A novel human glycosylsulfotransferase expressed in high endothelial cells (GST-3) and polypeptides related thereto, as well as nucleic acid compositions encoding the same, are provided. The subject polypeptides and nucleic acid compositions find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications. Also provided are methods of inhibiting selectin mediated binding events and methods of treating disease conditions associated therewith.

INTRODUCTION
 1. Field of the Invention
 The field of the invention is cell adhesion, particularly selectin mediated
 cell adhesion, as well as the treatment of disease conditions related
 thereto.
 2. Background of the Invention
 Sulfotransferases are enzymes that catalyze the transfer of a sulfate from
 a donor compound to an acceptor compound, usually placing the sulfate
 moiety at a specific location on the acceptor compound. There are a
 variety of different sulfotransferases which vary in activity, i.e. with
 respect to the donor and/or acceptor compounds with which they work. Known
 sulfotransferases include those acting on carbohydrate: heparin/heparan
 sulfate N-sulfotransferase (NST); chondroitin 6/keratan 6 sulfate
 sulfotransferase (C6ST/KSST); galactosylceramide 3'-sulfotransferase;
 heparan sulfate 2-sulfotransferase (Iduronic acid); 14NK-1
 sulfotransferase (3-glucuronic acid); heparan sulfate D-glucosamino
 3-O-sulfotransferase (3-OST);etc., as well as those acting on phenols,
 steroids and xenobiotics: aryl sulfotransferase I & II, hydroxy-steroid
 sulfotransferases I, II & III, dehydroepiandrosterone (DHEA); etc.
 Sulfotransferases play a central role in a variety of different
 biochemical mechanisms, as the presence of a sulfate moiety on a
 particular ligand is often required for a particular activity, e.g.
 binding.
 The presence of a sulfate moiety on selectin ligands has been shown to be
 important for selectin binding to occur. See Imai et al., Nature (1993)
 361:555-557 and Imai et al., Glycoconjugate J. (1993) 10:34-39, as well as
 U.S. Pat. No. 5,695,752. Several selectin ligands have, to date, been
 identified. The L-selectin endothelial ligands in mouse that have been
 identified are: CD34, GlyCAM-1, MAdCAM-1 and sgp200. In addition, PSGL-1
 has been identified as a leukocyte ligand for P-, E-, and L-selectin.
 Endothelial ligands for L-selectin in humans are still poorly defined, but
 include CD34 and podocalyxin.
 Selectin mediated binding plays an important and prominent role in a
 variety of biological processes. Selectins are lectin like cell adhesion
 molecules that mediate leukocyte-endothelial, leukocyte-leukocyte,
 leukocyte-platelet, platelet-endothelial and platelet-platelet
 interactions. One critical biological process in which selectin mediated
 binding plays a role is the maintenance of immune surveillance.
 Maintenance of immune surveillance depends on the constant recirculation of
 lymphocytes from the blood through the vascular wall into the tissues and
 eventually back into the blood. Lymphocyte recruitment from the blood into
 all secondary lymphoid organs (except the spleen) as well as into many
 sites of chronic inflammation is mediated by a specialized postcapillary
 venule called a high endothelial venule. These vessels are defined by the
 distinct, cuboidal morphology of their endothelial cells and their luminal
 presentation of ligands for the leukocyte adhesion molecule, L-selectin.
 This lectin-like adhesion molecule is expressed on all classes of
 leukocytes in the blood and is responsible for the initial tethering and
 rolling of a leukocyte on the endothelium prior to subsequent integrin
 mediated firm arrest and transmigration.
 Although selectin mediated binding events play a critical role in normal
 physiological processes, disease conditions do exist for which it is
 desired to regulate or modulate, e.g. limit or prevent, the amount of
 selectin mediated binding that occurs. Such conditions include: acute or
 chronic inflammation; autoimmune and related disorders, tissue rejection
 during transplantation, and the like.
 As the above conditions all result from selectin mediated binding events,
 there is great interest in the elucidation of the mechanisms underlying
 such binding events. There is also great interest in the identification of
 treatment methodologies for these and related disease conditions, as well
 the identification of active agents for use therein.
 As such, there is continued interest in the identification of participants
 in the selectin binding mechanism, including enzymatic agents, and the
 elucidation of their role(s) in selectin mediated binding events, as well
 as the development of therapies for disease conditions arising from such
 binding events.
 Relevant Literature
 Chondroitin-6-sulfotransferase is disclosed in EP 821 066, as well as in
 Fukuta et al., "Molecular Cloning and Characterization of Human Keratan
 Sulfate Gal-6-Sulfotransferase," J. Biol. Chem. (Dec. 19, 1997) 272:
 32321-32328; Habuchi et al., "Enzymatic Sulfation of Galactose Residue of
 Keratan Sulfate by Chondroitin 6-Sulfotransferase," Glycobiology (January
 1996) 6:51-57; llabuchi et al., "Enzymatic Sulfation of Galactose Residue
 of Keratan Sulfate by Chondroitin 6-Sulfate by Chondroitin
 6-Sulfotransferase," Glycobiology (January 1996) 6:51-57; Fukuta et al.,
 "Molecular Cloning and Expression of Chick Chondrocyte Chondroitin
 6-Sulfotransferase," J. Biol. Chem. (1995) 270: 18575-18580; and Habuchi
 et al., "Purification of Chondroitin 6-Sulfotransferase Secreted from
 Cultured Chick Embryo Chondrocytes," J. Biol. Chem. (1993) 268:
 21968-21974.
 References providing background information on selectin mediated binding
 include: Baumhueter et al., "Binding of L-Selectin to the Vascular
 Sialomucin CD34," Science (Oct. 15, 1993): 436-438; Boukerche et al., "A
 Monoclonal Antibody Directed Against a Granule Membrane Glycoprotein
 (GMP-140/PADGEM, .beta.-selectin, CD62P) Inhibits Ristocetin-Induced
 Platelet Aggregation," Br. J. Haematology (1996) 92: 442-451; Celi et al.,
 "Platelet-Leukocyte-Endothelial Cell Interaction on the Blood Vessel
 Wall," Seminars in Hematology (1997) 34: 327-335; Frenette et al.,
 "Platelets Roll on Stimulated Endothelium In Vivo: An Interaction Mediated
 by Endothelial .beta.-selectin," Proc. Natl. Acad. Sci. USA (August 1995)
 52:7450-7454; Girard & Springer, "High Endothelial Venules (HEVs):
 Specialized Endothelium for Lymphocyte Migration," Immun. Today (1995) 16:
 449-457; Hemmerich et al., "Sulfation Dependent Recognition of High
 Endothelial Venules (HEV)-Ligands by L-Selectin and Meca79, and
 Adhesion-Blocking Monoclonal Antibody," J. Exp. Medicine (December 1994)
 180: 2219-2226; 262 Lasky et al., "An Endothelial Ligand for L-Selectin is
 a Novel Mucin-Like Molecule," Cell (Jun. 12, 1992) 69:927-938; Rosen &
 Bertozzi, "The Selectins and Their Ligands," Current Opinion in Cell
 Biology (1994) 6: 663-673; and Sawada et al., "Specific Expression of a
 Complex Sialyl Lewis X Antigen On High Endothelial Venules of Human Lymph
 Nodes: Possible Candidate for L-selectin Ligand," Biochem. Biophys. Res.
 Comm. (May 28, 1993) 193: 337-347; as well as U.S. Pat. No. 5,580,862.
 U.S. Pat. No. 5,695,752 describes methods of treating inflammation through
 administration of sulfation inhibitors.
 SUMMARY OF THE INVENTION
 A novel human glycosyl sulfotransferase (GST-3) and polypeptides related
 thereto, as well as nucleic acid compositions encoding the same, are
 provided. The subject polypeptide and nucleic acid compositions find use
 in a variety of applications, including research, diagnostic, and
 therapeutic agent screening applications, as well as in treatment
 therapies. Also provided are methods of inhibiting selectin mediated
 binding events and methods of treating disease conditions associated
 therewith.

DETAILED DESCRIPTION OF THE INVENTION
 A novel human glycosyl transferase expressed in high endothelial cells
 (HEC) and polypeptides related thereto, as well as nucleic acid
 compositions encoding the same, are provided. The subject polypeptide
 and/or nucleic acid compositions find use in a variety of different
 applications, including research, diagnostic, and therapeutic agent
 screening/discovery/preparation applications. Also provided are methods of
 inhibiting selectin mediated binding events and methods of treating
 disease conditions associated therewith.
 Before the subject invention is further described, it is to be understood
 that the invention is not limited to the particular embodiments of the
 invention described below, as variations of the particular embodiments may
 be made and still fall within the scope of the appended claims. It is also
 to be understood that the terminology employed is for the purpose of
 describing particular embodiments, and is not intended to be limiting.
 Instead, the scope of the present invention will be established by the
 appended claims.
 In this specification and the appended claims, the singular forms "a,"
 "an," and "the" include plural reference unless the context clearly
 dictates otherwise. Unless defined otherwise, all technical and scientific
 terms used herein have the same meaning as commonly understood to one of
 ordinary skill in the art to which this invention belongs.
