by
each cDNA (normalized for transfection efficiency) is
indicated in the column labeled "Sp. Act. (cpms/ g/hr)".
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Figure lb shows the results of a Western blot analysis of
the eolypeptides expressed in COS-7 cells by cDNAs 3, 5, 6,
10, and 14. The extracts used in the c(1,3)fucosyltransferase
assays shown in Figure la, above, were also subjected to
Western blot analysis using an antigen-affinity purified anti-
Fuc-TVII antibody. The amounts of protein analyzed from each
type of transfecteci cell extract were varied to achieve
normalization to the transfection efficiencies, exactly as
10 inaicated above in Figure !a for the rZow cytometry and
a(1,3)fucosyltransf erase activity analysels. Cell extracts
were fractionated by SDS-polyacrylamide gel electrophoresis,
alectro-blotted to a PVDF membrane, and the Fuc-TVII
expression vector-encoded polypeptides were identified by
probing with an antigen affinity-purified rabbit anti-Fuc-TV'II
antibody, goat anti-rabbit IgG-peroxidase conjugate, and a
commercially available enhanced chemiluminescence reaaent
(ECL, Amersham), as described in the rxperimentai Procedures
in the Examples;
Figure 2 shows the nucleotide and deduced amino acid
sequence of the isolated mouse Fuc-TVII gene as per SEQ ID NO:1. The DNA
sequence of the isolated mouse Fuc-TVII gene. The DNA
sequence was derived from a phage containing the murine
Fuc-TVII locus. DNA sequence present in cDNAs (Figure la) is
shown in upper case, whereas DNA sequence corresponding to
intronic positions is displayed in lower case. Amino acid
sequences predicted by the cDNA sequences are shown in single
letter code. As discussed in detail in the text, alternative
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splicing events vield different cDNAs that :nay ;n turn encode
three different aolypeptides. One protein is predicted to
initiate at the methionine codon localized to nucleotide
positions 996-998 (389 residues, 44,492 Da; cDNA S; Fig. 1).
A second protein is predicted to initiate at the methionine
codon localized to nucleotide positions 1947-1949 (342
residues, 39,424 Da; cDNAs 6, 10, and 14; Fig. 1). The third
protein is predicted to initiate at the methionine codon
localizeci to nucleotide positions 2126-2128 (318 residues,
36,836 Da; =DNA 3, as well as all other cDNAs; Fig. 1);
Figures 3a and b show the tissue specific expression
patterns of the murine Fuc-TVII gene. Oligo-dT purified mRNA
(5 g) purified from murine tissues and cell lines was
fractionated by agarose gel electrophoresis, blotted to a
nylon hybridization membrane, and was probed with a 979 base
pair DNA seqaent derived from the codina region cf the mouse
Fuc-TVII locus (nucleotides 2228-3207; see Fig. 2 and SEQ ID NO:1 and the
Experimental Procedures in the Examples). RNA molecular size
standards, in kb, are indicated at the left in each panel.
Each blot was subsequently stripped and re-probed with a
radiolabeled chicken glyceraidehyde 3-phosphate dehydrogenase
probe to confirm that RNA samples were intact and loaded in
equivalent amounts (see the Experimental Procedures ~.n the
Examples);
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Figure 3a shows the polyadenylated RNA isolated from
mouse tissues, and from the murine T-lymphocyte cell line 14-
7fd (14FD).
Figure 3b shows the polyadenylated RNA isolated from
mouse bone marrow and spleen, and from cultured murine
leukocyte cell lines. Cell lines represent the following
lineages: MEL, murine erythroleukemia cell line; P388 and RAW
(RAW 264.7), macrophage; EL4, T cell; S107, 63 (TH2.54.63) and
180.1, B cell lines (hybridomas);
Figure 4 shows the results of an in'situ hybridization
analysis of Fuc-TVII transcripts in murine lymph nodes and
Peyer's Patches. Sequential 10 micron thick frozen sections
of an axillary lymph node (column labeled Peripheral Lymph
Node; panels A, B, C), a mesenteric lymph node (column labeled
Mesenteric Lymph Node, panels D, E, F), and Peyer's patches
(column labeled Peyer's Patches; panels G, H, I), were stained
with hematoxylin and eosin (row labeled H & E; panels A, D, G,
photograph at 5X magnification using bright field
illumination), or were processed for in situ hybridization, as
described in the Experimental Procedures in the Examples.
Adjacent sections subjected to in situ hybridization were
probed with an ''S-labeled anti-sense RNA probe derived from
base pairs 2196-2497 of the murine Fuc-TVII locus (row labeled
antisense; panels B, E, H), or were probed with an 35S-labeled
negative control sense RNA probe derived from base pairs 2196-
2497 of the murine Fuc-TVII locus (row labeled sense; panels
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C, F, I). Sections processed for in situ hybridization were
photographed at 5X magnification using dark field
illumination. The white areas in panels B, E, and H
correspond to sites (i.e. high endothelial venular endothelial
cells in panels B and E; high endothelial venular endothelial
cells and lumenally-positioned cells [the white-stained "caps"
on the lymphoid aggregates] in panel H ) where the Fuc-TVII
antisense probe identifies Fuc-TVII transcripts (see text for
details);
Figures 5a and b show the immunohiAochemical co-
localization of expression of Fuc-TVII, MECA-79, and
L-selectin ligands, in lymphoid aggregate high endothelial
venular endothelial cells.
Figure 5a shows the co-localized expression of Fuc-TVII,
MECA-79, and L-selectin ligands in HEV. Sequential 10 micron-
thick frozen sections of axillary lymph nodes (column labeled
Peripheral Lymph Node), mesenteric lymph nodes (column labeled
Mesenteric Lymph Node), and Peyer's patches (column labeled
Peyer's Patches) were stained with an antigen affinity
purified rabbit polyclonal anti-murine Fuc-TVII (row labeled
Fuc-4'VII), with the monoclonal antibody MECA-79 (row labeled
MECA-79), with a murine L-selectin/human IgM chimera (row
labeled L-sel/IgM Chimera), or with the L-selectin/IgM chimera
stained in the presence of EDTA (row labeled L-sel/IgM Chimera
+ EDTA). Detection of section-bound primary
immunohistochemical reagents was subsequently accomplished
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using secondary immunochemical reagents labeled with
flurochromes (FITC-conjugated [green] anti-rabbit IgG for Fuc-
TVII; FITC-conjugated [green] anti-human IgM for L-
selectin/IgM; rhodamine-conjugated [red] anti-rat IgM for
MECA-79), as described in the Experimental Procedures in the
Examples. These colored detection schemes yield white area on
the black and white reproductions of the original photographs
of these experiments. Thus, the "green" and "red" regions
appear as white regions. Photomicrographs were taken at 40X
magnification, using fluorescent microscopic procedures as
described in the Experimental Procedures in the Examples.
Fiqure 5b is a high power magnification of peripheral
lymph node HEV staining with anti-Fuc-TVII. Light gray areas
in panel A of Figure 5b correspond to MECA-79 staining. White
areas in panel A of Figure 5b correspond to staining with
anti-Fuc-TVII antibody. A 10 micron-thick frozen section of
an axillary lymph node was stained simultaneously with the
antigen affinity purified anti-Fuc-TVII antibody and with
anti-MECA-79 antibody (panel labeled "immune antibodies").
Section-bound antibodies were detected with fluorochrome-
conjugated secondary antibody reagents exactly as described in
the description of Figure 5a, and in the Experimental
Procedures in the Examples. The immediately adjacent 10
micron-thick frozen section of the same axillary lymph node
was instead stained simultaneously with a pair of negative
control antibodies (normal rabbit IgG, and normal rat IgM;
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panel labeled "non-immune ant_bodies"), and developed in a
manner identical to that used in the "immune antibodies"
panel. Photomicrographs were taken at 40oX magnification,
using fluorescent microsccmic procedures described in the
Experimental Procedures 4n the Examples.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Thus, in a first embodiment, the present invention
10 provides novel enzymes, murine a (1, 3) fucosyltransf erases,
murine Fuc-TVIIs. Suitably, the enzyme has one cf the two
amino acid secruences corresponding to the polypeptide encoded
by the gene shown in Fiqure la using one of two different ATG
initiation codons. Thus, in a first embodiment, the present
enzyme has the amino acid sequence corresponding to that
encoded by the DNA corresponding to cDNA 5 in Figure 1 and
nucleotide positions 996 to 1149, and 2067 to 3079 in SEQ ID NO:1. In another
embodiment, the enzyme has an amino acid sequence corresponding to that
encoded by the cDNAs 6, 10, or 14 in Figure 1 and having a nucleotide
sequence corresponding nucleotide positions 1947 to 1959, and 2067 to 3079 of
SEQ ID NO:1. In these two embodiments, it is to be understood that the amino
acid sequences correspond to:
(1) that obtained by linking the C terminus of the amino acid sequence
encoded by nucleotide positions 996 to 1149 of SEQ ID NO:1 to the N terminus
of the amino acid sequence encoded by nucleotide positions 2067 to 3079 of
SEQ ID NO:1; and
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(2) that obtained by linking the C terminus of the amino acid sequence
encoded by nucleotide positions 1947 to 1959 of SEQ ID NO:1 to the N terminus
of the amino acid sequence encoded by nucleotide positions 2067 to 3079 of
SEQ ID NO:1.
The present enzyme may also have one of the amino acid
sequences described above and in which up to 5 amino acid
sequences have been added, deleted, or substituted at the
amino terminus of the protein, provided that the enzyme
retains its activity. In this context an enzyme is considered
to retain its activity if it retains at least -10%, preferably
at least 'h, more preferably at least of the specific
activity of the native enzyme to transfer fucose from GDP-
fucose to 3'-sialyl-N-acetyllactosamine as described in the
examples below.
The present invention also provides novel fusion proteins
in which any of the enzymes of the present 'nvention are fused
to a polypeptide such as protein A, streptavidin, =racrments of
c-myc, maltose binding protein, IgG, 1gM, amino acid tag, etc.
Preferably, the polypeptide fused to the present invention is
fused to the amino terminus of the present enzyme. In
addition, it is preferred that the polypeptide fused to the
enzyme of the present invention is chosen to facilitate the
release of the fusion protein from a prokaryotic cell or a
eukaryotic cell, into the culture medium, and to enable its
(affinity) purification and possibly immobilization on a solid
phase matrix.
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Examples of such fusion proteins include those in which the polypeptide,
e.g., protein A, is fused to the glycine residue encoded by nucleotide
positions
2067 to 2069 in SEQ ID NO:1 or to the amino group of the praline residue
encoded by nucleotide positions 2162 to 2164 in SEQ ID NO:1.
In another embodiment, the present invention provides novel DNA
sequences which encode murine Fuc-TVII, or a fusion protein according to the
present invention. Suitably, the present DNA sequence is any which encodes
the two amino acid sequences described above. Thus, in a first embodiment, the
DNA sequence is any encoding a polypeptide having the amino acid sequence
corresponding to that encoded by the cDNA 5 in Figure 1 and having the
sequence corresponding to nucleotide positions 996 to 1149, and 2067 to 3079
in SEQ ID NO:1. In another embodiment, the DNA sequence is any which
encodes a protein having the sequence corresponding to that encoded by the
cDNAs 6, 10, or 14 in Figure 1 and having the sequence corresponding to
nucleotide positions 1947 to 1959, and 2067 to 3079 of SEQ ID NO:1.
Of course, the DNA sequence may encode a protein which corresponds
to any of those described above but in which up to 5 amino acid residues have
been added, deleted, or substituted at the amino terminus of the protein,
provided that the protein retains its activity. In addition, the DNA sequence
may
encode any of the present fusion proteins.
In a preferred embodiment, the DNA sequence has the nucleotide
sequence corresponding to from position 996 to 1149, and 2067 to 3079 of SEQ
ID NO:1. In another preferred embodiment, the DNA sequence has the
nucleotide sequence corresponding to from position 1947 to 1959, and 2067 to
3079 in the nucleotide sequence of SEQ ID NO:1.
In these preferred DNA sequences, it is to be understood that the DNA
sequence is:
(1) that obtained by linking the 3' terminus of the sequence
corresponding to nucleotide positions 996 to 1149 in SEQ ID NO:1 to the 5'
terminus of the sequence corresponding to nucleotide positions 2067 to 3079 in
SEQ ID NO:1; and
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(2) that obtained by linking the 3' terminus of the sequence
corresponding to nucleotide positions 1947 to 1959 in SEQ ID NO:1 to the 5'
terminus of the sequence corresponding to nucleotide positions 2067 to 3079 in
SEQ ID NO:1.
Another set of preferred DNA sequences includes the DNA having a
sequence corresponding to nucleotide positions 996 to 3079 in SEQ ID NO:1
and the DNA having a sequence corresponding to nucleotide positions 1947 to
3079 in SEQ ID NO:1.
The cloning of the full length DNA sequence shown in SEQ ID NO:1 is
described in great detail in the Examples below. The shorter length fragments
of
this sequence as well as the other DNA sequences of the present invention may
be obtained by conventional techniques, such as solid state DNA synthesis or
site-directed mutagenesis (Maniatis, T., et al, Molecular
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Cloning: A Laboratory Manual, cold Spring Harbor Press, Cold
Spring Harbor, NY (1989) and Ausebel, F.M., et al, Current
Protocols in Molecular Biology, John Wiley & Sons, NY (1989)).
In yet another embodiment, the present invention provides
novel plasmids or vectors which contain a DNA sequence
according to the present invention. The present plasmid may
be either a cloning vector or an expression plasmid. Suitable
plasmids or vectors are those obtained by inserting a DNA
sequence of the present invention into a plasmid or vector
such as pCDM8, pcDNA1, pREPB, pCEP4, PTZ18, etc. In the case
of an expression plasmid, the DNA sequence of the present
invention is preferably inserted into the plasmid downstream
from a promoter and in the correct reading frame and
transcriptional orientation. The insertion of a DNA sequence
according to the present invention into any conventional
expression plasmid in the correct reading frame and
transcriptional orientation and the insertion of a DNA
sequence of the present invention into a conventional cloning
vector can easily be accomplished by the skilled artisan using
conventional recombinant DNA technology.