 Polypeptide Compositions
 A novel human glycosylsulfotransferase expressed in high endothelial cells
 (HEC), as well as polypeptide compositions related thereto, are provided.
 The term polyeptide composition as used herein refers to both the full
 length human protein as well as portions or fragments thereof. Also
 included in this term are variations of the naturally occurring human
 protein, where such variations are homologous or substantially similar to
 the naturally occurring protein, as described in greater detail below, as
 well as corresponding homologs from non-human species, such as other
 mammalian species. In the following description of the subject invention,
 the term GST-3 is used to refer not only to the human form of this novel
 sulfotransferase, but also to homologs thereof expressed in non-human
 species.
 The novel human glycosylsulfotransferase enzyme of the subject invention
 has been named human glycosyl sulfotransferase 3 or huGST-3. huGST-3 is a
 type 2 membrane protein having a relatively short transmembrane domain and
 a short amino-terminal cytoplasmic tail. huGST-3 has a 31% amino acid
 sequence identity with CS6T/KSST (Habuchi et al., J. Biol. Chem. (1995)
 240:4172-4179) as measured by using the "GAP" program (part of the
 Wisconsin Sequence Analysis Package available through the Genetics
 Computer Group, Inc. (Madison Wis.)), where the parameters are: Gap
 weight:12; length weight:4. huGST-3 is capable of sulfating selectin
 ligands, particularly L-selectin ligands, e.g. GlyCAM-1. By sulfating
 selectin ligands is meant that huGST-3 is capable of catalyzing the
 transfer of a sulfate group from a donor compound to a position on a
 selectin ligand precursor as acceptor compound. Donor compounds from which
 huGST-3 obtains sulfate groups for transfer to acceptor ligand compounds
 include 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the like.
 Selectin ligands capable of being sulfated through huGST-3 action include
 E-, P- and L-selectin ligands, particularly L-selectin ligands, such as
 GlyCAM-1, CD34, MAdCAM-1, Sgp200, podocalyxin, and the like. huGST-3 is
 strongly predicted to have Gat-6-O sulfotransferase activity.
 Human GST-3 is a 386 amino acid protein having an amino acid sequence as
 shown in FIG. 2 and identified as SEQ ID NO:02. huGST-3 has a molecular
 weight based on its amino acid of about 45 kDa to 46 kDa, and more
 specifically from about 45100 to 45200 dalton, and specifically 45104
 dalton (using DNA Strider 1.2 software). Since GST-3 is glycosylated, its
 true molecular weight is greater, and is likely to be in the range from
 about 45 to 85 kDa, and more likely from about 50 kDa to 65 kDa.
 Expression of GST-3 in humans is highly restricted. For example, huGST-3
 is expressed in HEC but not tonsillar lymphocytes, or primary cultured
 human umbilical vein endothelial cells (HUVEC).
 In addition to the huGST-3, also provided are GST-3 proteins that are have
 the same expression pattern in humans and huGST-3, i.e. are highly
 restricted and expressed in HEC but not HUVEC or lymphocytes. huGST-3
 homologs or proteins (or fragments thereof) from nonhuman species are also
 provided, including mammals, such as: rodents, e.g. mice, rats; domestic
 animals, e.g. horse, cow, dog, cat; and humans, as well as non-mammalian
 species, e.g. avian, and the like. By homolog is meant a protein having at
 least about 35%, usually at least about 40% and more usually at least
 about 60% amino acid sequence identity to the huGST-3 protein.
 Also provided are GST-3 proteins that are substantially identical to the
 huGST-3 protein, where by substantially identical is meant that the
 protein as an amino acid sequence identity to the sequence of huGST-3 of
 at least about 35%, usually at least about 40% and more usually at least
 about 60%.
 The GST-3 proteins of the subject invention (e.g. huGST-3 or a homolog
 thereof) are present in a non-naturally occurring environment, e.g. are
 separated from their naturally occurring environment. In certain
 embodiments, the subject GST-3 is present in a composition that is
 enriched for GST-3 as compared to GST-3 in its naturally occurring
 environment. As such, purified GST-3 is provided, where by purified is
 meant that GST-3 is present in a composition that is substantially free of
 non-GST-3 proteins, where by substantially free is meant that less than
 90%, usually less than 60% and more usually less than 50% of the
 composition is made up of non-GST-3 proteins. The GST-3 of the subject
 invention may also be present as an isolate, by which is meant that the
 GST-3 is substantially free of both non-GST-3 proteins and other naturally
 occurring biologic molecules, such as oligosaccharides, polynucleotides
 and fragments thereof, and the like, where substantially free in this
 instance means that less than 70%, usually less than 60% and more usually
 less than 50% of the composition containing the isolated GST-3 is a
 non-GST-3 naturally occurring biological molecule. In certain embodiments,
 the GST-3 is present in substantially pure form, where by substantially
 pure form is meant at least 95%, usually at least 97% and more usually at
 least 99% pure.
 In addition to the naturally occurring GST-3 protein, GST-3 polypeptides
 which vary from the naturally occurring GST-3 protein are also provided.
 By GST-3 polypeptide is meant an amino acid sequence encoded by an open
 reading frame (ORF) of the GST-3 gene, described in greater detail below,
 including the full length GST-3 protein and fragments thereof,
 particularly biologically active fragments and/or fragments corresponding
 to functional domains, e.g. acceptor binding site (postulated to be the
 most 5' consensus region A (see experimental section infra), the donor
 binding site, e.g. VRYEDL, and the like; and including fusions of the
 subject polypeptides to other proteins or parts thereof. Fragments of
 interest will typically be at least about 10 aa in length, usually at
 least about 50 aa in length, and may be as long as 300 aa in length or
 longer, but will usually not exceed about 1000 aa in length, where the
 fragment will have a stretch of amino acids that is identical to GST-3 of
 at least about 10 aa, and usually at least about 15 aa, and in many
 embodiments at least about 50 aa in length.
 The subject GST-3 proteins and polypeptides may be obtained from naturally
 occurring sources or synthetically produced. Where obtained from naturally
 occurring sources, the source chosen will generally depend on the species
 from which the GST-3 is to be derived. For example, huGST-3 is generally
 derived from endothelial cells of high endothelial venules (HEV) of human
 secondary lymphoid organs, such as tonsils. The subject GST-3 may also be
 derived from synthetic means, e.g. by expressing a recombinant gene
 encoding GST-3 in a suitable host, as described in greater detail below.
 Any convenient protein purification procedures may be employed, where
 suitable protein purification methodologies are described in Guide to
 Protein Purification, (Deuthser ed.) (Academic Press, 1990). For example,
 a lysate may prepared from the original source, e.g. HEC or the expression
 host, and purified using HPLC, exclusion chromatography, gel
 electrophoresis, affinity chromatography, and the like.
 Nucleic Acid Compositions
 Also provided are nucleic acid compositions encoding GST-3 proteins or
 fragments thereof. By nucleic acid composition is meant a composition
 comprising a sequence of DNA having an open reading frame that encodes
 GST-3, i.e. a GST-3 gene, and is capable, under appropriate conditions, of
 being expressed as GST-3. Also encompassed in this term are nucleic acids
 that are homologous or substantially similar or identical to the nucleic
 acids encoding GST-3 proteins. Thus, the subject invention provides genes
 encoding huGST-3 and homologs thereof. The human GST-3 gene has the
 nucleic acid sequence shown in FIG. 1 and identified as SEQ ID NO:01,
 infra.
 The source of homologous genes may be any species, e.g., primate species,
 particularly human; rodents, such as rats and mice, canines, felines,
 bovines, ovines, equines, yeast, nematodes, etc. Between mammalian
 species, e.g., human and mouse, homologs have substantial sequence
 similarity, e.g. at least 75% sequence identity, usually at least 90%,
 more usually at least 95% between nucleotide sequences. Sequence
 similarity is calculated based on a reference sequence, which may be a
 subset of a larger sequence, such as a conserved motif, coding region,
 flanking region, etc. A reference sequence will usually be at least about
 18 nt long, more usually at least about 30 nt long, and may extend to the
 complete sequence that is being compared. Algorithms for sequence analysis
 are known in the art, such as BLAST, described in Altschul et al. (1990),
 J. Mol. Biol. 215:403-10 (using default settings). The sequences provided
 herein are essential for recognizing GST-3-related and homologous proteins
 in database searches.
 Nucleic acids encoding the GST-3 protein and GST-3 polypeptides of the
 subject invention may be cDNA or genomic DNA or a fragment thereof. The
 term "GST-3 gene" shall be intended to mean the open reading frame
 encoding specific GST-3 proteins and polypeptides, and GST-3 introns, as
 well as adjacent 5' and 3' non-coding nucleotide sequences involved in the
 regulation of expression, up to about 20 kb beyond the coding region, but
 possibly further in either direction. The gene may be introduced into an
 appropriate vector for extrachromosomal maintenance or for integration
 into a host genome.
 The term "cDNA" as used herein is intended to include all nucleic acids
 that share the arrangement of sequence elements found in native mature
 mRNA species, where sequence elements are exons and 3' and 5' non-coding
 regions. Normally mRNA species have contiguous exons, with the intervening
 introns, when present, being removed by nuclear RNA splicing, to create a
 continuous open reading frame encoding a GST-3 protein.
 A genomic sequence of interest comprises the nucleic acid present between
 the initiation codon and the stop codon, as defined in the listed
 sequences, including all of the introns that are normally present in a
 native chromosome. It may further include the 3' and 5' untranslated
 regions found in the mature mRNA. It may further include specific
 transcriptional and translational regulatory sequences, such as promoters,
 enhancers, etc., including about 1 kb, but possibly more, of flanking
 genomic DNA at either the 5' or 3' end of the transcribed region. The
 genomic DNA may be isolated as a fragment of 100 kbp or smaller; and
 substantially free of flanking chromosomal sequence. The genomic DNA
 flanking the coding region, either 3' or 5', or internal regulatory
 sequences as sometimes found in introns, contains sequences required for
 proper tissue and stage specific expression.
 The nucleic acid compositions of the subject invention may encode all or a
 part of the subject GST-3 protein. Double or single stranded fragments may
 be obtained from the DNA sequence by chemically synthesizing
 oligonucleotides in accordance with conventional methods, by restriction
 enzyme digestion, by PCR amplification, etc. For the most part, DNA
 fragments will be of at least 15 nt, usually at least 18 nt or 25 nt, and
 may be at least about 50 nt.
 The GST-3 genes are isolated and obtained in substantial purity, generally
 as other than an intact chromosome. Usually, the DNA will be obtained
 substantially free of other nucleic acid sequences that do not include a
 GST-3 sequence or fragment thereof, generally being at least about 50%,
 usually at least about 90% pure and are typically "recombinant", i.e.
 flanked by one or more nucleotides with which it is not normally
 associated on a naturally occurring chromosome.
 Preparation of GST-3 Polypeptides
 In addition to the plurality of uses described in greater detail in
 following sections, the subject nucleic acid compositions find use in the
 preparation of all or a portion of the GST-3 polypeptides, as described
 above. For expression, an expression cassette may be employed. The
 expression vector will provide a transcriptional and translational
 initiation region, which may be inducible or constitutive, where the
 coding region is operably linked under the transcriptional control of the
 transcriptional initiation region, and a transcriptional and translational
 termination region. These control regions may be native to a GST-3 gene,
 or may be derived from exogenous sources.
 Expression vectors generally have convenient restriction sites located near
 the promoter sequence to provide for the insertion of nucleic acid
 sequences encoding heterologous proteins. A selectable marker operative in
 the expression host may be present. Expression vectors may be used for the
 production of fusion proteins, where the exogenous fusion peptide provides
 additional functionality, i.e. increased protein synthesis, stability,
 reactivity with defined antisera, an enzyme marker, e.g. -galactosidase,
 etc.
 Expression cassettes may be prepared comprising a transcription initiation
 region, the gene or fragment thereof, and a transcriptional termination
 region. Of particular interest is the use of sequences that allow for the
 expression of functional epitopes or domains, usually at least about 8
 amino acids in length, more usually at least about 15 amino acids in
 length, to about 25 amino acids, and up to the complete open reading frame
 of the gene. After introduction of the DNA, the cells containing the
 construct may be selected by means of a selectable marker, the cells
 expanded and then used for expression.
 GST-3 proteins and polypeptides may be expressed in prokaryotes or
 eukaryotes in accordance with conventional ways, depending upon the
 purpose for expression. For large scale production of the protein, a
 unicellular organism, such as E. coli, B. subtilis, S. cerevisiace, insect
 cells in combination with baculovirus vectors, or cells of a higher
 organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may
 be used as the expression host cells. In some situations, it is desirable
 to express the GST-3 gene in eukaryotic cells, where the GST-3 protein
 will benefit from native folding and post-translational modifications.
 Small peptides can also be synthesized in the laboratory. Polypeptides
 that are subsets of the complete GST-3 sequence may be used to identify
 and investigate parts of the protein important for function.
 Uses of the Subject GST-3 Polypeptide and Nucleic Acid Compositions
 The subject polypeptide and nucleic acid compositions find use in a variety
 of different applications, including research, diagnostic, and therapeutic
 agent screening/discovery/preparation applications, as well as therapeutic
 compositions.
 Research Applications
 The subject nucleic acid compositions find use in a variety of research
 applications. Research applications of interest include: the
 identification of huGST-3 homologs; as a source of novel promoter
 elements; the identification of GST-3 expression regulatory factors; as
 probes and primers in hybridization applications, e.g. PCR; the
 identification of expression patterns in biological specimens; the
 preparation of cell or animal models for GST-3 function; the preparation
 of in vitro models for GST-3 function; etc.
 Homologs of GST-3 are identified by any of a number of methods. A fragment
 of the provided cDNA may be used as a hybridization probe against a cDNA
 library from the target organism of interest, where low stringency
 conditions are used. The probe may be a large fragment, or one or more
 short degenerate primers. Nucleic acids having sequence similarity are
 detected by hybridization under low stringency conditions, for example, at
 50.degree. C. and 6.times.SSC (0.9 M sodium chloride/0.09 M sodium
 citrate) and remain bound when subjected to washing at 55.degree. C. in
 1.times.SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequence
 identity may be determined by hybridization under stringent conditions,
 for example, at 50.degree. C. or higher and 0.1.times.SSC (15 mM sodium
 chloride/01.5 mM sodium citrate). Nucleic acids having a region of
 substantial identity to the provided GST-3 sequences, e.g. allelic
 variants, genetically altered versions of the gene, etc., bind to the
 provided GST-3 sequences under stringent hybridization conditions. By
 using probes, particularly labeled probes of DNA sequences, one can
 isolate homologous or related genes.
 The sequence of the 5' flanking region may be utilized for promoter
 elements, including enhancer binding sites, that provide for developmental
 regulation in tissues where GST-3 is expressed. The tissue specific
 expression is useful for determining the pattern of expression, and for
 providing promoters that mimic the native pattern of expression. Naturally
 occurring polymorphisms in the promoter region are useful for determining
 natural variations in expression, particularly those that may be
 associated with disease.
 Alternatively, mutations may be introduced into the promoter region to
 determine the effect of altering expression in experimentally defined
 systems. Methods for the identification of specific DNA motifs involved in
 the binding of transcriptional factors are known in the art, e.g. sequence
 similarity to known binding motifs, gel retardation studies, etc. For
 examples, see Blackwell et al. (1995), Mol. Med. 1:194-205; Mortlock et
 al. (1996), Genome Res. 6:327-33; and Joulin and Richard-Foy (1995), Eur.
 J. Biochem. 232:620-626.
 The regulatory sequences may be used to identify cis acting sequences
 required for transcriptional or translational regulation of GST-3
 expression, especially in different tissues or stages of development, and
 to identify cis acting sequences and transacting factors that regulate or
 mediate GST-3 expression. Such transcription or translational control
 regions may be operably linked to a GST-3 gene in order to promote
 expression of wild type or altered GST-3 or other proteins of interest in
 cultured cells, or in embryonic, fetal or adult tissues, and for gene
 therapy.
 Small DNA fragments are useful as primers for PCR, hybridization screening
 probes, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for
 production of the encoded polypeptide, as described in the previous
 section. For use in amplification reactions, such as PCR, a pair of
 primers will be used. The exact composition of the primer sequences is not
 critical to the invention, but for most applications the primers will
 hybridize to the subject sequence under stringent conditions, as known in
 the art. It is preferable to choose a pair of primers that will generate
 an amplification product of at least about 50 nt, preferably at least
 about 100 nt. Algorithms for the selection of primer sequences are
 generally known, and are available in commercial software packages.
 Amplification primers hybridize to complementary strands of DNA, and will
 prime towards each other.
 The DNA may also be used to identify expression of the gene in a biological
 specimen. The manner in which one probes cells for the presence of
 particular nucleotide sequences, as genomic DNA or RNA, is well
 established in the literature. Briefly, DNA or mRNA is isolated from a
 cell sample. The mRNA may be amplified by RT-PCR, using reverse
 transcriptase to form a complementary DNA strand, followed by polymerase
 chain reaction amplification using primers specific for the subject DNA
 sequences. Alternatively, the mRNA sample is separated by gel
 electrophoresis, transferred to a suitable support, e.g. nitrocellulose,
 nylon, etc., and then probed with a fragment of the subject DNA as a
 probe. Other techniques, such as oligonucleotide ligation assays, in situ
 hybridizations, and hybridization to DNA probes arrayed on a solid chip
 may also find use. Detection of mRNA hybridizing to the subject sequence
 is indicative of GST-3 gene expression in the sample.
 The sequence of a GST-3 gene, including flanking promoter regions and
 coding regions, may be mutated in various ways known in the art to
 generate targeted changes in promoter strength, sequence of the encoded
 protein, etc. The DNA sequence or protein product of such a mutation will
 usually be substantially similar to the sequences provided herein, i.e.
 will differ by at least one nucleotide or amino acid, respectively, and
 may differ by at least two but not more than about ten nucleotides or
 amino acids. The sequence changes may be substitutions, insertions,
 deletions, or a combination thereof. Deletions may further include larger
 changes, such as deletions of a domain or exon. Other modifications of
 interest include epitope tagging, e.g. with the etc. For studies of
 subcellular localization, fusion proteins with green fluorescent proteins
 (GFP) may be used.