The present invention also provides transformed cells
which contain a plasmid or vector according to the present
invention. Suitable host cells include any mammalian cell.
Preferred host cells include Chinese hamster ovary cells, COS
cells, etc. The transformation of such host cells with a
plasmid or vector according to the present invention may be
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carried out using ccnventional techniques 41-,usebel, F.M., et
al, Current Protocols in Molecular Bioloav, John Wiley & Sons,
NY (1989).
In a further embodiment, the present invention provides a
method for producing an enzyme of the present invention by
culturing in a culture medium a transformed cell according to
the present invention for a time sufficient to produce the
enzyme. Preferably, the cell has been transformed with an
expression plasmid such as cDNA5, cDNA6, cDNA10, or cDNA 14,
shown in Figure la, into which one embodiment of the present
DNA has been inserted. The particular culture conditions,
such as temperature, medium, etc., will depend on the type and
identity of the transfcrmed cell. However, the selection of
appropriate conditions is well within the abilities of the
skilled artisan. For example, suitable culture conditions and
media for a variety of cell types are taught in Ausebel, F.M.,
et al, Current Protccols in Molecuiar Biology, John Wiley &
Sons, NY (1989).
In another embodiment, the present invention provides
novel monoclonal or polyclonal antibodies which specifically
bind to the present enzymes. Such antibodies may be produced
using conventional methods such as described in Harlow, E. et
al, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1988). Preferably, the present
antibodies are monoclonal antibodies. The present antibodies
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may be labelled with any conventional label, such as a
radiolabel, a chromophore (e.g., a fluorescent label), or an
enzyme (e.g., horseradish peroxidase).
The present invention also provides novel immunoassays
for the detection and/or quantitation of the present enzymes
in a sample. The present immunoassays utilize one or more of
the present monoclonal or polyclonal antibodies which
specifically bind to the present enzymes. Preferably the
present immunoassays utilize a monoclonal antibody. The
present immunoassay may be a competitive assay, a sandwich
assay, or a displacement assay, such as those described in
Harlow, E. et al, Antibodies. A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY (1988) and may
rely on the signal generated by a radiolabel, a chromophore,
or an enzyme, such as horseradish peroxidase.
The DNA sequences, enzymes, plasmids, cells, and methods
of the present invention have a number of uses. Thus, the
present invention provides previously undiscovered murine DNA
sequences that encode specific and heretofore undiscovered
protein sequences capable of functioning as a GDP-
Fuc:NeuNAcac(2,3)-S-D-Gal(1,4)-D-G1cNAc c(1,3)-
fucosyltransferase. These enzymes, when expressed by the
present DNA sequence, function within mammalian cells to
generate de novo expression of specific cell surface
glycoconjugate structures on those cells. These structures
are recognized by an antibody against the sialyl Lewis x
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determinant {NeuAcc(2,3)Ga1B(1,4)[Fuca(1,3)]G1cNAc-R}, where R
is an underlying glycoconjugate-glycoprotein, glycolipid, free
oligosaccharide, or hydroxyl group. It has been demonstrated
that these enzymes do not generate de novo expression of
specific cell surface glycoconjugate structures known as the
stage specific embryonic antigen I (SSEA-1 or Lewis x;
structure Ga1B(1,4)[Fuc c(1,3)]G1cNAc), the Lewis a structure,
or the sialyl Lewis a structure. This enzyme, when expressed
by the cloned DNA sequence described here, has also been shown
to function in the enzymatic manner implied in its name, when
assayed in extracts prepared from cells that express the DNA
sequence. The oligosaccharide products of this enzyme
represent fucose linked in alpha 1,3 configuration to the
G1cNAc residue of an a(2,3)sialylated "type II" lactosamine
acceptor. This product is hereinafter referred to as the
sialyl Lewis x determinant. For convenience, this reaction is
shown in the equation given below:
NeuNAca(2,3)Ga1A(1,4)G1cNAc ----* NeuNAca(2,3)Ga1p(1,4)[Fuca(1,3)]G1cNAc
Fuc-TVII
and sialyl Lewis X
GDP-Fuc(ose)
The transfer of GDP-fucose to NeuNAca(2,3)Gal [SO4-
6]G1cNAc proceeds analogously.
This enzyme may also be able to utilize other
oligosaccharide precursors similar or identical to those shown
below, wherein the underlined Gal or Ga1NAc moiety is
substituted with another molecule (mono- or oligosaccharide, a
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modified sialic acid moiety, or sulfate, for example) and
transfer fucose in c(1,3)linkage to the G1cNAc residues on
these molecules also referred to hereinafter as sialyl Lewis X
for simplicity's sake:
Gala(1,3)Ga1B(1,4)G1cNAc-R
NeuNAca(2,3)[Ga1NAcB(1,4)]Ga1B(1,4)G1cNAc-R
NeuNAca(2,3)Ga1NAcB(1,4)G1cNAc-R
N-glycolylNeuoc(2,3)Ga1B(1,4)GlcNAc-R
SO4-3-Ga B(1,4)G1cNAc-R
NeuNAca(2,3)[SO4-6]Gal (1,4)G1cNAc-R
NeuNAcoc(2,3)[SO4-6]Gal (1,4)[SO4-6]G1cNAc-R
wherein R is a N-linked or 0-linked oligosaccharide moiety or
backbone.
The location of the catalytic domain of the present
enzyme is inferred by comparison to the catalytic domain of
several structurally similar enzymes. Specific utilities
include:
i. Construction of animal cell lines with specific
capabilities with respect to post-translational modification
of the oligosaccharides on cell-surface, intracellular, or
secreted proteins or lipids by sialyl Lewis x determinants
that represent the products of this enzyme (for the production
of diagnostics and therapeutics by the biotechnology
industry).
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24
Specifically, the cloned DNA sequences described here may
be introduced by standard technologies into a mammalian cell
line that does not normally express the cognate enzyme cr its
product (sialyl Lewis x determinants on oligosaccharides), but
which does maintain expression of the appropriate
oligosaccharide precursors(s), and GDP-fucose, and transcribed
in that cell in the "sense" direction, to yield a cell line
capable of expressing sialyl Lewis x determinants on
oligosaccharides cn cell-surface, intracellular, or secreted
proteins or lipids. Alternatively, these cloned DNA sequences
may be introduced by standard technologies into a mammalian
cell line that does express the cognate enzyme and its product
(sialyl Lewis x determinants), and transcribed in that cell in
the "anti-sense" direction, to yield a cell line incapable of
expressing sialyl Lewis x determinants on cell-surface,
intracellular, or secreted proteins or lipids. The
introduct=on and "anti-sense" transcription cf the nresent DNA
sequences in a cell may be carried out using conventional
methods as described in Maniatis, T., et al, Molecular
Cloning: A Laboratory Manual, Cold spring Harbor Laboratory
Press, Cold Spring Harbor, NY (1989). Alternatively, the endogenous GDP-
Fuc:NeuNAca(2,3)-S-D-Gal(1,4)-D-GlcNAc a(1,3)-
fucosyltransferase gene(s) , in a mammalian cell expressing the
cognate enzyme(s) , might be inactivated with the present DNA
sequence by homologous recombination techniques, or by "anti-
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sense" oligonucleotide approaches using the present DNA
sequences, or by dominant negative mutant fucosyltransferase
sequences that inactivate endogenous GDP-Fuc:NeuNAca(2,3)-8-D-
Gal(1,4)-D-G1cNAc a(1,3)-fucosyltransferase(s), derived via
mutagenesis and genetic selection schemes, from the present
DNA sequences, or from structurally related but otherwise wild
type a(1,3)fucosyltransferases.
This method may be used to construct animal cell lines
that are suitable host cells for the production of diagnostic
or therapeutic materials whose usefulness or efficacy depends
upon the specific post-translational modification determined
by the present cloned DNA sequences and their cognate enzymes.
For example, it is known that the biological effectiveness of
many therapeutic proteins or peptides, recombinant or
otherwise, depends critically upon the oligosaccharide
structure(s) that are covalently attached to them. The
structure of these oligosaccharides is primarily a function of
the number and kind of glycosyltransferase enzymes that are
found in the cell used to produce these therapeutic products.
Animal cells and some yeasts are competent to perform these
glycosylation reactions; however, not all glycosyltransferase
enzymes are produced by every animal cell or yeast, and
therefore, some oligosaccharide structures (including sialyl
Lewis x determinants generated by the present enzyme) are not
produced by them. The converse is also true, namely, that
producing cells may express a glycosyltransferase analogous
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to, or identical to, the GDP-Fuc:NeuNAcc(2,3)-f3-D-Gal(1,4)-D-
G1cNAc a(1,3)-fucosyltransferase encoded by the DNA sequence
described here. It is likely that sialyl Lewis x determinants
may alter the bioactivity (for better or for worse) of natural
or recombinant therapeutic or diagnostic agents (glycoproteins
or glycolipids) produced by mammalian or other eukaryotic
hosts. Eukaryotic host cells that the biotechnology industry
uses to produce these recombinant agents may be altered with
the DNA sequence information and related information described
in this invention, to add sialyl Lewis x determinants to the
oligosaccharides on recombinant products by expressing all or
part of the cloned sequences described here in the desired
host. Alternatively, sialyl Lewis x determinants may be
eliminated from the product produced in these host cells by
the use of transfected '!anti-sense" vector constructs,
recombination-based gene inactivation, "anti-sense"
oligonucleotide approaches, or dominant negative mutant
fucosyltransferases, outlined above.
The old "methods" used for this process include an
empirical approach to identify a cell line that does or does
not express this particular enzyme or an enzyme that functions
in a similar or identical manner, for the production of the
appropriately modified recombinant or natural product. This
is not always optimal since cell lines with this particular
post-translation modification capabilities may not exist
naturally, or may not be especially suited to high level
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production of an appropriately modified product.
Alternatively, unwanted sialyl Lewis x determinants present on
a therapeutic material produced by an empirically identified
animal cell line must be removed chemically or enzymatically,
a process that may be costly or inefficient. The advantages
of using the cloned, functional present DNA sequences in
conjunction with the technologies outlined above, relative to
these older methods include the ability to construct lines
that specifically lack the capability to generate sialyl Lewis
x determinants on the oligosaccharides of glycoproteins or
glycolipids. Properly constructed, these cell lines will
eliminate any need for chemical or enzymatic treatment of a
therapeutic or diagnostic material to remove unwanted sialyl
Lewis x determinants. Moreover, in the event that sialyl
Lewis x determinants are found to be desirable for a
particular diagnostic or therapeutic product produced by
animal cells, cell lines may be engineered with the present
cloned DNA sequence to generate these residues.
ii. Isolation of reagents suitable for efficient
enzymatic synthesis and production of oligosaccharides (in
enzyme reactors, for example).
Oligosaccharides may have therapeutic utility as
immunomodulatory reagents in the field of organ
transplantation. In particular, soluble and solid-phase
oligosaccharides may find use as therapeutic agents with which
to block or ameliorate antibody-mediated organ transplant
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rejection in cases involving incompatibility due to
differences in the major blood group antigen systems of the
organ donor and the recipient, including the Lewis blood group
system. Likewise, soluble oligosaccharides may find use as
therapeutic agents that function by blocking attachment of
bacterial, viral, or parasitic pathogens to glycoconjugate
"receptors" found on the surface of the animal tissues that
these pathogens invade. For example, there is evidence that
portions of the Lewis blood group oligosaccharide antigens
serve as "receptors" for some forms of uropathogenic bacteria.
Moreover, such glycoconjugates have been implicated in
modulating adhesive events between cells, and between cells
and their environment during developmental and differentiation
processes. These events include binding of spermatozoa to
eggs, and the initial events that mediate attachment of
fertilized ova to the uterine wall at the beginning of
implantation. These observations suggest, for example, the
possibility that contraceptive uses for (biologically
"natural") oligosaccharide molecules might exist. In
addition, specific glycoconjugates containing sialyl Lewis x
determinants have been implicated as ligands for the Selectin
family of adhesion molecules, that play important roles in
mediating adhesion between cells of the immune system, and
some tumor cells, and the surfaces of the endothelial cells
that line the vascular tree. Published observations confirm
that the cloned fucosyltransferase sequence described here
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could be used to construct oligosaccharide-type molecules,
with pharmaceutical properties possessing anti-inflammatory
and anti-tumor metastatic functions, and that can prevent
life-threatening tissue damage in acute lung disease (ARDS)
and in tissue re-perfusion, after myocardial infarction, for
example (see, e.g., Buerke, M., et al, J. Clin. Invest., vol.
93, pp. 1140-1148 (1994); Mulligan, M.S., et al, Nature, vol.
364, pp. 149-151 (1993); and Mulligan, M.S., et al, J. Exn.
Med., vol. 178, pp. 623-631 (1993)).
Currently, pharmaceutically useful amounts of
oligosaccharides containing sialyl Lewis x determinants are
produced by chemical synthesis (a procedure that is
inefficient and costly), by isolation from natural sources
(using costly and inefficient procedures that often require
the processing of large quantities of animal or plant
material, and the purification of the desired oligosaccharide
from other contaminating oligosaccharides), or by
glycosyltransferase-assisted synthetic procedures, using other
recombinant a-(1,3)fucosyltransferases. The invention
described here provides a mechanism to synthesize abundant
quantities of purified GDP-Fuc:NeuNAca(2,3)-B-D-Gal(1,4)-D--
G1cNAc a(1,3)-fucosyltransferase. This could be used to
construct an enzyme bioreactor (enzyme in solution or
immobilized on a solid phase matrix, for example via the
protein-A moiety fused to the catalytic domain of the enzyme,
as described in Ball, et al, J. Am. Chem. Soc., vol. 114, pp.