 Techniques for in vitro mutagenesis of cloned genes are known. Examples of
 protocols for site specific mutagenesis may be found in Gustin et al.
 (1993), Biotechniques 14:22; Barany (1985), Gene 37:111-23; Colicelli et
 al. (1985), Mol. Gen. Genet. 199:537-9; and Prentki et al. (1984), Gene
 29:303-13. Methods for site specific mutagenesis can be found in Sambrook
 et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.
 15.3-15.108; Weiner et al. (1993), Gene 126:35-41; Sayers et al. (1992),
 Biotechniques 13:592-6; Jones and Winistorfer (1992), Biotechniques
 12:528-30; Barton et al. (1990), Nucleic Acids Res 18:7349-55; Marotti and
 Tomich (1989), Gene Anal. Tech. 6:67-70; and Zhu (1989), Anal Biochem
 177:120-4. Such mutated genes may be used to study structure-function
 relationships of GST-3, or to alter properties of the protein that affect
 its function or regulation.
 The subject nucleic acids can be used to generate transgenic, non-human
 animals or site specific gene modifications in cell lines. Transgenic
 animals may be made through homologous recombination, where the normal
 gst-3 locus is altered. Alternatively, a nucleic acid construct is
 randomly integrated into the genome. Vectors for stable integration
 include plasmids, retroviruses and other animal viruses, YACs, and the
 like.
 The modified cells or animals are useful in the study of gst-3 function and
 regulation. For example, a series of small deletions and/or substitutions
 may be made in the host's native gst-3 gene to determine the role of
 different exons in oncogenesis, signal transduction, etc. Of interest are
 the use of gst-3 to construct transgenic animal models for cancer, where
 expression of GST-3 is specifically reduced or absent. Specific constructs
 of interest include anti-sense gst-3, which will block GST-3 expression,
 expression of dominant negative gst-3 mutations, and over-expression of
 GST-3 genes. Where a gst-3 sequence is introduced, the introduced sequence
 may be either a complete or partial sequence of a gst-3 gene native to the
 host, or may be a complete or partial gst-3 sequence that is exogenous to
 the host animal, e.g., a human GST-3 sequence. A detectable marker, such
 as lac Z may be introduced into the gst-3 locus, where upregulation of
 gst-3 expression will result in an easily detected change in phenotype.
 One may also provide for expression of the gst-3 gene or variants thereof
 in cells or tissues where it is not normally expressed, at levels not
 normally present in such cells or tissues, or at abnormal times of
 development.
 DNA constructs for homologous recombination will comprise at least a
 portion of the human GST-3 gene or of a gst-3 gene native to the species
 of the host animal, wherein the gene has the desired genetic
 modification(s), and includes regions of homology to the target locus. DNA
 constructs for random integration need not include regions of homology to
 mediate recombination. Conveniently, markers for positive and negative
 selection are included. Methods for generating cells having targeted gene
 modifications through homologous recombination are known in the art. For
 various techniques for transfecting mammalian cells, see Keown et al.
 (1990), Meth. Enzymol. 185:527-537.
 For embryonic stem (ES) cells, an ES cell line may be employed, or
 embryonic cells may be obtained freshly from a host, e.g. mouse, rat,
 guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder
 layer or grown in the presence of leukemia inhibiting factor (LIF). When
 ES or embryonic cells have been transformed, they may be used to produce
 transgenic animals. After transformation, the cells are plated onto a
 feeder layer in an appropriate medium. Cells containing the construct may
 be detected by employing a selective medium. After sufficient time for
 colonies to grow, they are picked and analyzed for the occurrence of
 homologous recombination or integration of the construct. Those colonies
 that are positive may then be used for embryo manipulation and blastocyst
 injection. Blastocysts are obtained from 4 to 6 week old superovulated
 females. The ES cells are trypsinized, and the modified cells are injected
 into the blastocoel of the blastocyst. After injection, the blastocysts
 are returned to each uterine horn of pseudopregnant females. Females are
 then allowed to go to term and the resulting offspring screened for the
 construct. By providing for a different phenotype of the blastocyst and
 the genetically modified cells, chimeric progeny can be readily detected.
 The chimeric animals are screened for the presence of the modified gene and
 males and females having the modification are mated to produce homozygous
 progeny. If the gene alterations cause lethality at some point in
 development, tissues or organs can be maintained as allogeneic or congenic
 grafts or transplants, or in in vitro culture. The transgenic animals may
 be any non-human mammal, such as laboratory animals, domestic animals,
 etc. The transgenic animals may be used in functional studies, drug
 screening, etc., e.g. to determine the effect of a candidate drug on GST-3
 activity.
 The availability of a number of components in the leukocyte trafficking
 mechanism, such as GlyCAM-1, L-selectin and the subject GST-3 enzyme, and
 the like, allows in vitro reconstruction of the mechanism, i.e. the
 production of an in vitro model.
 Diagnostic Applications
 Also provided are methods of diagnosing disease states based on observed
 levels of GST-3 or the expression level of the GST-3 gene in a biological
 sample of interest. Samples, as used herein, include biological fluids
 such as semen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis
 fluid and the like; organ or tissue culture derived fluids; and fluids
 extracted from physiological tissues. Also included in the term are
 derivatives and fractions of such fluids. The cells may be dissociated, in
 the case of solid tissues, or tissue sections may be analyzed.
 Alternatively a lysate of the cells may be prepared.
 A number of methods are available for determining the expression level of a
 gene or protein in a particular sample. Diagnosis may be performed by a
 number of methods to determine the absence or presence or altered amounts
 of normal or abnormal GST-3 in a patient sample. For example, detection
 may utilize staining of cells or histological sections with labeled
 antibodies, performed in accordance with conventional methods. Cells are
 permeabilized to stain cytoplasmic molecules. The antibodies of interest
 are added to the cell sample, and incubated for a period of time
 sufficient to allow binding to the epitope, usually at least about 10
 minutes. The antibody may be labeled with radioisotopes, enzymes,
 fluorescers, chemiluminescers, or other labels for direct detection.
 Alternatively, a second stage antibody or reagent is used to amplify the
 signal. Such reagents are well known in the art. For example, the primary
 antibody may be conjugated to biotin, with horseradish
 peroxidase-conjugated avidin added as a second stage reagent.
 Alternatively, the secondary antibody conjugated to a flourescent
 compound, e.g. fluorescein, rhodamine, Texas red, etc. Final detection
 uses a substrate that undergoes a color change in the presence of the
 peroxidase. The absence or presence of antibody binding may be determined
 by various methods, including flow cytometry of dissociated cells,
 microscopy, radiography, scintillation counting, etc.
 Alternatively, one may focus on the expression of GST-3. Biochemical
 studies may be performed to determine whether a sequence polymorphism in a
 GST-3 coding region or control regions is associated with disease. Disease
 associated polymorphisms may include deletion or truncation of the gene,
 mutations that alter expression level, that affect the activity of the
 protein, etc.
 Changes in the promoter or enhancer sequence that may affect expression
 levels of GST-3 can be compared to expression levels of the normal allele
 by various methods known in the art. Methods for determining promoter or
 enhancer strength include quantitation of the expressed natural protein;
 insertion of the variant control element into a vector with a reporter
 gene such as .beta.-galactosidase, luciferase, chloramphenicol
 acetyltransferase, etc. that provides for convenient quantitation; and the
 like.
 A number of methods are available for analyzing nucleic acids for the
 presence of a specific sequence, e.g. a disease associated polymorphism.
 Where large amounts of DNA are available, genomic DNA is used directly.
 Alternatively, the region of interest is cloned into a suitable vector and
 grown in sufficient quantity for analysis. Cells that express GST-3 may be
 used as a source of mRNA, which may be assayed directly or reverse
 transcribed into cDNA for analysis. The nucleic acid may be amplified by
 conventional techniques, such as the polymerase chain reaction (PCR), to
 provide sufficient amounts for analysis. The use of the polymerase chain
 reaction is described in Saiki, et al. (1985), Science 239:487, and a
 review of techniques may be found in Sambrook, et al. Molecular Cloning: A
 Laboratory Manual, CSH Press 1989, pp.14.2-14.33. Alternatively, various
 methods are known in the art that utilize oligonucleotide ligation as a
 means of detecting polymorphisms, for examples see Riley et al. (1990),
 Nucl. Acids Res. 18:2887-2890; and Delahunty et al. (1996), Am. J. Hum.
 Genet. 58:1239-1246.
 A detectable label may be included in an amplification reaction. Suitable
 labels include fluorochromes, e.g fluorescein isothiocyanate (FITC),
 rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein
 (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),
 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein
 (HIEX), 5-carboxyfluorescein (5-FAM) or
 N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g.
 .sup.32 P, .sup.35 S, .sup.3 H; etc. The label may be a two stage system,
 where the amplified DNA is conjugated to biotin, haptens, etc. having a
 high affinity binding partner, e.g. avidin, specific antibodies, etc.,
 where the binding partner is conjugated to a detectable label. The label
 may be conjugated to one or both of the primers. Alternatively, the pool
 of nucleotides used in the amplification is labeled, so as to incorporate
 the label into the amplification product.
 The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by
 one of a number of methods known in the art. The nucleic acid may be
 sequenced by dideoxy or other methods, and the sequence of bases compared
 to a wild-type GST-3 sequence. Hybridization with the variant sequence may
 also be used to determine its presence, by Southern blots, dot blots, etc.
 The hybridization pattern of a control and variant sequence to an array of
 oligonucleotide probes immobilized on a solid support, as described in
 U.S. Pat. No. 5,445,934, or in WO 95/35505, may also be used as a means of
 detecting the presence of variant sequences. Single strand conformational
 polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis
 (DGGE), and heteroduplex analysis in gel matrices are used to detect
 conformational changes created by DNA sequence variation as alterations in
 electrophoretic mobility. Alternatively, where a polymorphism creates or
 destroys a recognition site for a restriction endonuclease, the sample is
 digested with that endonuclease, and the products size fractionated to
 determine whether the fragment was digested. Fractionation is performed by
 gel or capillary electrophoresis, particularly acrylamide or agarose gels.
 Screening for mutations in GST-3 may be based on the functional or
 antigenic characteristics of the protein. Protein truncation assays are
 useful in detecting deletions that may affect the biological activity of
 the protein. Various immunoassays designed to detect polymorphisms in
 GST-3 proteins may be used in screening. Where many diverse genetic
 mutations lead to a particular disease phenotype, functional protein
 assays have proven to be effective screening tools. The activity of the
 encoded GST-3 protein may be determined by comparison with the wild-type
 protein.
 Diagnostic methods of the subject invention in which the level of GST-3
 expression is of interest will typically involve comparison of the GST-3
 nucleic acid abundance of a sample of interest with that of a control
 value to determine any relative differences, where the difference may be
 measured qualitatively and/or quantitatively, which differences are then
 related to the presence or absence of an abnormal GST-3 expression
 pattern. A variety of different methods for determine the nucleic acid
 abundance in a sample are known to those of skill in the art, where
 particular methods of interest include those described in: Pietu et al.,
 Genome Res. (June 1996) 6: 492-503; Zhao et al., Gene (Apr. 24, 1995) 156:
 207-213; Soares , Curr. Opin. Biotechnol. (October 1997) 8: 542-546;
 Raval, J. Pharmacol Toxicol Methods (November 1994) 32: 125-127; Chalifour
 et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz & Tuan, Mol.
 Biotechnol. (December 19960 6: 225-230; Hong et al., Bioscience Reports
 (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143: 298. Also of
 interest are the methods disclosed in WO 97/27317, the disclosure of which
 is herein incorporated by reference.
 Screening Assays
 The subject GST-3 polypeptides find use in various screening assays
 designed to identify therapeutic agents. Thus, one can use a cell model
 such as a host cell, e.g. COS7 cell, which has been cotransfected with a
 selectin ligand cDNA, e.g. GlyCAM-1 and a GST-3 vector. One can then label
 the transfectants with a labeled sulfate, e.g. .sup.35 S-labeled sulfate,
 and compare the amount of sulfate incorporation into GlyCAM-1 in the
 presence and absence of a candidate inhibitor compound. Alternatively, in
 a cell-free enzyme activity assay, recombinant GST-3 polypeptide may be
 combined with .sup.35 S-labeled sulfate donor such as [.sup.35 S]-PAPS,
 candidate inhibitor compound, and an acceptor molecule, which may be a
 synthetic carbohydrate mimicking structures found in mature and/or
 immature L-selectin ligands, or a simple nucleophile capable of accepting
 sulfate (such as phenolic compunds, and the like). The amount of [.sup.35
 S]-sulfate transferred to the receptor by the candidate agent is then
 determined by counting the acceptor-associated radioactivity or product
 quantitation with an antibody specific for the sulfated acceptor, or in a
 suitable scintillation proximity assay format. Alternatively, the
 candidate inhibitor compound may also be combined with a selectin, a
 non-sulfated selectin ligand precursor, GST-3 and a sulfate donor compound
 under physiological conditions and the resultant amount of ligand which is
 capable of binding to the selectin is determined. Depending on the
 particular method, one or more of, usually one of, the specified
 components may be labeled, where by labeled is meant that the components
 comprise a detectable moiety, e.g. a fluorescent or radioactive tag, or a
 member of a signal producing system, e.g. biotin for binding to an
 enzyme-streptavidin conjugate in which the enzyme is capable of converting
 a substrate to a chromogenic product.
 A variety of other reagents may be included in the screening assay. These
 include reagents like salts, neutral proteins, e.g. albumin, detergents,
 etc that are used to facilitate optimal protein--protein binding and/or
 reduce non-specific or background interactions. Reagents that improve the
 efficiency of the assay, such as protease inhibitors, nuclease inhibitors,
 anti-microbial agents, etc. may be used.
 The above screening methods may be designed a number of different ways,
 where a variety of assay configurations and protocols may be employed, as
 are known in the art. For example, one of the components may be bound to a
 solid support, and the remaining components contacted with the support
 bound component. The above components of the method may be combined at
 substantially the same time or at different times. Incubations are
 performed at any suitable temperature, typically between 4 and 40.degree.
 C. Incubation periods are selected for optimum activity, but may also be
 optimized to facilitate rapid high-throughput screening. Typically between
 0.1 and 1 hours will be sufficient. Following the contact and incubation
 steps, the subject methods will generally, though not necessarily, further
 include a washing step to remove unbound components, where such a washing
 step is generally employed when required to remove label that would give
 rise to a background signal during detection, such as radioactive or
 fluorescently labeled non-specifically bound components. Following the
 optional washing step, the presence of bound selectin-ligand complexes
 will then be detected.
 A variety of different candidate agents may be screened by the above
 methods. Candidate agents encompass numerous chemical classes, though
 typically they are organic molecules, preferably small organic compounds
 having a molecular weight of more than 50 and less than about 2,500
 daltons. Candidate agents comprise functional groups necessary for
 structural interaction with proteins, particularly hydrogen bonding, and
 typically include at least an amine, carbonyl, hydroxyl or carboxyl group,
 preferably at least two of the functional chemical groups. The candidate
 agents often comprise cyclical carbon or heterocyclic structures and/or
 aromatic or polyaromatic structures substituted with one or more of the
 above functional groups. Candidate agents are also found among
 biomolecules including peptides, saccharides, fatty acids, steroids,
 purines, pyrimidines, derivatives, structural analogs or combinations
 thereof.
 Candidate agents are obtained from a wide variety of sources including
 libraries of synthetic or natural compounds. For example, numerous means
 are available for random and directed synthesis of a wide variety of
 organic compounds and biomolecules, including expression of randomized
 oligonucleotides and oligopeptides. Alternatively, libraries of natural
 compounds in the form of bacterial, fungal, plant and animal extracts are
 available or readily produced. Additionally, natural or synthetically
 produced libraries and compounds are readily modified through conventional
 chemical, physical and biochemical means, and may be used to produce
 combinatorial libraries. Known pharmacological agents may be subjected to
 directed or random chemical modifications, such as acylation, alkylation,
 esterification, amidification, etc. to produce structural analogs.
 GST-3 Nucleic Acid and Polypeptide Therapeutic Compositions
 The nucleic acid compositions of the subject invention also find use as
 therapeutic agents in situations where one wishes to enhance GST-3
 activity in a host. The GST-3 genes, gene fragments, or the encoded GST-3
 protein or protein fragments are useful in gene therapy to treat disorders
 associated with GST-3 defects. Expression vectors may be used to introduce
 the GST-3 gene into a cell. Such vectors generally have convenient
 restriction sites located near the promoter sequence to provide for the
 insertion of nucleic acid sequences. Transcription cassettes may be
 prepared comprising a transcription initiation region, the target gene or
 fragment thereof, and a transcriptional termination region. The
 transcription cassettes may be introduced into a variety of vectors, e.g.
 plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the
 vectors are able to transiently or stably be maintained in the cells,
 usually for a period of at least about one day, more usually for a period
 of at least about several days to several weeks.
 The gene or GST-3 protein may be introduced into tissues or host cells by
 any number of routes, including viral infection, microinjection, or fusion
 of vesicles. Jet injection may also be used for intramuscular
 administration, as described by Furth et al. (1992), Anal Biochem
 205:365-368. The DNA may be coated onto gold microparticles, and delivered
 intradermally by a particle bombardment device, or "gene gun" as described
 in the literature (see, for example, Tang et al. (1992), Nature
 356:152-154), where gold microprojectiles are coated with the DNA, then
 bombarded into skin cells.