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5449-5451 (1992) capable of enzymatic synthesis of structures
containing sialyl Lewis x determinants. This may be more
efficient than approaches involving chemical synthesis of
structures containing sialyl Lewis x determinants or their
purification from natural sources, or by using other known
ac(1,3)fucosyltransferases, for a variety of reasons. One, the
only chemicals necessary would be the enzyme substrates; these
are easily obtained or synthesized. Two, enzymatic synthesis
of such structures will produce only the desired product and
the nucleotide diphosphate product of substrate hydrolysis.
This latter chemical is found as the natural by-product of
these reactions in animal cells, is relatively non-toxic, and
may be easily separated from the oligosaccharide synthetic
product. By contrast, chemical synthetic procedures typically
generate numerous products of side reactions which must be
removed, and which may be toxic as'well. Similarly,
purification of oligosaccharides from natural sources requires
the removal of other contaminating oligosaccharides present in
the natural material. Three, enzymatic catalysis is
extraordinarily efficient; nearly complete conversion of
substrate to product can be achieved. By contrast, chemical
synthesis of sialyl Lewis x determinants on oligosaccharides
is a multi-step process; yields at each step may be much less
than 100%, and the cumulative efficiency of current chemical
synthesis procedures does not approach the efficiency possible
with enzymatic synthesis. Similarly, purification of
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oligosaccharides with sialyl Lewis x determinants from natural
materials can entail significant losses inherent to the
purification procedures required to separate the desired
oligosaccharide from contaminating, irrelevant
oligosaccharides, with inefficient isolation of the desired
oligosaccharide. The GDPFuc:NeuNAca(2,3)-f3-D-Gal(1,4)-D-
G1cNAc a(1,3)-fucosyltransferase encoded by the present DNA
sequence has never been previously identified in animal
tissues. In theory, however, this activity may be partially
purified from animal tissues for synthetic use. These
purifications are themselves typically inefficient, however,
primarily because such enzymes are typically present in very
low abundance. This invention provides two mechanisms that
may provide for the abundant production of this enzyme.
First, this may be done through the construction and selection
of animal cells that produce relatively large quantities of
the enzymes. Alternatively, present nucleic acid sequences
may then be used with standard recombinant DNA technologies to
produce large quantities of glycosyltransferases in mammalian
host cells, fungi, yeasts, or using other eukaryotic cell-
based systems (i.e. baculovirus mammalian host), or in
prokaryotic hosts. Furthermore, the sequence encoding this
enzyme may be modified via standard molecular cloning schemes
or mutagenesis to yield a recombinant fucosyltransferase with
novel properties that make it more desirable than the wild-
type enzyme. For example, the modifications might be made to
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the enzyme that make it more stable, more suitable for
immobilization in a bioreactor, or more catalytically
efficient for a particular substrate.
iii. Isolation of reagents suitable for producing
recombinant GDP-Fuc:NeuNAca(2,3)-B-D-Gal(1,4)-D-G1cNAc a(1,3)-
fucosyltransferase to be used directly as a research reagent,
or to be used to generate antibodies against the GDP-
Fuc:NeuNAca(2,3)-f3-D-Gal(1,4)-D-G1cNAc a(1,3)-
fucosyltransferase, for research applications.
The present invention provides two methods for producing
large quantities of the present enzymes (see ii. above - i.e.
specially constructed animal cells, or via natural or
synthetic genes encoding these enzymes) which may be used as a
research tool with which to study the structures and functions
of oligosaccharides and glycoproteins. Likewise, the enzymes
produced by this method, or the nucleic acid sequences and
derived protein sequences provided by this method, may be used
to generate antibodies to this enzyme (via synthetic peptides
or recombinant protein). These antibodies might also be used
as research reagents to study the biosynthesis and processing
of these enzymes, and might be used as an aid in their
purification for all the uses described herein.
iv. Antibodies to glycosyltransferases as diagnostic
reagents.
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Aberrant expression of a(1,3)fucosyltransferases has been
associated with malignancy in humans, indicating that this
enzyme may serve as a tumor marker for early detection of
malignancy involving a number of human tissues. Enzyme tumor
markers have typically been assayed in body fluids by activity
assays, which may be subject to non-specificity due to
competing glycosyltransferase activity. These assays may also
be insensitive since it is possible that inactive enzymes
might be useful as tumor markers but would not be detected by
enzyme activity assays. This invention provides a mechanism
for generating antibodies to this enzyme (monoclonal and
polyclonal antibodies against synthetic peptides constructed
from information derived from cloned DNA sequence encoding
GDP-Fuc:NeuNAca(2,3)-S-D-Gal(1,4)-D-G1cNAc c(1,3)-
fucosyltransferase, or against the recombinant enzyme produced
by eukaryotic or prokaryotic hostsj. Antibodies specific for
this GDP-Fuc:NeuNAcac(2,3)-B-D-Gal(1,4)-D-G1cNAc a(1,3)-
fucosyltransferase so produced may be used to detect and
quantitate this glycosyltransferases in body fluids, with
specificity and sensitivity exceeding enzyme activity assays,
serving as a method for the early detection of malignancy.
v. Recombinant enzyme for use in screening natural and
synthetic compounds for fucosyltransferase inhibitors or
inactivators.
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It is known that the sialyl Lewis x determinant is an
essential component of counterreceptors for the E-selectin and
P-selectin. Related compounds are candidate ligands for L-
selectin. These receptor-counter-receptor pairs operate to
enable leukocytes (neutrophils, monocytes, eosinophils,
lymphocytes in general (in recirculatary events), and some
kinds of T lymphocytes) to leave the vascular tree and
participate in normal inflammatory events in humans, or in
pathological inflammatory events in humans (like ARDS, tissue-
reperfusion injury, and a host of other such events). Since
~
the present GDP-Fuc:NeuNAcac(2,3)-B-D-Gal(1,4)-D-G1cNAc c(1,3)-
fucosyltransferase gene is expressed in human leukocytes and
plays a central role in sialyl Lewis x biosynthesis,
pharmacologic inhibitors of this enzyme will diminish
leukocyte sialyl Lewis x expression and thus act as anti-
inflammatory pharmaceutical agents for use in humans or other
animals in acute and chronic selectin-dependent inflammatory
states. The GDP-Fuc:NeuNAcac(2,3)-B-D-Gal(1,4)-D-G1cNAc
a(1,3)-fucosyltransferase described here represents a tool for
identifying compounds that inhibit this enzyme, either through
"screening" methods to identify such compounds in natural
product or chemical libraries (using recombinant enzyme or
cell lines expressing this enzyme, in screening assays), or
through "rational drug design" strategies (via solution of the
enzyme's tertiary structure with the aid of recombinant
enzyme, followed by design or identification of molecules that
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inhibit the enzyme's catalytic activity or other essential
function).
A number of studies have noted an association between
increased numbers of cell surface sialyl Lewis x determinants
on oligosaccharides of a cell and the ability of that cell to
metastasize in a malignant fashion. If there is a causal
relationship here, then drugs that inhibit the present enzyme
may be used as anti-tumor agents. The reagents described in
this disclosure may prove useful for screening compounds for
antifucosyltransferase activity, since the cloned sequence may
~
be used with standard techniques to produce relatively large
amounts of pure fucosyltransferase. This will aid in
screening since the effects of potential inhibitors will be
tested on a pure enzyme, without the confounding effects that
may occur in whole cell extracts or with partially purified
enzyme.
vi. Engineering of glycosyltransferase substrate
specificity to generate novel glycoconjugate structures on
secreted or cell-associated glycoconjugates.
The present invention provides a cloned GDP-
Fuc:NeuNAca(2,3)-6-D-Gal(1,4)-D-G1cNAc a(1,3)-
fucosyltransferase gene that, when used with appropriate
mutagenesis and genetic selection schemes, may allow the
generation of mutant GDP-Fuc:NeuNAca(2,3)-B-D-Gal(1,4)-DG1cNAc
a(1,3)-fucosyltransferases that generate glycosidic linkages
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different from that generated by the wild-type enzyme. These
novel linkages may or may not be naturally occurring, and
could find utility as moieties that enhance bioactivity of the
molecules to which they are attached. Directed mutagenesis
procedure may also be considered since this enzyme maintains
primary sequence similarity to other
a(1,3)fucosyitransferases, yet exhibits a distinct set of
acceptor substrate utilization properties. Alternatively,
mutagenesis and selection approaches may be used to generate
mutant GDP-Fuc:NeuNAca(2,3)-B-D-Gal(1,4)-D-G1cNAc a(1,3)-
fucosyltransferases that act in a dominant negative fashion.
The dominant negative mutants so generated may be used to
inactivate endogenous glycosyltransferase activities when the
product(s) of such an enzyme are not desired. Mutant GDP-
Fuc:NeuNAca(2,3)-B-D-Gal(1,4)-D-G1cNAc a(1,3)-
fucosyltransferases may also be generated, for example, that
function as fucosidases that hydrolyze various sugar linkages
(fucose, mannose, or others) from oligosaccharides in vitro
and in vivo.
vii. Genotyping individuals at this fucosyltransferase
locus.
Absence of a fucosyltransferase similar to the one
encoded by the DNA sequence detailed here has been found in
several families. Should such absence be associated with a
detrimental phenotype, DNA sequence polymorphisms, including
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restriction fragment length polymorphisms, within or linked to
the gene corresponding to this cloned gene segment may be used
to genotype individuals at this locus, for the purpose of
genetic counseling. Likewise, the molecular basis for such
detrimental phenotypes might be elucidated via the study of
the gene segment described here, should it be causally-related
to such phenotypes.
viii. Identification and production of inhibitors that
operate as anti-inflammatory pharmaceuticals.
Much effort is being exerted trying to identify compounds
that will inhibit sialyl Lewis x formation in human white
cells, since such molecules, if non-toxic, will act as anti-
inflammatory pharmaceuticals by preventing the synthesis of
sialyl Lewis x in white cells, thus rendering the cells unable
to bind to inflamed vascular endothelium (via selectins)
during inflammation, and thus unable to participate in
extravascular inflammatory activities. One candidate target
for such molecules is the Fuc-TVII enzyme. This is a better
candidate than any of the other known fucosyltransferases
since it is the only known fucosyltransferase that is
expressed in meaningful quantities in white cells and in high
endothelial venules in lymphoid aggregates, that can also
synthesize the sialyl Lewis x determinant. Furthermore, the
leukocytes of mice genetically engineered for a deficiency in
this enzyme are deficient in expression of functional ligands
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for E-selectin and P-selectin, and exhibit a concomitant
immune deficiency characterized by a deficit in leukocyte
mobilization to inflammatory sites. These observations
demonstrate that Fuc-TVII controls leukocyte selectin ligand
expression, and indicate that inhibition of this enzyme by
pharmaceutical agents or maneuvers will represent an anti-
inflammatory approach that may have therapeutic benefit in
human disease where selectin-dependent inflammation is
pathologic. The availability of recombinant Fuc-TVII will
facilitate identification of such inhibitory compounds since
it may be used in high throughput assays to screen chemical
libraries and natural product libraries for inhibitors.
Moreover, the recombinant form of the enzyme, or a derivative
of the enzyme made via recombinant techniques, may be used to
determine its tertiary conformation, including the shape of
functionally important surfaces (i.e. GDP-fucose binding
pocket, metal binding pocket, oligosaccharide acceptor
substrate binding pocket, etc.). One could then use "rational
drug design" approaches to identify or synthesize molecules
with morphological complementarity that act as inhibitors.
ix. A "one-pot", in vitro synthesis of the sialyl Lewis
x tetrasaccharide.
The sialyl Lewis x tetrasaccharide, and some derivatives,
have been shown to operate as anti-inflammatory molecules in
animal models of selectin-dependent inflammation (see, e.g.,
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M. Buerke et al, J. Clin. Invest., vol. 93, pp. 1140-1148
(1994); M.S. Mulligan et al, Nature, vol. 364, pp. 149-151
(1993); and M.S. Mulligan et al, J. Exp. Med., vol. 178, pp.
623-631 (1993)). These molecules are now in clinical trials.
Chemical synthesis of the sialyl Lewis x molecule is extremely
expensive. Glycosyltransferase-assisted synthetic procedures
have therefore been developed, in which recombinant
glycosyltransferases are used to effect specific and efficient
synthesis of the sialyl Lewis x moiety. These systems involve
additional enzymes (not shown in the schemes below) that
continuously form the nucleotide sugar substrates necessary to
the glycosyltransferases (see Ichikawa et al, J. Am. Chein.
Soc., vol. 114, p. 9283 (1992). Because of the nature of the
previously existing enzymes, the conventional synthetic scheme
had to proceed in a "two-pot" synthesis (see below), where the
sialyated product of the first pot would have to be separated
from the non-sialylated intermediate product (of Rxn #1), and
then added to a second "pot" where Rxn #3 could take place.
This was necessary in order to avoid a competing reaction
(Rxn#4), since the available fucosyltransferases (i.e Fuc-
TIII, Fuc-TV, and Fuc-TVI) would transfer to Ga1Q(1,4)G1cNAc
(to form Lewis x) and also to NeuNAca(2,3)Ga10(1,4)G1cNAc (to
form sialyl Lewis x). Since Lewis x cannot be sialylated by
any known a(2,3)sialyltransferases (Rxn #5), the formation of
Lewis x would effectively diminish the yield by depleting
substrate for Rxn #2.