 Methods of Modulating Selectin Mediated Binding Events
 Also provided are methods of regulating, including modulating and
 inhibiting, selectin mediated binding events. The selectin receptor of the
 selectin mediated binding event will generally be a receptor which binds
 to a sulfated ligand under physiological conditions and is a member of the
 selectin family of receptors that have an amino terminal C-type lectin
 domain followed by an EFG-like domain, a variable number of short
 consensus repeats known as SCR, CRP or sushi domains, and share greater
 than 50% homology in their lectin and EFG domains. Of interest is the
 modulation of selectin binding events in which the selectin is L-, P-, or
 E-selectin. Of particular interest are L-selecting mediated binding
 events.
 Where the selectin mediated binding event occurs in vivo in a host, an
 effective amount of active agent that modulates the activity, usually
 reduces the activity, of GST-3 in vivo, is administered to the host. The
 active agent may be a variety of different compounds, including a
 naturally occurring or synthetic small molecule compound, an antibody,
 fragment or derivative thereof, an antisense composition, and the like.
 Naturally occurring or synthetic small molecule compounds of interest
 include numerous chemical classes, though typically they are organic
 molecules, preferably small organic compounds having a molecular weight of
 more than 50 and less than about 2,500 daltons. Candidate agents comprise
 functional groups necessary for structural interaction with proteins,
 particularly hydrogen bonding, and typically include at least an amine,
 carbonyl, hydroxyl or carboxyl group, preferably at least two of the
 functional chemical groups. The candidate agents often comprise cyclical
 carbon or heterocyclic structures and/or aromatic or polyaromatic
 structures substituted with one or more of the above functional groups.
 Candidate agents are also found among biomolecules including peptides,
 saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,
 structural analogs or combinations thereof.
 Also of interest as active agent are antibodies that at least reduce, if
 not inhibit, GST-3 activity in the host. Suitable antibodies are obtained
 by immunizing a host animal with peptides comprising all or a portion of
 GST-3 protein. Suitable host animals include mouse, rat sheep, goat,
 hamster, rabbit, etc. The origin of the protein immunogen may be mouse,
 human, rat, monkey etc. The host animal will generally be a different
 species than the immunogen, e.g. human GST-3 used to immunize mice, etc.
 The immunogen may comprise the complete protein, or fragments and
 derivatives thereof. Preferred immunogens comprise all or a part of GST-3,
 where these residues contain the post-translation modifications, such as
 glycosylation, found on the native GST-3. Immunogens comprising the
 extracellular domain are produced in a variety of ways known in the art,
 e.g. expression of cloned genes using conventional recombinant methods,
 isolation from HEC, etc.
 For preparation of polyclonal antibodies, the first step is immunization of
 the host animal with GST-3, where the GST-3 will preferably be in
 substantially pure form, comprising less than about 1% contaminant. The
 immunogen may comprise complete GST-3, fragments or derivatives thereof.
 To increase the immune response of the host animal, the GST-3 may be
 combined with an adjuvant, where suitable adjuvants include alum, dextran,
 sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's
 adjuvant, Freund's complete adjuvant, and the like. The GST-3 may also be
 conjugated to synthetic carrier proteins or synthetic antigens. A variety
 of hosts may be immunized to produce the polyclonal antibodies. Such hosts
 include rabbits, guinea pigs, rodents, e.g. mice, rats, sheep, goats, and
 the like. The GST-3 is administered to the host, usually intradermally,
 with an initial dosage followed by one or more, usually at least two,
 additional booster dosages. Following immunization, the blood from the
 host will be collected, followed by separation of the serum from the blood
 cells. The Ig present in the resultant antiserum may be further
 fractionated using known methods, such as ammonium salt fractionation,
 DEAE chromatography, and the like.
 Monoclonal antibodies are produced by conventional techniques. Generally,
 the spleen and/or lymph nodes of an immunized host animal provide a source
 of plasma cells. The plasma cells are immortalized by fusion with myeloma
 cells to produce hybridoma cells. Culture supernatant from individual
 hybridomas is screened using standard techniques to identify those
 producing antibodies with the desired specificity. Suitable animals for
 production of monoclonal antibodies to the human protein include mouse,
 rat, hamster, etc. To raise antibodies against the mouse protein, the
 animal will generally be a hamster, guinea pig, rabbit, etc. The antibody
 may be purified from the hybridoma cell supernatants or ascites fluid by
 conventional techniques, e.g. affinity chromatography using GST-3 bound to
 an insoluble support, protein A sepharose, etc.
 The antibody may be produced as a single chain, instead of the normal
 multimeric structure. Single chain antibodies are described in Jost el al.
 (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding the
 variable region of the heavy chain and the variablc region of the light
 chain are ligated to a spacer encoding at least about 4 amino acids of
 small neutral amino acids, including glycine and/or serine. The protein
 encoded by this fusion allows assembly of a functional variable region
 that retains the specificity and affinity of the original antibody.
 For in vivo use, particularly for injection into humans, it is desirable to
 decrease the antigenicity of the antibody. An immune response of a
 recipient against the blocking agent will potentially decrease the period
 of time that the therapy is effective. Methods of humanizing antibodies
 are known in the art. The humanized antibody may be the product of an
 animal having transgenic human immunoglobulin constant region genes (see
 for example International Patent Applications WO 90/10077 and WO
 90/04036). Alternatively, the antibody of interest may be engineered by
 recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains,
 and/or the framework domain with the corresponding human sequence (see WO
 92/02190).
 The use of Ig cDNA for construction of chimeric immunoglobulin genes is
 known in the art (Liu el al. (1987) P.N.A.S. 84:3439 and (1987) J.
 Immunol. 139:3521). mRNA is isolated from a hybridoma or other cell
 producing the antibody and used to produce cDNA. The cDNA of interest may
 be amplified by the polymerase chain reaction using specific primers (U.S.
 Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and
 screened to isolate the sequence of interest. The DNA sequence encoding
 the variable region of the antibody is then fused to human constant region
 sequences. The sequences of human constant regions genes may be found in
 Kabat et al. (1991) Sequences of Proteins of Immunological Interest,
 N.I.H. publication no. 91-3242. Human C region genes are readily available
 from known clones. The choice of isotype will be guided by the desired
 effector functions, such as complement fixation, or activity in
 antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,
 IgG3 and IgG4. Either of the human light chain constant regions, kappa or
 lambda, may be used. The chimeric, humanized antibody is then expressed by
 conventional methods.
 Antibody fragments, such as Fv, F(ab').sub.2 and Fab may be prepared by
 cleavage of the intact protein, e.g. by protease or chemical cleavage.
 Alternatively, a truncated gene is designed. For example, a chimeric gene
 encoding a portion of the F(ab').sub.2 fragment would include DNA
 sequences encoding the CH1 domain and hinge region of the H chain,
 followed by a translational stop codon to yield the truncated molecule.
 Consensus sequences of H and L J regions may be used to design
 oligonucleotides for use as primers to introduce useful restriction sites
 into the J region for subsequent linkage of V region segments to human C
 region segments. C region cDNA can be modified by site directed
 mutagenesis to place a restriction site at the analogous position in the
 human sequence.
 Expression vectors include plasmids, retroviruses, YACs, EBV derived
 episomes, and the like. A convenient vector is one that encodes a
 functionally complete human CH or CL immunoglobulin sequence, with
 appropriate restriction sites engineered so that any VH or VL sequence can
 be easily inserted and expressed. In such vectors, splicing usually occurs
 between the splice donor site in the inserted J region and the splice
 acceptor site preceding the human C region, and also at the splice regions
 that occur within the human CH exons. Polyadenylation and transcription
 termination occur at native chromosomal sites downstream of the coding
 regions. The resulting chimeric antibody may be joined to any strong
 promoter, including retroviral LTRs, e.g. SV-40 early promoter, (Okayama
 et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus LTR (Gorman et
 al. (1982) P.N.A.S. 79:6777), and moloney murine leukemia virus LTR
 (Grosschedl et al. (1985) Cell 41:885); native Ig promoters, etc.
 In yet other embodiments of the invention, the active agent is an agent
 that modulates, and generally decreases or down regulates, the expression
 of GST-3 in the host. Antisense molecules can be used to down-regulate
 expression of GST-3 in cells. The anti-sense reagent may be antisense
 oligonucleotides (ODN), particularly synthetic ODN having chemical
 modifications from native nucleic acids, or nucleic acid constructs that
 express such anti-sense molecules as RNA. The antisense sequence is
 complementary to the mRNA of the targeted gene, and inhibits expression of
 the targeted gene products. Antisense molecules inhibit gene expression
 through various mechanisms, e.g. by reducing the amount of mRNA available
 for translation, through activation of RNAse H, or steric hindrance. One
 or a combination of antisense molecules may be administered, where a
 combination may comprise multiple different sequences.
 Antisense molecules may be produced by expression of all or a part of the
 target gene sequence in an appropriate vector, where the transcriptional
 initiation is oriented such that an antisense strand is produced as an RNA
 molecule. Alternatively, the antisense molecule is a synthetic
 oligonucleotide. Antisense oligonucleotides will generally be at least
 about 7, usually at least about 12, more usually at least about 20
 nucleotides in length, and not more than about 500, usually not more than
 about 50, more usually not more than about 35 nucleotides in length, where
 the length is governed by efficiency of inhibition, specificity, including
 absence of cross-reactivity, and the like. It has been found that short
 oligonucleotides, of from 7 to 8 bases in length, can be strong and
 selective inhibitors of gene expression (see Wagner et al. (1996), Nature
 Biotechnol. 14:840-844).