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Rxn #1 Rxn #2
/3(1,4)Gal-T a(2,3)sialyl-T
Pot #1 G1cNAc -----> Gal/3(1,4)G1cNAc ------>NeuNAca(2,3)Galp(1,4)G1cNAc
Rxn #4 (Undesirable: avoided by doing 2 pot synthesis)
a(1,3)fucosyl-T
Galp(1,4)G1cNAc ------>GalA(1,4)(Fuca(1,3)]G1cNAc
Lewis x
Rxn #5 (does not produce a product)
a(2,3)sialyl-T
Galp(1,4)[Fuca(1,3)]G1cNAc -----> No product
Rxn #3
a(1,3)fucosyl-T
Pot #2 NeuNAca(2,3)Galp(1,4)GlcNAc ---->
NeuNAca(2,3)Galp(1,4)[Fuca(1,3)]GlcNAc
sialyl Lewis x
This problem can be circumvented with Fuc-TVII. This
enzyme cannot operate on Galp(1,4)GlcNAc, so it can be
included in the first pot. It will not operate until the
product of Rxn #2 is made, at which time it will complete
rxn #3. Thus, Fuc-TVII makes possible the one-pot
synthesis shown below:
One-pot synthesis
Rxn #1 Rxn #2
p(1,4)Gal-T a(2,3)sialyl-T
GIcNAc -----> Gal(3(1,4)G1cNAc ----- >NeuNAca(2,3)Ga1p(1,4)G1cNAc
Rxn #3
a(l,3)fucosyl-T
(Fuc-TVII)
NeuNAca(2,3)Galp(1,4)(Fuca(1,3)]G1cNAc
sialyl Lewis x
Other features of the invention will become apparent
in the course of the following descriptions of exemplary
embodiments which are given for illustration of the
invention and are not intended to be limiting thereof.
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EXAMPLES
EXPERIMENTAL PROCEDURES
Cell Culture. The sources and growth conditions for
COS-7 cells (K.M. Gersten et al, J. Biol. Chem., vol. 270,
pp. 25047-25056 (1995)), CHO-Tag cells (P.L. Smith et al,
J. Biol. Chem., vol. 269, pp. 15162-15171 (1994)), and
cultured murine blood cell lines (B cell line S107, (M.L.
Atchison et al, Cell, vol. 48, pp. 121-128 (1987)); T cell
line EL4, (L.J. Old et al, Cancer Res., vol. 25, pp. 813-
819 (1965)); B cell hybridoma line TH2.54.63, (T. Hamano et
al, J. Immunol., vol. 130, pp. 2027-2d32 (1983)); B cell
hybridoma line 180.1, (M. Hummel et al, J. Innnunol., vol.
138, pp. 3539-3548 (1987)); Friend murine erythroleukemia
cell line MEL, (D. Singer et al, Proc. Natl. Acad. Sc;.
U.S.A., vol. 71, pp. 2668-2670 (1974); B.L. Weber et al,
Science, vol. 249, pp. 1291-1293 (1990)); macrophage cell
line RAW264.7, (P. Ralph et al, J. Immunol., vol. 119, pp.
950-954 (1977); W.C. Raschke et al, Cel , vol. 15, pp. 261-
267 (1978)); macrophage cell line P388D1, (H.S. Koren et
al, J. Immunol., vol. 114, pp. 894-897 (1975)); and the
cytotoxic T-cell line 14-7fd, (T.J. Braciale et al, J. Exn.
Med., vol. 153, pp. 910-923 (1981); M.E. Andrew et al, J.
Immunol., vol. 132, pp. 839-844 (1984)).
Antibodies. The sources of the monoclonal antibodies
used here have been described previously (anti-Lewis
x/anti-SSEA-1, (D. Solter et al, Proc. Natl. Acad. Sci.
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U.S.A., vol. 75, pp. 5565-5569 (1978)); anti-H and anti-
Lewis a, (K.M. Gersten et al, J. Biol. Chem., vol. 270, pp.
25047-25056 (1995)); anti-sialyl Lewis x/CSLEX,
(K. Fukushima et al, Cancer Res.. vol. 44, pp. 5279-5285
(1984)); anti-sialyl Lewis a, (D. Chia et al, Cancer Res.,
vol. 45, pp. 435-437 (1985)); anti-VIM-2 antibody, (B.A.
Macher et al, J. Biol. Chem., vol. 263, pp. 10186-10191
(1988)); fluorescein-conjugated goat anti-mouse IgM and IgG
antibodies; Sigma). MECA-79 (P.R. Streeter et al, J. Cell
Biol., vol. 107, pp. 1853-1862 (1988)) was the generous
gift of Drs. Louis Picker and Eugene Butcher (Stanford
University).
cDNA cloning. Mouse Fuc-TVII cDNAs were isolated from
a cDNA library constructed from the mouse cytotoxic T cell
line 14-7fd (P.L. Smith et al, J. Biol. Chem., vol. 269,
pp. 15162-15171 (1994)), using colony hybridization
procedures (T. Maniatis et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY (1982)) and a segment of the mouse Fuc-
TVII gene (Fig. 2) corresponding to nucleotides 2053-2285.
Murine genomic library screening. Approximately
1.0 x 106 recombinant lambda phage from a genomic library
prepared from mouse 3T3 cell DNA (Stratagene) were screened
by plaque hybridization, using a 324 bp segment of the
human Fuc-TIII gene (nucleotides 571-894), and low
stringency hybridization procedures described previously
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(K.M. Gersten et al, 7. Biol. Chem., vol. 270, pp. 25047-
25056 (1995); K.M. Gersten et al, Doctoral Thesis, pp. 66-
98, University of Michigan, Ann Arbor, Michigan (1995)).
DNA from a phage with a unique restriction pattern was
digested with SacI and a 2.6 kb fragment which cross-
hybridized with the human Fuc-TIII probe was gel purified
and cloned into the Sacl site of pTZ19R (Pharmacia LKB
Biotechnology, Inc.). A representative subclone containing
a single ?nsert was designated pMFuc-TVII. The DNA
sequence of the 2.6 r:b insert was determined by the dideoxy
uhain termination method (F. Sanger et al, Proc. Nat1.
Acad. Sci. 'J.S.A., vol. 74, pp. 5463-5467 (1977)) using T7
DNA polymerase (Sequenasn~* United States Biochemical Corp.)
and oligonucleotide primers synthesized according to
flanking plasmid seauences. Sequence data was used to
design additional svnthetic primers which were then
utilized to sequence the remaining portion of the SacI
insert ;n cMFuc-TVII. Sequence analysis was perfcrmed
using the sequence analysis software package of the
University of Wisconsin Genetics Computer Group (GCG)
(J. Devereux et al, Nucleic Acids Res., vol. 12, pp. 387-
395 (1984)) and the MacVector version of the IBI Pustell
Sequence Analysis Software package (IBI). Sequence
alignments were assembled with the Gap function of the GCG
package.
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Transfection and analysis of COS-7 cells and CHO-Taa
cells. COS-7 cells were transfected with various Fuc-TVII
expression vectors using a DEAE-dextran transfection
procedure previously described (J.F. Kukowska-Latallo et
al, Genes & Dev., vol. 4, pp. 1288-1303 (1990); T. Maniatis
et al, Molecular Clonina= A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY (1982)). CHO-Tag
cells were transfected with plasmid DNAs using a liposome-
based reagent (N-(2,3-dioleoyloxy)propyl]-N,N,N-
trimethylammonium methylsulfate, DOTAP, Boehringer
Mannheim), as modified previously (P.L. Smith et al, Js
Biol. Chem., vol. 269, pp. 15162-15171 (1994)).
Transiently transfected cells were harvested 72 hours
after transfection, and were stained with monoclonal
antibodies diluted in staining media, as previously
described (B.W. Weston et al, J. Biol. Chem., vol. 267, pp.
24575-24584 (1992)). Anti-Lewis a, anti-H, and anti-sialyl
Lewis x antibodies were used at 10 g/ml. Anti-Lewis x was
used at a dilution of 1:1000. Anti-sialyl Lewis a was used
at a dilution of 1:500. Anti-VIM-2 antibody was used at a
dilution of 1:200. Cells were then stained with
fluorescein isothiocyanate-conjugated goat anti-mouse IgM
or anti-mouse IgG and subjected to analysis on a FACScan
(Becton-Dickinson) as previously described (B.W. Weston et
al, J. Biol. Chem., vol. 267, pp. 24575-24584 (1992)).
Cells were also co-transfected with the plasmid pCDM8-CAT
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(P.L. Smith et al, J. Biol. Chem., vol. 269, pp. 15162-
15171 (1994)), and extracts prepared from these cells were
subjected to chloramphenicol acetyltransferase activity
assays (P.L. Smith et al, J. Biol. Chem., vol. 269, pp.
15162-15171 (1994)), to allow for normalization of flow
cytometry and Western blot data to transfection efficiency.
Fucosyltransferase assays. COS-7 cells transiently
transfected with Fuc-TVII expression vectors were harvested
72 hours after transfection, and extracts were prepared
from these cells, exactly as previously described (J.F.
Kukowska-Latallo et al, Genes & Dev., vol. 4, pp. 1288-1303
(1990); B.W. Weston et al, J. Biol. Chem., vol. 267, pp.
4152-4160 (1992)). These extracts were subjected to
a(1,3)fucosyltransferase assays (B.W. Weston et al, J.
Biol. Chem., vol. 267, pp. 24575-24584 (1992)), assembled
in a total volume of 20 i. Reaction mixtures contained 3
M GDP-[14C,fucose, 20 mM acceptor (N-acetyllactosamine,
lactose, lacto-N-biose I, 2'-fucosyllactose (Sigma), or
3' sialyl N-acetyllactosamine (Oxford Glycosystems)), 50 mM
cacodylate buffer, pH 6.2, 5 mM ATP, 10 mM L-fucose, 15 mM
MnC12 and a quantity of cell extract protein sufficient to
yield approximately linear reaction conditions (consumption
of less than 15% of the GDP-fucose substrate) during the
course of the reaction (1 h). Control reactions were
prepared by omitting the acceptor in the reaction mixture,
and values obtained with these reactions were subtracted
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from the corresponding acceptor-replete reaction. This
background radioactivity reproducibly represented less than
1$ of the total radioactivity in the assays, and
corresponds to free [14C]fucose present in the
GDP-L14C]fucose as obtained from the manufacturer.
Identical enzyme prenarations were used in assays for the
determination cf enzyme activity with different accemtor
substrates.
Reactions containing neutral accentors
(N-acetyllactcsamine, lactose, lacto-N-biose I,
2'-fucosyllactcse, all from Sigma) were terminated by the
addition of 20 l ethanol and 560 l water. Samples were
centrifuged at 15,000 x g for 5 minutes and a 50 l aliquot
was subjected to scintillation counting to determine the
total amount of radioactivity in the reaction. An aliquot
of 200 ul was applied to a column containing 400 p1 of
Dowex*1X2-400, ornate form (J.F. riukowska-Latallo et al,
Genes & Dev., vol. 4, pp. :288-1303 (1990); B.W. Weston et
al, J. Biol. ''hem., vol. 267, pp. 4152-4160 (1992)). The
column was washed with 2 ml of water and the radioactive
reaction product, not retained by the column, was
quantitated by scintillation counting. Reactions with the
acceptor NeuNAca2--3Ga101--4G1cNAc (Oxford Glycosystems,
Inc.) were terminated by adding 980 "l of 5.0 mM sodium
phosphate buffer, pH 6.8. Samples were then centrifuged at
15,000 x g fcr 5 minutes, and a 500 41 aliquot applied onto
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a Dowex 1x8-200 column (1 ml) prepared in the phosahate
form. The reaction product -was collected and quantitated
as previously described (B.W. Weston et al, J. Biol. Chem.,
vol. 267, pp. 24575-24584 (1992)).
Generation of rabbit anti-Fuc-TVII antibodv. The PCR
was used to amplify a segment of the murine Fuc-?'VII gene
corresponding to the enzyme's "stem" and catalytic domains
(J.B. Lowe, Seminars in Cell Biolocrv, vol. 2, pp. 289-307
(1991)), using PCR primers corresponding to base pairs
2194-2224 and 3053-3085; Fig.2; 5' primer
gcgcgQatccCACCATCCTTATCTGGCACTGGCCTTTCACC (SEQ ID NO:3);
3'primer gcgcaaatccAGTTCAAGCCTGGAACCAGCTTTCAAGGTCTTC (SEQ ID
NO: 4); BamHl sites underlined). The PCR was completed using twenty rounds of
amplification consisting of a 1.5 minute 94 C denaturation
step and a 2.0 minute 72 C annealing/extension step. The
PCR product was subsequently cloned into the Bamf:I site of
the T7 Esc:eric::ia co2i expression vector pET-3b (F.W.
Studier et al, Methods Enzvmol., vol. 185, pp. 60-89
(1990)). The insert in one clone (termed pET-3b-Fuc-
TVIlstem/cat) containing a single insert in the correct
orientation was sequenced to confirm that no errors were
introduced during DNA amplification. The recombinant Fuc-
TVII fusion protein was produced by inducing mid-log phase
E. coli (BL21 Lys S) carrying pET-3b-Fuc-TVIIstem/cat with
0.4 mM IPTG for three hours (K.M. Gersten et al, Doctoral
Thesis, pp. 66-98, University of Michigan, Ann Arbor,
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Michigan (1995); F.W. Studier et al, Methods Enzvmol., vol.
185, pp. 60-89 (1990)). The bacteria were subsequently
harvested, and lysed by freezing and then thawing the
bacterial suspension. Bacterial genomic DNA was sheared by
sonication, followed by separation of soluble and insoluble
material by centrifugation. The Fuc-TVII protein was found
in the insoluble fraction, as determined by SDS-
polyacrylamide electrophoresis (E. Harlow et al,
Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1988)).