 A specific region or regions of the endogenous sense strand mRNA sequence
 is chosen to be complemented by the antisense sequence. Selection of a
 specific sequence for the oligonucleotide may use an empirical method,
 where several candidate sequences are assayed for inhibition of expression
 of the target gene in an in vitro or animal model. A combination of
 sequences may also be used, where several regions of the mRNA sequence are
 selected for antisense complementation.
 Antisense oligonucleotides may be chemically synthesized by methods known
 in the art (see Wagner et al. (1993), supra, and Milligan et al., supra.)
 Preferred oligonucleotides are chemically modified from the native
 phosphodiester structure, in order to increase their intracellular
 stability and binding affinity. A number of such modifications have been
 described in the literature, which alter the chemistry of the backbone,
 sugars or heterocyclic bases.
 Among useful changes in the backbone chemistry are phosphorothioates;
 phosphorodithioates, where both of the non-bridging oxygens are
 substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and
 boranophosphates. Achiral phosphate derivatives include
 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate,
 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic
 acids replace the entire ribose phosphodiester backbone with a peptide
 linkage. Sugar modifications are also used to enhance stability and
 affinity. The .alpha.-anomer of deoxyribose may be used, where the base is
 inverted with respect to the natural .beta.-anomer. The 2'-OH of the
 ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars,
 which provides resistance to degradation without comprising affinity.
 Modification of the heterocyclic bases must maintain proper base pairing.
 Some useful substitutions include deoxyuridine for deoxythymidine;
 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine.
 5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown
 to increase affinity and biological activity when substituted for
 deoxythymidine and deoxycytidine, respectively.
 As an alternative to anti-sense inhibitors, catalytic nucleic acid
 compounds, e.g ribozymes, anti-sense conjugates, etc. may be used to
 inhibit gene expression. Ribozymes may be synthesized in vitro and
 administered to the patient, or may be encoded on an expression vector,
 from which the ribozyme is synthesized in the targeted cell (for example,
 see International patent application WO 9523225, and Beigelman et al.
 (1995), Nucl. Acids Res. 23:4434-42). Examples of oligonucleotides with
 catalytic activity are described in WO 9506764. Conjugates of anti-sense
 ODN with a metal complex, e.g. terpyridylCu(II), capable of mediating mRNA
 hydrolysis are described in Bashkin et al. (1995), Appl. Biochem.
 Biotechnol. 54:43-56.
 As mentioned above, an effective amount of the active agent is administered
 to the host, where "effective amount" means a dosage sufficient to produce
 a desired result. Generally, the desired result is at least a reduction in
 the amount of selectin binding as compared to a control.
 In the subject methods, the active agent(s) may be administered to the host
 using any convenient means capable of resulting in the desired inhibition
 of selectin binding. Thus, the agent can be incorporated into a variety of
 formulations for therapeutic administration. More particularly, the agents
 of the present invention can be formulated into pharmaceutical
 compositions by combination with appropriate, pharmaceutically acceptable
 carriers or diluents, and may be formulated into preparations in solid,
 semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,
 granules, ointments, solutions, suppositories, injections, inhalants and
 aerosols.
 As such, administration of the agents can be achieved in various ways,
 including oral, buccal, rectal, parenteral, intraperitoneal, intradermal,
 transdermal, intracheal, etc., administration.
 In pharmaceutical dosage forms, the agents may be administered in the form
 of their pharmaceutically acceptable salts, or they may also be used alone
 or in appropriate association, as well as in combination, with other
 pharmaceutically active compounds. The following methods and excipients
 are merely exemplary and are in no way limiting.
 For oral preparations, the agents can be used alone or in combination with
 appropriate additives to make tablets, powders, granules or capsules, for
 example, with conventional additives, such as lactose, mannitol, corn
 starch or potato starch; with binders, such as crystalline cellulose,
 cellulose derivatives, acacia, corn starch or gelatins; with
 disintegrators, such as corn starch, potato starch or sodium
 carboxymethylcellulose; with lubricants, such as talc or magnesium
 stearate; and if desired, with diluents, buffering agents, moistening
 agents, preservatives and flavoring agents.
 The agents can be formulated into preparations for injection by dissolving,
 suspending or emulsifying them in an aqueous or nonaqueous solvent, such
 as vegetable or other similar oils, synthetic aliphatic acid glycerides,
 esters of higher aliphatic acids or propylene glycol; and if desired, with
 conventional additives such as solubilizers, isotonic agents, suspending
 agents, emulsifying agents, stabilizers and preservatives.
 The agents can be utilized in aerosol formulation to be administered via
 inhalation. The compounds of the present invention can be formulated into
 pressurized acceptable propellants such as dichlorodifluoromethane,
 propane, nitrogen and the like.
 Furthermore, the agents can be made into suppositories by mixing with a
 variety of bases such as emulsifying bases or water-soluble bases. The
 compounds of the present invention can be administered rectally via a
 suppository. The suppository can include vehicles such as cocoa butter,
 carbowaxes and polyethylene glycols, which melt at body temperature, yet
 are solidified at room temperature.
 Unit dosage forms for oral or rectal administration such as syrups,
 elixirs, and suspensions may be provided wherein each dosage unit, for
 example, teaspoonful, tablespoonful, tablet or suppository, contains a
 predetermined amount of the composition containing one or more inhibitors.
 Similarly, unit dosage forms for injection or intravenous administration
 may comprise the inhibitor(s) in a composition as a solution in sterile
 water, normal saline or another pharmaceutically acceptable carrier.
 The term "unit dosage form," as used herein, refers to physically discrete
 units suitable as unitary dosages for human and animal subjects, each unit
 containing a predetermined quantity of compounds of the present invention
 calculated in an amount sufficient to produce the desired effect in
 association with a pharmaceutically acceptable diluent, carrier or
 vehicle. The specifications for the novel unit dosage forms of the present
 invention depend on the particular compound employed and the effect to be
 achieved, and the pharmacodynamics associated with each compound in the
 host.
 The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
 carriers or diluents, are readily available to the public. Moreover,
 pharmaceutically acceptable auxiliary substances, such as pH adjusting and
 buffering agents, tonicity adjusting agents, stabilizers, wetting agents
 and the like, are readily available to the public.
 Where the agent is a polypeptide, polynucleotide, analog or mimetic
 thereof, e.g. antisense composition, it may be introduced into tissues or
 host cells by any number of routes, including viral infection,
 microinjection, or fusion of vesicles. Jet injection may also be used for
 intramuscular administration, as described by Furth et al. (1992), Anal
 Biochem 205:365-368. The DNA may be coated onto gold microparticles, and
 delivered intradermally by a particle bombardment device, or "gene gun" as
 described in the literature (see, for example, Tang et al. (1992), Nature
 356:152-154), where gold microprojectiles are coated with the GST-3 DNA,
 then bombarded into skin cells.
 Those of skill will readily appreciate that dose levels can vary as a
 function of the specific compound, the severity of the symptoms and the
 susceptibility of the subject to side effects. Preferred dosages for a
 given compound are readily determinable by those of skill in the art by a
 variety of means.
 The subject methods find use in the treatment of a variety of different
 disease conditions involving selectin binding interactions, particularly
 L-, E- or P- selectin, and more particularly L-selectin mediated binding
 events. Such disease conditions include those disease conditions
 associated with or resulting from the homing of leukocytes to sites of
 inflammation, the normal homing of lymphocytes to secondary lymph organs;
 and the like. Accordingly, specific disease conditions that may be treated
 with the subject methods include: acute or chronic inflammation;
 autoimmune and related disorders, e.g. systemic lupus erythematosus,
 rheumatoid arthritis, polyarteritis nodosa, polymyositis and
 dermatomyositis, progressive systemic sclerosis (diffuse scleroderma),
 glomerulonephritis, myasthenia gravis, Sjogren's syndrome, Hashimoto's
 disease and Graves' disease, adrenalitis, hypoparathyroidism, and
 associated diseases; pernicious anemia; diabetes; multiple sclerosis and
 related demyelinating diseases; uveitis pemphigus and pemphigoid;
 cirrhosis and other diseases of the liver; ulcerative colitis;
 myocarditis; regional enteritis; adult respiratory distress syndrome;
 local manifestations of drug reactions (dermatitis, etc.);
 inflammation-associated or allergic reaction patterns of the skin; atopic
 dermatitis and infantile eczema; contact dermatitis, psoriasis lichen
 planus; allergic enteropathies; atopic diseases, e.g. allergic rhinitis
 and bronchial asthma; transplant rejection (heart, kidney, lung, liver,
 pancreatic islet cell, others); hypersensitivity or destructive responses
 to infectious agents; poststreptococcal diseases e.g. cardiac
 manifestations of rheumatic fever, etc.; tissue rejection during
 transplantation; and the like.