Recombinant, E. coli-derived Fuc4I'VII was fractionated
by SDS-polyacrylamide gel electrophoresis, and segments of
the gel containing Fuc-TVII were excised and used
subsequently as antigen for rabbit immunizations. Rabbit
immunization services were purchased (Pel-Freeze
Biologicals; Rogers, AZ). Each of three rabbits were
initially immunized subcutaneously with a total of
approximately 200 gg of Fuc-TVII in pulverized
polyacrylamide gel slices, mixed with complete Freund's
adjuvant. Subsequent immunizations were completed in an
essentially identical manner, at 14 day intervals, except
that antigen was administered in incomplete Freund's
adjuvant. Antisera were harvested ten days following the
last of a total of approximately 6 secondary immunizations.
Antigen affinity purification of anti-Fuc-TVII
antibody. The insert in pET-3b-Fuc-TVIIstem/cat was
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released by digestion with Bam.HI and was cloned between the
BamHI sites in the E. coli expression vector pATH10 (T.J.
Koerner et al, Methods Enzvmol., vol. 194, pp. 477-490
(1991)), to yield a fusion protein derived from the E. coli
anthranilate synthase sequence, fused in frame to Fuc-TVII
sequence. This recombinant fusion protein was expressed in
E. coli strain DH5a (induction for 6 hr with 0.0125$
indoleacrylic acid in M9 medium). The bacteria were
harvested, washed, and disrupted by treating with lysozyme
(3 mg/ml) in 50 mM Tris HCl pH 7.5, 5 mM EDTA, with 0.65%
NP-40, 0.38M NaCl, followed by sonication for 20 sec at the
maximal microtip setting (Vibracell, Sonics and Materials,
Inc., Danbury, CT) (T.J. Koerner et al, Methods Enzvmol.,
vol. 194, pp. 477-490 (1991)). Inclusion bodies were
washed twice with 50 mM Tris HC1 pH 7.5, 5 mM EDTA, were
solubilized by heating to 100 C in 1% SDS, 12 mM Tris HC1,
5% glycerol, 1% 2-mercaptoethanol, and were subjected to
SDS-polyacrylamide gel elect:oohoresis. The fractionated
proteins were transferred to PVDF membrane (Bio-Rad
Laboratories, Hercules, CA) by electroblotting (1 mA/cm2).
The membrane was then blocked for 4-6 hr at 4 C with PBS
containing 10% bovine serum albumin, and 0.2% TwEZn 20. A
strip of membrane containing the recombinant Fuc-TVII
fusion protein was incubated overnight at 4 C with 0.5 ml
rabbit anti-mouse Fuc-TVII antiserum, diluted with 2.5 ml
of PBS containing 3% bovine serum albumin and 0.2%
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Tween-20. The membrane was then washed at room temperature
in PBS, 0.05% Tween-20, sliced into small pieces, and the
bound antibody was eluted by incubating the membrane
fragments on ice for 10 min in 450 l Tris-Glycine pH 2.5.
The supernatant was collected and immediately neutralized
with 100 ul of 1M Tris-HC1 pH 8Ø The elution procedure
was completed a second time, and the two eluates were
pooled and used subsequently for immunohistochemical
procedures.
Western blot analysis. Cell extracts were prepared
from transfected COS-7 cells 72 h after transfection.
Extracts contained 50 mM Tris-HC1 (pH 6.8), 1% SDS, and 10%
glycerol. Extracts were boiled for 3 min immediately after
preparation, and were stored frozen until use. Protein
content was determined using the BCA reagent procedure.
Extracts were prepared for SDS-PAGE by adding
dithiothreitol to a final concentration of 0.1 M, and
bromophenol blue to a final concentration of 0.05%.
Samples were then boiled, and fractionated by
electrophoresis through a 10% SDS-polyacrylamide gel.
After electrophoresis, the proteins were electrotransferred
to a PVDF membrane (Biorad). The membrane was rinsed, and
then blocked for 12-14 h at 4 C in phosphate buffered
saline, pH 7.4, containing 10% bovine serum albumin and
0.2% Tween-20. The blot was washed at room temperature in
phosphate buffered saline, pH 7.4, 0.2% Tween-20, and was
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probed with a. 1:200 dilution of antigen affinity purified
rabbit anti-tuc-TVII antibody. The blot was then washed
and probed with a 1:2500 dilution of a horse radish
peroxidase-conjugated anti-rabbit immunoglobulin (Sigma).
The blot was then rinsed, exposed to ECL reagent (Amersham,
UK), and subjected to autoradiography.
Northern blot analysis. Total RNA was prepared from
mouse (FVB/N) tissues and cultured cell lines, using
published prccedures (T. Maniatis et al, Molecular C!oninc:
A Laboratory Manual, Cold Spring Harbcr Laboratory, Cold
Spring Harbor, NY (1982)). Oligo-dT-purified poly A+ RNA
samples were electrophoresed through 1.0% agarose gels
containing formaldehyde, and were transferred to a nylon
membrane (Hybond-N*Amersham). Northern blots were
prehybridized for 2 hours at 42 C in IX PE, 5X SSC, 0.5%
sodium dodecyl sulfate, and 150 g/mi sheared salmon sperm
DNA. Blots were hybridized for 18 hours at 42 C ?n
prehybridization solution containing cf3zP]-labeled 974 bp
EBgI-EcoRI 'ragment isolated from the insert in pMFuc-TVII.
The EagI site is located at nucleotides 2228-2233 while the
EcoRI site spans base pairs 3202-3207. Blots were stripped
in boiling 0.1% SDS and re-hybridized with a chicken
glyceraldehvde 3-phosphate dehydrogenase probe (A.
Dugaiczyk et al, Biochemistry, vol. 22, pp. 1605-1613
(1983)) to confirm that RNA samples were ?ntact and loaded
in equivalent amounts.
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Construction of a mouse L-Selectin/IaM chimera
histochemical probe. A mouse L-selectin cDNA (M.H.
Siegelman et al, Science, vol. 243, pp. 1165-1172 (1989))
was kindly provided by Dr. Mark Siegelman at the University
of Texas Southwestern Medical Center, Dallas, TX. The
extra-cellular domain was truncated at the junction of its
transmembrane domain with an Hph I digest followed by the
litigation of an adaptor. A human IgM cDNA containing the
CH2, CH3, and CH4 domains (kindly provided by Dr. Ernie
Kawasaki, Procept Inc.) was ligated to the adaptor modified
end of the L-selectin sequence in a manner that fuses the
open reading frame encoding L-selectin to the open reading
frame encoding the CH2, CH3, and CH4 domains of human IqM.
This fragment was inserted into the vector SRa-PCDM8
immediately downstream of the SRa promoter in the sense
orientation with respect to the SRa promoter. This vector
was introduced into COS-7 cells using the DEAE dextran
transfection method (J.F. Kukowska-Latallo et al, Genes &
0ev., vol. 4, pp. 1288-1303 (1990); T. Maniatis et al,
Molecular Clonina= A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1982)). Media was
harvested from the transfected cells 3 days after the
transfection, and was replaced with fresh media (DMEM, 10%
FCS, P/S, Q) that was collected 4 days later. The L-
selectin/IgM chimera was purified and concentrated
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approximately 40-fold by affinity chromatcgraphy on goat
anti-human IgM agarose.
Immunohiszcche*nistry nrocedures. Peripheral
(axillary) and mesenteric lymph nodes, and Peyer's patches
were isolated from mice immediately after sacrifice. These
lymphoid tissues were embedded in OCT medium (Tissue-Tek,
MILES, Elkhart, IN), sectioned with a Leica 2800N criostat,
and collected cn glass microscope slides.
Sections to be stained with anti-Fuc-TVII were fixed
in 2$ paraforaaidehyde in phosphate buffered saline for
min on ice. The sections were rinsed with phosphate
buffered saline at room temperature, were quenched with
50 mM NH4C1 in phosphate buffered saline at room
temperature, and then rinsed briefly with water. The
tissues were then permeabilized with 100% methanol for
20 min an ice, rehydrated in phosphate buffered saline, and
then incubated for :0 min at room temperature with blockina
solution A(phosphate buffered saline containing 2% goat
serum, 0.05% Triton X-100, 0.05% Tween 20) The blocking
20 solution was aspirated, and the sections were incubated
overnight at 7 C with antigen affinity purified anti-Fuc-
TVII, used at a final concentration of 5 g/ml, in blocking
solution A. After the overnight incubation, the anti-Fuc-
TVII/blocking solution was removed, the slides were washed
with phosphate buffered saline, and were incubated for 1 hr
at room temperature with a FITC-conjugated goat anti-rabbit
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IgG reagent (Sigma), diluted 1:200 in blocking solution A.
The slides were then washed at room temperature in
phosphate buffered saline, mounted with citifluor
(Citifluor Products, Chemical Laboratory, The University,
Canterbury, Kent CT2 7NH), and examined by
immunofluorescence microscopy (Leitz DM RB microscope).
Sections to be stained with the monoclonal antibody
MECA-79 (P.R. Streeter et al, J. Cell Biol., vol. 107, pp.
1853-1862 (1988)) were fixed on ice for 20 min in 2%
paraformaldehyde in phosphate buffered saline, washed at
room temperature with phosphate buffered saline, and were
quenched for 20 min at room temperature with 50 mM NH4C1 in
phosphate buffered saline. The slides were then rinsed
briefly in water, permeabilized with 100% methanol for
20 min on ice, rehydrated in phosphate buffered saline, and
then incubated overnight at room temperature with blocking
solution A (phosphate buffered saline containing 2% goat
serum, 0.05% Triton X-100, 0.05% Tween 20). The blocking
solution was then aspirated, and the sections were
incubated for 1 hr at 7 C with MECA-79 at a concentration
of 5 g/ml in blocking solution A. Sections were then
washed extensively with phosphate buffered saline at room
temperature. The washed sections were then incubated for
1 hr at room temperature with a TRITC-conjugated goat anti-
rat IgM reagent (Jackson ImmunoResearch, Pennsylvania),
used at a dilution 1:200 in blocking solution A. The
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slides were then washed 3 times at room temperature with
phosphate buffered saline, mounted with citifluor, and
examined.
Sections to be stained with the L-selectin/IgM chimera
were fixed in 1% paraformaldehyde, O.1M cacodylate, pH 7.1,
for 20 min on ice, and were then washed with Tris-buffered
saline, pH 7.4. The L-selectin/IgM chimera was applied to
the sections at a concentration 60 g/ml, in blocking
solution B (Tris-buffered saline, pH 7.4, containing 2%
goat serum), supplemented with either 3 mM CaC12, or with
~
mM EDTA, and were allowed to incubate overnight at 7 C.
Sections were then washed extensively with ice cold Tris-
buffered saline supplemented with 3 mM CaC12. Sections
were then incubated for 1 hr at 7 C with a biotinylated
goat anti-human IgM.reagent (Sigma), diluted 1:200 in
blocking solution B, and supplemented either with 3 mM
CaC121 or with 5 mM EDTA. The sections were then washed
with ice cold Tris-buffered saline supplemented with 3 mM
CaC12, and were incubated for 1 hr at 7 C with a FITC-
conjugated streptavidin reagent (Vector Labs, Burlingame,
CA) diluted 1:200 in blocking solution B supplemented with
3 mM CaC12. The slides were washed with ice cold Tris-
buffered saline supplemented with 3 mM CaC121 mounted with
citifluor, and examined by immunofluorescence microscopy
(Leitz DM RB microscope).
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In situ hybridization nrocedures. In situ
hybridization procedures were completed using a
modification of published procedures (D.G. Wilkinson Ia
Situ Hybridization: A Practical Approach, IRL Press, Oxford
University Press, Oxford, UK). Fresh murine axillary lymph
nodes, mesenteric lymph nodes and Peyer's patches were
embedded in OCT medium (Tissue-Tek, MILES, Elkhart, IN) and
quick-frozen in isopentane on liquid nitrogen. Cryostat
sections (10 M) were collected on Superfrost/Plus
microscope slides (Fisher Scientific, Pittsburgh, PA),
fixed in freshly prepared 4% paraformaldehyde in PBS for 30
min on ice, washed twice in PBS and digested for 5 min at
room temperature with 1 g/ml proteinase K in 50 mM Tris-
HC1 pH 7.5, 5 mM EDTA. The slides were then washed in PBS,
fixed again in 4% paraformaldehyde, rinsed in water, and
treated with 0.25% acetic anhydride in 0.1M triethanolamine
pH 8.0, for 10 min at room temperature. Acetylation was
followed by room temperature washes in PBS, and then in
0.85% NaCl. The slides were then dehydrated in a graded
series of solutions of ethanol in water (30%, 50%, 80%,
95%, 100% ethanol). Air dried sections were overlaid with
a hybridization solution containing 35S-labeled RNA, in
sense or antisense orientation. RNA probes were derived by
in vitro transcription procedures, using recombinant T7 or
Sp6 RNA polymerases, initiating on the T7 or Sp6 promoter
sequences flanking a DNA segment derived from the coding
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region of the mouse Fuc-TVII gene (base pairs 2197-2494;
Fig. 2), as subcloned into the vector pCDNAI (Invitrogen).
The hybridizaticn solution contained 50% deionized
formamide, 0.3 M NaC 1, 20 mM Tris HC1 pH 8.0, 5 mM EDTA, 10
mM phosphate buffer pH 8.0, 10% dextran sulphate,
1X Denhardt's solution (T. Maniatis et al, Molecular
CloninQ: A Laboratorv Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1982)), 0.5 mg/ml yeast
tRNA, 10 mM dithiothreitol, and 107cpm/ml of radiolabeled
probe. The hybridization solution was sealed over the
sections with a coverslip and DPX mounting media (BDH Lab
Supplies, Poole, England). Hybridization was carried out
for 16 hours at 55 C in a sealed container humidified with
5X Standard Saline Citrate (SSC). After hybridization, the
DPX mounting media seal was removed, and slides were washed
at 55 C for 30 min in 5X SSC, 10 mM dithiothreitol, and
then at 65 C for 20 min in for=amide wash buffer (50%
formamide, 2X SSC, 20 mM dithiothreitol). The slides were
then washed 4 times at 37 C in 0.5 M NaC1, 10 mM Tris HC1
pH 7.5, 5 mM EDTA. Slides were then digested with RNAseA
(1 g/ml) for 30 min at 37 C, and were washed in formamide
buffer, then in 2X SSC, and then in 0.1X SSC. Slides were
dehydrated in a graded series of solutions of ethanol in
0.3 M ammonium acetate (30$, 50%, 80%, 95%, 100% ethanol),
air 4-ried, and were coated with NTB2 I-iquid emulsion
*
(Kodak). Following a two to three week exposure time, the
* trademark
1
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emulsion was developed using procedures suggested by the
manufacturer. Sections were then stained with hematoxylin
and eosin, and examined and photographed with bright field
and dark field modalities, using a Leitz DM RB microscope.
RESULTS
A hybridization screen identifies a novel murine
a(1 3)fucosyltransferase locus. In an effort to isolate
novel murine c(1,3)fucosyltransferase genes, a murine
genomic DNA phage library was screened with a probe
corresponding to the catalytic domain"of the human Lewis
ar(1,3/1,4)fucosyltransferase (Fuc-TIII) (J.F. Kukowska-
Latallo et al, Genes & Dev., vol. 4, pp. 1288-1303 (1990)),
using low stringency hybridization conditions (see the
Experimental Procedures). One phage was isolated that
contained an insert with a translational reading frame
sharing approximately 40% amino acid sequence similarity
with the amino acid sequences encoded by four previously
cloned members of the human e(1,3)fucosyltransferase family
(Fuc-TIII-VI) (J.F. Kukowska-Latallo et al, Genes & Dev.,
vol. 4, pp. 1288-1303 (1990); B.W. Weston et al, J. Biol.
C em , vol. 267, pp. 24575-24584 (1992); B.W. Weston et al,
J. Biol. Chem., vol. 267, pp. 4152-4160 (1992); J.B. Lowe
et al, J. Biol. Chem., vol. 266, pp. 17467-17477 (1991);
S.E. Goelz et al, Cell, vol. 63, pp. 1349-1356 (1990);
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R. Kumar et al, J. Biol. Chem., vol. 266, pp. 21777-21783
(1991)).
To identify transcripts corresponding to this genomic
sequence, a segment of the phage insert representative of
the open reading frame was used to probe Northern blots
prepared from mouse cell lines and tissues. Transcripts
corresponding to this probe were identified in the murine
cytotoxic T-cell line 14-7fd (T.J. Braciale et al, a, EXR.
Med., vol. 153, pp. 910-923 (1981); M.E. Andrew et al, J.
Immunol., vol. 132, pp. 839-844 (1984)). A cDNA library
constructed from this cell line (P.L. tmith et al, J. Biol.
Chem., vol. 269, pp. 15162-15171 (1994)) was screened by
hybridization with a segment of the phage insert, yielding
16 hybridization positive colonies. The sequences of all
16 cDNA clones were determined, as was the sequence of the
corresponding genomic DNA (Figure la). Analysis of this
sequence data indicates that this locus yields multiple,
structurally-distinct transcripts derived from alternative
splicing events, and possibly also from alternative
transcription initiation events. Five classes of cDNAs
were identified (Figure 1). Analysis of these cDNA
sequences identifies three methionine codons that may
function to initiate translation of an open reading frame
with amino acid sequence similarity to human Fuc-TIII, Fuc-
TIV, Fuc-TV, and Fuc-TVI (Figure 2). The positions of
these methionine codons predicts the synthesis of
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a(1,3)fucosyltransferases with diff erent cytosolic domains
(encoded by exons 2 and/or 3), but with identical Golgi-
localized catalytic domains (encoded by exon 4). One
relatively abundant class of cDNAs (represented by cDNA 14)
maintains an open reading frame initiating at the
methionine codon at nucleotide 1947. This reading frame
predicts a 342 residue, 39,424 Da type II transmembrane
protein, with a hydrophobic, transmembrane segment derived
10 from amino acids 9-31 (Figure 2 and SEQ ID NO:2). An in-frame methionine
codon at nucleozide 2126 predicts a 318 residue, 36,836 Da
polypeptide that initiates within the'-hydrophobic,
transmembrane segment of the polypeptide predicted by the
longer reading frame initiated at nucleotide 1947. A
similar structural arrangement is found in two other cDNA
classes, represented by cDNAs 6 and 10. However, these two
cDNAs differ from cDNA 14 in that they contain an
additional upstream exon with a methionine codon
20 corresponding to nucleotide 996. The translational reading
frame initiated by this methionine codon is truncated by a
termination codon in exon 2 at a position proximal to the
methionine codon at nucleotide 1947, and thus cannot
generate a polypeptide that shares similarity to the human
a(1,3)fucosyltransferases. However, in cDNA 5, absence of
exon 2 allows the translational reading frame generated by
the methionine codon at nucleotide 996 to continue in frame
30 with sequence in exon 4. This arrangement predicts the
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synthesis of a 389 residue, 44,492 Da type II transmembrane protein, with the
same putative transmembrane segment defined for the protein predicted by
cDNA 14 (Figure 2 and SEQ ID NO:1). Finally, cDNA 3 is representative of a
relatively abundant class of cDNAs that each initiate between the splice
acceptor site of exon 4 and the methionine codon at
nucleotide 2126. This class of cDNAs predicts a 318
residue, 36,836 Da polypeptide that initiates within the
transmembrane secnaent predicted for the proteins
corresponding to the other cDNA classes.
Because the polypeptides predicted by these murine
cDNAs share primary sequence similarity to the four human
a(1,3)fucosyltransferases known at the time (Fuc-TIII, IV,
V, and VI), we anticipated that one or more of them would
function as an c(1,3)fucosyltransferase. However, because
the murine peptide sequence shares approximately equivalent
sequence similarity to each of these human enzymes, we
expected that it did not represent the murine homologue of
any of them, and consequently named it Fuc-TVII. This
appellation has been justified by subsequent work in which
this murine gene has been used to isolate cDNAs encoding
the human Fuc-TVII (S. Natsuka et al, J. Biol. Chem., vol.
269, pp. 16789-16794 (1994)).
None of the three putative initiation codons are
embedded in a sequence context consistent with Kozak's
rules for translation initiation (Figure 2 and SEQ ID NO:1) (M. Kozak,
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Cell, vol. 44, pp. 283-292 (1986)). To determine which, if
any, of these initiation codons and cognate cDNAs function
to encode the predicted polypeptide(s), and to confirm that
this locus encodes an a(1,3)fucosyltransferase, COS-7 cells
were transfected with a cDNA representative of each class,
and the transfectants were subjected to assays to (i)
identify cDNA-determined cell surface-localized fucosylated
oligosaccharide antigens, (ii) identify and quantitate the
polypeptides encoded by cDNAs, and (iii) identify and
partially characterize cDNA-determined
ac(1,3)fucosyltransferase activity in transfectant cell
extracts using in vitro ac(1,3)fucosyltransferase activity
assays.
eDNAs representative of three of the five classes
(cDNAs 6, 10, and 14) (Figure 1) each determine relatively
high levels of cell surface-localized sLeX expression
(35.1%, 21.8%, and 16.5%, respectively, above a 2%
background) when introduced into COS-7 cells by
transfection. cDNA 5 also directs cell surface sLeX
expression in COS-7 cells, but at a level (9% positive
cells) that is lower than the sLeX expression levels
determined by cDNAs 6, 10, and 14. By contrast, none of
these four cDNAs directs expression of Lewis x, Lewis a, or
sialyl Lewis a determinants. These results indicate that
one or both of the two potential methionine initiator
codons in each cDNA can efficiently direct translation to
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yield c(1,3)fucosyltransferase activity. These
observations further indicate that this
cz(1,3)fucosyltransferase activity can utilize
a(2,3)sialylated lactosamine-based glycan structures to
form sLeX determinants, but indicate that the activity does
not efficiently utilize neutral type II oligosaccharide
Lewis x precursors, nor neutral or a(2,3)sialylated type I
precursors to the Lewis a isomers. Since all four of these
cDNAs direct qualitatively identical cell surface antigen
profiles in COS-7 cells, it seems likely that individually,
or together, each directs the expression of polypeptides
that individually, or together, maintain essentially
identical acceptor substrate specificities (at least for
the four antigens examined).
In contrast to the results obtained with cDNAs 5, 6,
10, and 14, cDNA 3 does not direct detectable sLeX
expression. This result suggests that the methionine codon
at nucleotide 2126 in this cDNA does not efficiently
promote initiation of translation of the cognate mRNA, and
thus does not encode functionally significant levels of
enzyme activity. Alternatively, this cDNA may encode a
polypeptide without ac(1,3)fucosyltransf erase activity.
Qualitatively identical results were obtained when
these five cDNAs were expressed in another cell line (CHO-
Tag cells) (P.L. Smith et al, J. Biol. Chem., vol. 269, pp.
15162-15171 (1994)) informative for expression of the Lewis
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x, and sLeX determinants. Unlike COS-7 cells, this cell
line is also capable of forming the internally fucosylated
VIM-2 determinant (NeuAca2,3Ga1p1,4G1cNAcF1,3Ga1Q1,4-
(Fuca1,3)G1cNAc-R) (B.W. Weston et al, J. Biol. Chem., vol.
267, pp. 24575-24584 (1992)). It was found that none of
the cDNAs directs expression of the VIM-2 epitope when
expressed in the CHO-Tag cells. Considered together, these
results indicate that some, though not all, of the cDNAs
can encode an a(1,3)fucosyltransferase activity that can
catalyze a(1,3)fucosylation of the N-acetylgalactosamine
moiety on a terminal a(2,3)sialylated lactosamine unit, but
not to internal N-acetylgalactosamine moieties on
a(2,3)sialylated polylactosamine precursors, nor to neutral
type II precursors.
To confirm that the sLex expression efficiency
characteristic of each cDNA correlates with the level of
expression of the corresponding protein, cell extracts of
the transfected COS-7 cells were subjected to Western blot
analysis using an affinity purified rabbit polyclonal
antibody generated against a recombinant form of the
predicted polypeptide (Figure lb). Cells transfected with
cDNAs 6, 10, and 14 express two major forms of the protein,
with molecular weights of 35 kDa and 37 kDa. Smaller
amounts of several other proteins are also evident in these
cells. The amount of immunoreactive protein generated by
these three cDNAs correlates with the level of sLex
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expression directed by each. This observation indicates
that the relative sLex expression level directed by each is
a function of the efficiency with which each corresponding
mRNA is translated, and thus the relative intracellular
accumulation of the cognate polypeptide.
Cells transfected with cDNA 5 also contain multiple
immunoreactive polypeptides (Figure ib). The most abundant
pair of these proteins migrate more rapidly than do the
proteins detected in cells transfected with cDNAs 6, 10,
and 14, yet are approximately similar in quantity to the
immunoreactive protein directed by cDNAs 6 and 10. As cDNA
directs lower levels of cell surface sLex expression than
these two cDNAs, it is therefore possible that the lower Mr
immunoreactive polypeptides found in cDNA 5-transfected
cells maintain substantially lower specific enzyme activity
than do the proteins encoded by cDNAs 6, 10, and 14, or are
otherwise less able to direct sLex expression in COS-7
cells. Finally, cells transfected with cDNA 3 do not
contain any detectable immunoreactive proteins. This
implies that the putative initiator codon at base pair 2126
in this cDNA does not initiate translation of an
immunoreactive product, and is consistent with the
observation that this cDNA does not yield sLex expression
following transfection into COS-7 cells.
Conclusions derived from the flow cytometry and
Western blot analyses summarized above are supported by the
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results of in vitro a(1,3)fucosyltransf erase assays
completed on the same cell extracts. These assays
demonstrate that cells transfected with cDNAs 5, 6, 10, and
14 contain enzyme activity that can transfer 14C-labelled
fucose from the nucleotide donor substrate GDP-fucose to
the low molecular weight acceptor 3'-sialyl-N-
acetyllactosamine (SLN; NeuNAca2,3Ga1p1,4G1cNAc) (Figure
la). For each cDNA, the product of this reaction co-elutes
with a radiolabeled sLeX tetrasaccharide standard when
fractionated by ion suppression amine adsorption HPLC. The
a(1,3)fucosyltransferase activity directed by each of these
four cDNAs does not utilize the neutral acceptor substrates
N-acetyllactosamine or lacto-N-biose I. And, extracts
prepared from cells transfected with cDNA 3 do not contain
detectable a(1,3)fucosyltransferase activity when tested
with 3'-sialyl-N-acetyllactosamine, nor when tested with
the neutral acceptor substrates N-acetyllactosamine or
lacto-N-biose I. These results are entirely consistent
with the flow cytometry data summarized above, and indicate
that this locus encodes an a(1,3)fucosyltransferase
activity that apparently requires type II acceptor
substrates that are terminally substituted with an a(2,3)-
linked sialic acid residue. Considered together, these
results suggest that differential splicing and/or
transcriptional initiation events can control the level of
a(1,3)fucosyltransferase activity, and thus cell surface
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sLex expression level, through mechanisms that depend on
the efficiency with which each transcript is translated.
Transcription of the mouse Fuc-TVII locus is
restricted to cells found in the bone marrow and the luna.
Northern blot analysis indicates that transcripts
corresponding to the Fuc-TVII locus accumulate to
detectable levels in only a few tissues in the adult mouse
(Figure 3). Abundant transcript accumulation is only
observed in the lung and in the bone marrow, with very
small amounts evident in the spleen, salivary gland, and
skeletal muscle. Northern blot analysis of cultured murine
blood cell-type cell lines indicates that the Fuc-TVII
transcript is relatively abundant in the mouse cytotoxic
T line 14-fd (used to clone the Fuc-TVII cDNAs), and in the
mouse T cell line EL4 (L.J. Old et al, Cancer Res., vol.
25, pp. 813-819 (1965)). Less abundant transcript
accumulation is evident in the murine macrophage-derived
lines RAW (P. Ralph et al, J. Immunol., vol. 119, pp. 950-
954 (1977); W.C. Raschke et al, Cell, vol. 15, pp. 261-267
(1978)) and P388 (H.S. Koren et al, J. Immunol., vol. 114,
pp. 894-897 (1975)). Fuc-TVII transcripts are not evident
in the murine erythroleukemia cell line MEL (D. Singer et
al, Proc. Natl. Acad. Sci. U.S.A., vol. 71, pp. 2668-2670
(1974); B.L. Weber et al, Science, vol. 249, pp. 1291-1293
(1990)), nor in three murine B-lymphocyte lineage cell
lines [S107, (M.L. Atchison et al, Cell, vol. 48, pp. 121-
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128 (1987)); TH2.54.63, (T. Hamano et al, J. Immunol., vol.
130, pp. 2027-2032 (1983)); 180.1, (M. Hummel et al, J.
Immunol., vol. 138, pp. 3539-3548 (1987))].
Both the marrow and lung maintain several differently-
sized transcripts, including two abundant transcripts of
approximately 1.6 and 2.2 kb in size, and a fainter
transcript at approximately 3.0 kb. These three
transcripts are similar in size to the three most abundant
transcripts observed in the murine 14-7fd cytotoxic T cell
line. These observations suggest that cells in the bone
marrow and lung yield alternatively sp'liced transcripts
similar in structure to those characterized by cDNA cloning
studies in the 14-7fd cells. These data also suggest that
in the marrow, the Fuc-TVII locus is transcribed in cells
assigned to the myclold and T-lymphoid lineages, but not in
B-lymphoid lineage cell types, and suggest that expression
of this fucosyltransferase correlates with selectin ligand
expression on myelold and T-lymphocyte lineage cell types.
Fuc-TVII is expressed in endothelial cells linina the
high endothelial venules in peripheral lvmph nodes,
mesenteric lymph nodes, and Pever's patches. The identity
of the cell types in the lung responsible for the Northern
blot signal in that organ was disclosed by in situ
hybridization analyses. These studies identified Fuc-TVII
transcripts in paratracheal lymph nodes within the
extirpated lung, but not in any other cell type. The
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pattern of expression in these nodes suggested that the
Fuc-TVII transcripts were localized to the high endothelial
venules within these nodes. When considered together with
recent observations suggesting that a sulfated derivative
of the sialyl Lewis x determinant represents a terminal
oligosaccharide moiety found on HEV-specific L-selectin
ligands (S. Heaunerich et al, Biochemistry, vol. 33, pp.
4820-4829 (1994); S. Hemmerich et al, Biochemistry,
vol. 33, pp. 4830-4835 (1994); S. Hemmerich et al, J. Biol.
Chem., vol. 270, pp. 12035-12047 (1995)), detection of Fuc-
~
TVII transcripts in these HEV suggests a possible role for
this locus in the synthesis of this fucosylated
oligosaccharide, and this in controlling lymphocyte homing.
To further characterize the HEV-specific expression of Fuc-
TVII, this was systematically evaluated using in situ
hybridization analysis of HEV in peripheral lymph nodes, in
mesenteric lymph nodes, and in Peyer's patches, where L-
selectin ligand expression has been well-characterized
(S.D. Rosen et al, Curr. 0gin. Cell Biol., vol. 6, pp. 663-
673 (1994)). These analyses (Figure 4) indicate that Fuc-
TVII transcripts accumulate to easily detectable levels in
the HEV of all three types of lymphoid aggregates. An in
situ hybridization signal is also obtained with the anti-
sense Fuc-TVII probe in a population of cells that line the
gut lumenal surface overlying the Peyer's patches.
Although these are presumed to be epithelial cells, and may
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represent so-called M cells (R.L. Owen, Gastroenterolocrv,
vol. 72, pp. 440-451 (1977)), their precise identity
remains unknown.
As noted above, not all Fuc-TVII-derived transcripts
yield a protein product. Immunohistochemical analyses were
therefore used to confirm that the Fuc-TVII transcripts
detected in HEV are accompanied by Fuc-TVII polypeptide
expression, and to confirm that such expression co-
localizes with L-selectin ligand expression. A rabbit
polyclonal antibody raised against the Fuc-TVII peptide
yields an intracellular staining pattern in the endothelial
cells within HEV in all three lymphoid aggregates (Fig. 5).
The perinuclear intracellular staining pattern seen with
the anti-Fuc-TVII antibody is consistent with the notion
that this enzyme is.localized to the Golgi apparatus, where
it may participate in the synthesis of fucosylated
oligosaccharides with L-selectin ligand activity. In each
of the three types of lymphoid aggregate, expression of
i.mmunoreactive Fuc-TVII co-localizes with expression of
epitopes recognized by the MECA-79 antibody, shown
previously to stain HEV, and to interfere with L-selectin
binding to HEV (P.R. Streeter et al, J. Cell Biol., vol.
107, pp. 1853-1862 (1988)). Fuc-TVII expression also co-
localizes with expression of L-selectin ligands on HEV, as
detected with a recombinant mouse L-selectin/human IgM
chimeric protein. These observations imply that Fuc-TVII
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may participate in the synthesis of the sialylated,
sulfated, and c(1,3)fucosylated candidate oligosaccharide
components of HEV-derived L-selectin ligands.
In an effort to understand the functions of cell
surface fucosylated oligosaccharides in animals, we have
established a program to isolate murine
a(1,3/4)fucosyltransferase genes, to be used initially as
reagents to characterize tissue-specific expression
patterns of the loci that control expression of cell
surface fucosylated oligosaccharides. These reagents, and
the information gathered from their application will be
used eventually with transgenic approaches to uncover
functions of their cognate cell surface fucosylated
oligosaccharides, by perturbing their expression patterns.
A cross-hybridization approach outlined here yielded a
novel genomic sequence that cross-hybridizes with segments
derived from the conserved portions of the human
Fuc-Ts III, V, and VI genes, in a position corresponding to
their catalytic domains. Following the isolation of this
murine genomic locus, functional analyses indicated that it
encoded an cz(1,3)fucosyltransferase, termed Fuc-TVII, with
structural features and catalytic activities that were, at
the time of its isolation, unique to the
ac(1,3)fucosyltransferase family. In particular, this locus
was the only c(1,3)fucosyltransferase known to maintain a
coding region distributed over more than one exon, and the
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first fucosyltransferase with multiple distinct initiation
codons with the potential to yield structurally distinct
polypeptides, characterized by different cytoplasmic
domains, but with essentially identical catalytic
activities. The catalytic activity of each Fuc-TVII
isoenzyme is characterized by an ability to utilize
a2,3sialylated type II N-acetyllactosamine precursors,
without the ability to utilize neutral type II, neutral
type I, or sialylated type I N-acetyllactosamine
substrates. Similar observations have been made for the
human homologue of Fuc-TVII isolated subsequently (S.
Natsuka et al, J. Biol. Chem., vol. 269, pp. 16789-16794
(1994); K. Sasaki et al, J. Biol. Chem., vol. 269, pp.
14730-14737 (1994)). This catalytic specificity, and the
leukocyte-specific expression pattern of this gene,
strongly suggest that it plays a pivotal role in the
biosynthetic scheme that yields the a2,3sialylated,
al,3fucosylated lactosaminoglycans essential to E- and P--
selectin ligand activity. Furthermore, the leukocytes of
mice genetically engineered for a deficiency in this enzyme
are deficient in expression of functional ligands for E-
selectin and P-selectin, and exhibit a concomitant immune
deficiency characterized by a deficit in leukocyte
mobilization to inflammatory sites. These observations
demonstrate that Fuc-TVII controls leukocyte selectin
ligand expression, and indicate that inhibition of this
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enzyme by pharmaceutical agents or maneuvers will represent
an anti-inflammatory approach that may have therapeutic
benefit in human disease where selectin-dependent
inflammation is pathologic.
Other observations made in the work described here
suggest a role for Fuc-TVII in directing synthesis of the
oligosaccharide components of the ligands for L-selectin.
As Rosen and colleagues have shown, L-selectin ligands on
HEV correspond to 0-linked carbohydrate determinants
displayed by the mucin-type glycoproteins GlyCAM-1, CD34,
and MAdCAM-1 (L.A. Lasky et al, Cell, vol. 69, pp. 927-938
(1992); S. Baumhueter et al, Science, vol. 262, pp. 436-438
(1993); E.L. Berg et al, Mature, vol. 366, pp. 695-698
(1993)). Their earlier biochemical analyses indicate that
the oligosaccharides relevant to L-selectin ligand activity
are sialylated, sulfated, and possibly fucosylated (Y. Imai
et al, J. Cell Biol., vol. 113, pp. 1213-1221 (1991)).
More recent structural analyses from the Rosen group are
consistent with the hypothesis that the capping groups on
such olioosaccharides correspond to sulfated versions of
the sialyl sLeX moiety, with sulfate attached via the 6-
hydroxyl of the terminal galactose moiety
[NeuNAcac2,3(SO46)Ga1Q1,4(Fucczl,3)G1cNAc-R], or via the 6-
hydroxyl of the subterminal N-acetyl-glucosamine moiety
[NeuNAcac2,3Ga1Q1,4(S046)(Fuco1,3)G1cNAc-R], or both
(S. Hemmerich et al, Biochemistry, vol. 33, pp. 4820-4829
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(1994); S. Hemmerich et al, Biochemistry, vol. 33,
pp. 4830-4835 (1994); S. Hemmerich et al, J. Biol. Chem.,
vol. 270, pp. 12035-12047 (1995)). Non-fucosylated forms
of these structures were also identified, however, and the
evidence that fucose is required for activity of
physiological L-selectin ligands remains circumstantial.
The identification of such non-fucosylated structures
suggests the possibility that these sialylated and sulfated
molecules represent acceptor substrates for
a(1,3)fucosyltransferases expressed in HEV endothelial
cells. Our observation that expression of the Fuc-TVII
locus co-localizes with L-selectin ligand expression in
such cells suggests that Fuc-TVII may operate in this
context. Furthermore, mice genetically engineered for a
deficiency in Fuc-TVII are deficient in L-selectin-
dependent lymphocyte homing to=lymph nodes, and are
deficient in expression of L-selectin ligand activity on
the high endothelial venules in peripheral and mesenteric
lymph nodes, and in Peyer's patches. These observations
demonstrate that Fuc-TVII controls HEV selectin ligand
expression, and indicate that inhibition of this enzyme by
pharmaceutical agents or maneuvers will represent an anti-
inflammatory approach that may have therapeutic benefit in
human disease where L-selectin-dependent lymphocyte
trafficking is pathologic. The notion that sulfated and
sialylated lactosamine moieties represent acceptor
SUBSTITUTE SHEET (RULE 26)
CA 02247966 1998-09-04
WO 97/32889 PCTIUS97/03184
-75-
substrates for enzymes like Fuc-TVII is supported by
studies suggesting that Fuc-TIII (E.V. Chandrasekaran et
al, Biochem. Biophvs. Res. Commun., vol. 201, pp. 78-89
(1994)) and Fuc-TV (P.R. Scudder et al, Glycobiology, vol.
4, pp. 929-933 (1994)) can utilize sialylated, sulfated
lactosamine-type acceptors. Indirect evidence derived from
studies.on the biosynthesis of G1yCAM-1 are also consistent
with this hypothesis (S. Hemmerich et al, Biochemistry,
vol. 33, pp. 4820-4829 (1994); D. Crommie et al, J. Biol.
Chem., vol. 270, 22614-22624 (1995)). However, our results
indicate that the non-sulfated entity'-
NeuNAca2,3Ga1p1,4G1cNAc is used, in vitro, and in vivo, by
Fuc-TVII. Biochemical analyses indicate that 6-0 sulfation
of the G1cNAc moiety on this sialylated molecule yields a
substance that is used effectively by mouse Fuc-TVII. It
therefore remains to be determined if this sulfated
molecule or others are utilized by Fuc-TVII, in HEV
endothelial cells. Thus, while the biosynthetic scheme for
such molecules remains to be defined, it is clear that Fuc-
TVII plays an essential role in this pathway.
There is evidence to suggest that L-selectin expressed
by granulocytes and other leukocytes mediates adhesion of
these cells to activated vascular endothelium through as
yet undefined extracellular endothelial cell counter-
receptors (M.L. Arbones et al, Immunity, vol. 1, pp. 247-
260 (1994); K. Ley et al, J. Exy. Med., vol. 181, pp. 669-
SUBSTITUTE SHEET (RULE 26)
CA 02247966 1998-09-04
WO 97/32889 PCT/US97/03184
-76-
675 (1995)). Since the chemical nature of this counter-
receptor(s) is not known, a role for Fuc-TVII in the
synthesis of such ligands remains speculative, and is a
subject of current exploration in this laboratory. In any
event, the demonstration here that Fuc-TVII is co-expressed
with L-selectin ligands on HEV, when considered together
with previous observations demonstrating that Fuc-TVII is
expressed in leukocytes and can direct synthesis of ligands
for E- and P-selectin, along with evidence that the Fuc-
TVII null mice are deficient in ligands for E-, P- and
L-selectin, indicates that Fuc-TVII ig-a master control
locus for the synthesis of ligands for all three selectins,
and thus for controlling selectin-dependent leukocyte
traf f icking.
Obviously, numerous modifications and variations of
the present invention are possible in light of the above
teachings. It is therefore to be understood that, within
the scope of the appended claims, the invention may be
practiced otherwise than as specifically described herein.
SUBSTITUTE SHEET (RULE 26)
CA 02247966 2006-09-28
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: THE REGENTS OF THE UNIVERSITY OF MICHIGAN
(B) STREET: 3003 S. State Street
(C) CITY: ANN ARBOR
(D) COUNTRY:USA
(F) ZIP CODE: MI 48104-1280
(ii) TITLE OF INVENTION: MURINE ALPHA (1,3) FUCOSYLTRANSFERASE
FUC-TVII, DNA ENCODING THE SAME, METHOD FOR PREPARING THE
SAME, ANTIBODIES RECOGNIZING THE SAME, IMMUNOASSAYS FOR
DETECTING THE SAME, PLASMIDS CONTAINING SUCH DNA
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Robic
(B) STREET: 1001 Square Victoria
(C) CITY: Montreal
(D) STATE: QC
(E) COUNTRY: Canada
(F) ZIP: H2Z 2B7
(G) TELEPHONE: 514-987-6242
(H) TELEFAX: 514-845-7874
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Disk 3.5" / 1.44 MB
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: TXT ASCII
(Vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,247,966
(B) FILING DATE: March 7, 1997
(Vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER: PCT/US97/03184
(B) FILING DATE: March 7, 1997
(A) APPLICATION NUMBER: US 085/613,098
(B) FILING DATE: MarCh 8, 1996
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3594
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
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CA 02247966 2006-09-28
ACAAACAGGA AGGACAGCAG GCTCTGGCAG CCAGAAGCCT GTGGCCCCAA GCTGGCAGGA 60
TGGCCCCCTT CCTGCAGGTC CCCCACAGCC TTCTGGGTTC CTGACACGAG AGAAGAGGTG 120
GGGCGGGGTG AAGTGAACTC TGAAGCCAAA ATGTGACTCT CCTGGGGTCA CCAGCTTGGG 180
GAGAGGTGAA GAAAGATGCC GGGGCGGAAA CAAAGGGGCA GATATCACTA TGGTTATCTT 240
ACTAAGCACA GAGTAACTGA AAAAGCAAGG GTACCGCTGC CCACCTCGTG CCCACCTTAC 300
GTTATACCTC AAACCAGCTA GATAGTTTCT GATGGCACCC ATACCCTCCC TTCCCCTTTA 360
GGCATTGCGC AAGCTCTCCA CCACAATCTG GAAGTTATAC CCTGCGAGGG GATGGGCAGG 420
GCACTTCTGA GGTGCCAATC AGCCTGCACT CGCCTCTGCC CTGGCCATGG CACTGCTGTC 480
AGTTTCTTGG TACCTGTCTC AACAGCAGCC TTGTCACGTG AGACTATGGC TGGCGGTGGG 540
GGTGGGGGCA GGAATCCTAG AAGCACAGGA GTGACATAGG GTCGGGTCGG GCAGAGCGAA 600
GTGTAGGAGG TGATCCCCAA AGGGATGCTG GGGACGATCT GGCCAACACT GTCCTCCCAT 660
TCAAAACTCC CAGTCTGGAG CTCTGGGACA TGGACAAGCC AGGCCTGCTA TTCTCCATAC 720
AGGGCTCCAT AGTGTCTGGC TCAGCAGAGT GGGGGATCTG GTGGGGATGG AGGAAGCTTA 780
GCTAAAAGCT TTGTATAGGC TGAAGCTCTG AGTGACCCTG CTGGGCCACC CTACCCTGGT 840
CTGGGCTGGG TCATTGCATC CCCAGATTGG AAGGCTTGGT GAGATGGAGA GGAACCTTGG 900
CTACAAGCTA TAGCTTTGCC CACCAGAGCC TGCTGGAGGG GAATCAAACA AGCCTGGACC 960
TGAGGCTGGG ACTAGCTTTC CTGTTTCTGG AGTGGATGCC AACCCCCTGC CCACCAGCCT 1020
GCCTGTCCAC GCCAGGGACA CACAGACTCC TTCCCTTTCC AGACTGGAAA GCCCCCTCCT 1080
GGGAGAGCAG GAAGGAAGCA ACCTGCAACT CTTCCAGCCC TGGACCTTGG GCTGAACCTA 1140
CAGTTCAAGG TTTGTATGCT CACAGGTCTT GGCAGGGAAA GATAAGAATC CCCAGGGCAC 1200
CCTCCCCCCC GCCCCCCAGT CCACTGCAGG TAGCTCCTGG GTCTGCCCTT CAGGGCAAGT 1260
GCTGACGCTC CATCAGACTG TGATGGGGCC CTTTTCTGAG GATGACAATT CTGAGAACAA 1320
GGCATTTTTC TAGAGGTGGC AGAACAGCAT TTTGTGATGC CCGAGGATCT GGGAGCACAG 1380
GTCCAGCTTA ATGAGGGATT GGAGGAAGTG GGTATCATCA TTACAGGGAG GGGCCTCTGT 1440
GGCCTCCTGG GAAAATGCAG TTGCTCTCTT TGGGTGGCCT GGGGTTGTGT GGTGGGCAGA 1500
GGACGGAGGT GCTCATTGGG GGAAGGGATC ACTTCTGCTC AGAGTGCTCG CAAGGGCCTT 1560
TCCTTTTCCT GAAGGCAAGC AGGCCTCCTC CTCCTCCTCT TCCTCCTTCT CCTCTTCCTC 1620
CTCTTTCTCC ATATGCCTAG CTGGTCATTT CTAGGGACCA GCATGGTTGG GAAGGGGGCC 1680
TTGTCTTGGC CTTCCTCTTG TCTCAATTCC CTCTTTGAGC AGAAGACGGG GTGGGTGGGG 1740
TAGGATTGGA TAGTGGTTGA TGCCAAAGAT TGAAGGGGTA GGGCGGGGCA GAAGTGGGAA 1800
GGTCCCTGGC TTCCTCACCT TGGTAGATGG TGAGGAGCCC CAGAGGTTGA GCTGAGCAGC 1860
AGCTGTGATT TCAGGGTGCC TCTGTTGGAG AGGCTGCTGT GATTTGAAAA TCTTCTTTCC 1920
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CA 02247966 2006-09-28
TTGGTGACAA TTCCAGAAGG CTCCAGATGA ATTGTATTGG TGAGTGCCTG GCCCTTAAGC 1980
AGTCCCAGCT GGGGATGATG GGGATTTATG GGTGTCCCTG AGCCTAGGGT GACAGGGCCT 2040
CTCCTTTTTT TTTTATTCTG CTTCAGGGTA CCACCCCACC AGGAGGCTGC GGGCCTGGGG 2100
CGGCCTAGCT GGAGGAGCAA CATTCATGGT AATTTGGTTT TTCTGGCTGT GGGGATCAGC 2160
TCCTGGAAGT GCCCCTGTGC CTCAGTCCAC ACTCACCATC CTTATCTGGC ACTGGCCTTT 2220
CACCAACCGG CCGCCAGAGC TACCTGGTGA CACCTGCACT CGCTATGGCA TGGCCAGCTG 2280
CCGTCTGAGT GCTAACCGGA GCCTGCTAGC CAGTGCTGAT GCTGTGGTCT TCCACCACCG 2340
TGAGCTGCAA ACCCGGCAAT CTCTCCTACC CCTGGACCAG AGGCCACACG GACAGCCTTG 2400
GGTCTGGGCC TCCATGGAAT CGCCCAGTAA TACCCATGGT CTCCATCGCT TCCGGGGCAT 2460
CTTCAACTGG GTGCTGAGCT ATCGGCGTGA TTCAGATATC TTTGTACCCT ACGGTCGCTT 2520
GGAGCCTCTC TCTGGGCCCA CATCCCCACT ACCGGCCAAA AGCAGGATGG CTGCCTGGGT 2580
GATCAGCAAT TTCCAGGAGC GGCAGCAGCG TGCAAAGCTG TACCGGCAGC TGGCCCCTCA 2640
TCTGCAGGTG GATGTGTTCG GTCGCGCCAG CGGACGGCCC CTATGCGCTA ATTGTCTGCT 2700
GCCCACTTTG GCCCGGTACC GCTTCTACCT GGCCTTTGAG AACTCACAGC ATCGGGACTA 2760
CATCACTGAG AAGTTCTGGC GCAATGCCCT GGCGGCTGGT GCTGTACCCG TGGCGCTGGG 2820
ACCTCCTCGG GCCACCTACG AGGCTTTTGT GCCACCAGAT GCCTTTGTAC ACGTGGACGA 2880
CTTCAGCTCT GCCCGTGAAC TGGCTGTCTT CCTCGTCAGC ATGAATGAGA GTCGTTATCG 2940
TGGCTTCTTT GCTTGGCGAG ACCGGCTCCG TGTGCGGCTC CTGGGTGACT GGAGGGAGCG 3000
CTTCTGCACC ATCTGTGCCC GCTACCCTTA CTTGCCCCGC AGCCAGGTCT ATGAAGACCT 3060
TGAAAGCTGG TTCCAGGCTT GAACTCCTGC TGCTGGGAGA GGCTGGATGG GTGGGAGACT 3120
GATGTTGAAA CCAAAGAGCT GGGCATCCAG GCTTTTGGTC ACCATGGCAC TACCCCAAGG 3180
CTTTTCCTGT TCAGTGAGCA GGAATTCAGG ATATAAGGAG AAGACTGGGC TGAGATACCC 3240
TGGTGGGCTT TAGAGTAGGG GCCCAGGATA AGAGACAATG AATTAATGAG GAGCATATGG 3300
GGAAGGTGGC TGAGGGTCCC TGACTTACCT TGACCCATGG CTGAAGGCTC CATGCCCATG 3360
GCTGGAGCTG GGACCCTACA CTTCTATAGT CAAGGTGCTT AGCCTCAAGG TTGCAGATGC 3420
ACCCTCTAGT ACTCTGGGTG CAGACTGTAC ACTGGGCGCA GGGGGTTGTG GAAGGACAGT 3480
GCAGATGATT CTGGGCTTTT GACACCACAG TTCCCCCAGG GAAAGAGGCA CTACTAATAA 3540
AAACACTGAC AGAAATCTCC TGGTCAAGTC TGTTAGGCAG CAGAGCTCGA ATTC 3594
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393
(B) TYPE: amino acid
(C) STRANDEDNESS: single
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CA 02247966 2006-09-28
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Pro Thr Pro Cys Pro Pro Ala Cys Leu Ser Thr Pro Gly Thr His
1 5 10 15
Arg Leu Leu Pro Phe Pro Asp Trp Lys Ala Pro Ser Trp Glu Ser Arg
20 25 30
Lys Glu Ala Thr Cys Asn Ser Ser Ser Pro Gly Pro Trp Ala Glu Pro
35 40 45
Thr Val Gln Met Asn Cys Ile Gly Tyr His Pro Thr Arg Arg Leu Arg
50 55 60
Ala Trp Gly Gly Leu Ala Gly Gly Ala Thr Phe Met Val Ile Trp Phe
65 70 75 80
Phe Trp Leu Trp Gly Ser Ala Pro Gly Ser Ala Pro Val Pro Gln Ser
85 90 95
Thr Leu Thr Ile Leu Ile Trp His Trp Pro Phe Thr Asn Arg Pro Pro
100 105 110
Glu Leu Pro Gly Asp Thr Cys Thr Arg Tyr Gly Met Ala Ser Cys Arg
115 120 125
Leu Ser Ala Asn Arg Ser Leu Leu Ala Ser Ala Asp Ala val Val Phe
130 135 140
His His Arg Glu Leu Gln Thr Arg Gln Ser Leu Leu Pro Leu Asp Gln
145 150 155 160
Arg Pro His Gly Gln Pro Trp Val Trp Ala Ser Met Glu Ser Pro Ser
165 170 175
Asn Thr His Gly Leu His Arg Phe Arg Gly Ile Phe Asn Trp Val Leu
180 185 190
Ser Tyr Arg Arg Asp Ser Asp Ile Phe Val Pro Tyr Gly Arg Leu Glu
195 200 205
Pro Leu Ser Gly Pro Thr Ser Pro Leu Pro Ala Lys Ser Arg Met Ala
210 215 220
Ala Trp Val Ile Ser Asn Phe Gln Glu Arg Gln Gln Arg Ala Lys Leu
225 230 235 240
Tyr Arg Gln Leu Ala Pro His Leu Gln Val Asp val Phe Gly Arg Ala
245 250 255
Ser Gly Arg Pro Leu Cys Ala Asn Cys Leu Leu Pro Thr Leu Ala Arg
260 265 270
Tyr Arg Phe Tyr Leu Ala Phe Glu Asn Ser Gln His Arg Asp Tyr Ile
275 280 285
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CA 02247966 2006-09-28
Thr Glu Lys Phe Trp Arg Asn Ala Leu Ala Ala Gly Ala Val Pro Val
290 295 300
Ala Leu Gly Pro Pro Arg Ala Thr Tyr Glu Ala Phe Val Pro Pro Asp
305 310 315 320
Ala Phe val His val Asp Asp Phe Ser Ser Ala Arg Glu Leu Ala val
325 330 335
Phe Leu val Ser Met Asn Glu ser Arg Tyr Arg Gly Phe Phe Ala Trp
340 345 350
Arg Asp Arg Leu Arg val Arg Leu Leu Gly Asp Trp Arg Glu Arg Phe
355 360 365
Cys Thr Ile Cys Ala Arg Tyr Pro Tyr Leu Pro Arg Ser Gln Val Tyr
370 375 380
Glu Asp Leu Glu Ser Trp Phe Gln Ala
385 390
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (artificial)
(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCGCGGATCC CACCATCCTT ATCTGGCACT GGCCTTTCAC C 41
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (artificial)
(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GCGCGGATCC AGTTCAAGCC TGGAACCAGC TTTCAAGGTC CTTC 44
Page 5