 By treatment is meant at least an amelioration of the symptoms associated
 with the pathological condition afflicting the host, where amelioration is
 used in a broad sense to refer to at least a reduction in the magnitude of
 a parameter, e.g. symptom, associated with the pathological condition
 being treated, such as inflammation and pain associated therewith. As
 such, treatment also includes situations where the pathological condition,
 or at least symptoms associated therewith, are completely inhibited, e.g.
 prevented from happening, or stopped, e.g. terminated, such that the host
 no longer suffers from the pathological condition, or at least the
 symptoms that characterize the pathological condition.
 A variety of hosts are treatable according to the subject methods.
 Generally such hosts are "mammals" or "mammalian," where these terms are
 used broadly to describe organisms which are within the class mammalia,
 including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,
 mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and
 monkeys). In many embodiments, the hosts will be humans.
 Kits with unit doses of the active agent, usually in oral or injectable
 doses, are provided. In such kits, in addition to the containers
 containing the unit doses will be an informational package insert
 describing the use and attendant benefits of the drugs in treating
 pathological condition of interest. Preferred compounds and unit doses are
 those described herein above.
 The following examples are offered primarily for purposes of illustration.
 It will be readily apparent to those skilled in the art that the
 formulations, dosages, methods of administration, and other parameters of
 this invention may be further modified or substituted in various ways
 without departing from the spirit and scope of the invention.
 EXPERIMENTAL
 I. Identification of GST-1 & GST-2
 Human ESTs that are related to the C6ST/KSST at the protein level were
 searched by using ASTN which compares a protein query sequence against
 a nucleotide sequence database translated in all 6 reading frames.
 (Karlin, Samuel and Stephen F. Altschul (1990). Methods for assessing the
 statistical significance of molecular sequence features by using general
 scoring schemes. Proc. Natl. Acad. Sci. USA 87:2264-68; Karlin, Samuel and
 Stephen F. Altschul (1993). Applications and statistics for multiple
 high-scoring segments in molecular sequences. Proc. Natl. Acad. Sci. USA
 90:5873-7). As shown in Table 1, several ESTs, ranging from 228 to 861
 bases, resulted in high scores. When compared over their entire length
 with the C6ST/KSST, the predicted amino acid identities ranged from 27% to
 57%.
 TABLE 1
 Human ESTs related to the chick chondroitin
 6/keratan sulfate sulfotransferase
 Identity
 Covering of AA
 AA of Sequences Contig
 No. mRNA source C6ST/KSST (%) Assignment
 1 infant brain 347-451 42 1
 2 infant brain 140-216 57 1
 3 adult heart 405-451 42 1
 4 fetal lung 89-375 30 2
 5 fetal liver/spleen 332-403 27 2
 6 teratocarcinoma 100-165 31 2
 The cDNA clones corresponding to each EST were obtained from the ATCC and
 Research Genetics, Inc., (Huntsville, Ala.) and sequenced in full to
 obtain further 3' information. Sequence alignment analysis revealed the
 presence of two distinct sequences ("contigs"), covering 74% (contig 1,
 starting at amino acid 137) and 78% (contig 2, starting at amino acid
 100). Contig sequences 1 and 2 are apparently both complete at the 3' end,
 since both contain poly A tracts at the end of their 3' untranslated
 regions (UTR).
 Expression of transcripts corresponding to the two contigs was examined in
 a number of human tissues by Northern analysis. Blots of poly A.sup.+ RNA
 (Clontech, Palo Alto, Calif.) were probed at high stringency with probes
 derived from the EST clones. A 3.1 kb band corresponding to contig 1 was
 detected in multiple human organs (heart, placenta, lung, liver, skeletal
 muscle, kidney, pancreas, spleen, lymph nodes, thymus) but most strongly
 in brain. Contig 2 was also broadly expressed in various organs (3.3 kb
 band), including lymph node and brain.
 Full-length cDNAs containing the two contigs and predicting CS6T/KSST
 homologs were obtained by screening a human fetal brain )ZAP cDNA library
 (Stratagene, La Jolla, Calif.) with labeled 700-800 bp restriction
 fragments (from EST 2 for contig 1 and from EST 5 for contig 2). Briefly,
 10.sup.6 plaque-forming units were used to infect E. coli, which were then
 distributed on 20 plates. Duplicate filter lifts were performed. The
 probes were labeled with .sup.32 P by random priming (Amersham), and
 hybridization was performed at 60.degree. C. with high stringency washing.
 In both screens, multiple positive spots were obtained in the first round.
 Single positive clones were obtained after either the second or third
 round of screening. Multiple clones were sequenced for each gene and the
 presence of the ESTs was confirmed. As will be described below, the cDNAs
 contain open reading frames that encode predicted proteins of high
 homology to CS6T/KSST. The proteins encoded by these cDNAs were designated
 as GST 1 and GST 2, where "GST" denotes "glycosylsulfotransferase." GST 1
 has been independently cloned by Fukuta et al., J. Biol. Chem. (1997) 272:
 32321-8.
 II. Identification of GST-3
 ESTs potentially coding for novel human glycosyl sulfotransferases other
 than GST-1 & 2 were identified through a secondary homology screen, in
 which the peptide sequences of GST-1 and GST-2 were used as template in
 two parallel ASTN searches against a public (dbest) and a private
 genomic database (Lifeseq, Incyte Pharmaceuticals, Palo Alto, Calif.).
 Only matches that produced alignments with smallest sum probabilities
 P(N)&lt;10.sup.-5 were selected from the output of the search, imported into
 a contig assembler (Sequencher 3.0, Gene Codes Corporation, Ann Arbor,
 Mich.) and assembled using the default settings of the program. The vast
 majority of these matches assembled into two contigs defined by GST-1 and
 GST-2. However, four particular ESTs found only in the private Lifeseq
 database did not assemble into either contig or with each other. These
 were termed GST-3 through GST-6.
 III. GST-3 is Expressed in High Endothelial Venules
 In order to investigate if any of thc above putative human glycosyl
 sulfotransferases or similar genes were expressed in high endothelial
 venules, an HEV-derived cDNA pool for use as template in homology
 polymerase chain reaction (PCR) was prepared. In order to clone HEV genes,
 an expression library from the aforementioned HEV-derived cDNA was also
 generated. Briefly, total RNA (45 .mu.g) was isolated from 10.sup.7 HEC.
 Since the amount of poly A.sup.+ RNA was too limited for preparation of a
 cDNA library by conventional procedures, the Capfinder (SMART.TM.) cDNA
 technology (CLONTECH) was used. In this technique, the reverse
 transcription reaction is primed by a modified oligo(dT) primer
 (containing a Not I site) and a "SMART" oligonucleotide which anneals to
 an oligo dC stretch added by reverse transcriptase (RT) at the 3' end of
 the first strand cDNA. The annealed oligonucleotide serves as a "switch"
 template for RT, resulting in the generation of single stranded cDNAs
 which are enriched for full length sequences and contain universal primer
 sites for subsequent long distance PCR amplification. This technology
 therefore makes it possible to generate high quality double stranded cDNA
 (from limiting amounts of RNA), which is sufficient to construct a
 library. According to the published test results for this technology,
 Capfinder cDNA is comparable to conventionally prepared cDNA in gene
 representation and is significantly enriched for full length cDNAs. The
 HEC cDNA generated by the Capfinder technology was evaluated by PCR for
 the presence of the following genes, which are known or suspected to be
 expressed in HEC: CD34 (Baumhueter et al., Science (1993) 262: 436-438),
 hevin (Girard & Springer, Immunity (1995) 2:113-123), fucosyltransferase
 VII (Maly et al., Cell (1996) 86: 643-653);
 .beta.-1,6-N-acetylglucosaminyl-transferase (C2GnT) (Bierhuizen & Fukuda.,
 Proc. Natl. Acad. Sci. USA (1992) 89: 9326-9330), and fractalkine (Schall,
 Immunology Today (1997) 18:147). By this analysis, all of these cDNAs were
 detected in the HEC cDNA, and at least two of them (CD34 and C2GnT) were
 full length. With this validation of the HEC cDNA, a library was generated
 as follows: the double-stranded cDNA was ligated to Eco RI adapters,
 digested with Not I and cloned into the Not I and Eco RI sites of pCDNA1.1
 (Invitrogen, Inc, Carlsbad, Calif.), which is a modified version of the
 eucaryotic expression vector pCDM8 (Aruffo et al., Proc. Natl. Acad. Sci.
 USA (1987) 84: 8753-8577). The resulting library has a complexity of
 500,000 independent clones and an average insert size of 1.1 kb, according
 to the characterization performed by CLONTECH.
 HEV-derived Capfinder cDNA was used as a template for homology PCR with
 degenerate primers. In-frame translations of GST-1 and GST-2 were aligned
 with other known sulfotransferase protein sequences retrieved from the
 public databases. See FIG. 3. Three putative consensus regions were
 identified, and the following degenerate primers were synthesized to
 encode within these consensus regions a maximal number of possible
 permutations at the amino-acid level in order to cover a maximal number of
 novel sulfotransferases that may fall into these patterns.

These primers were (I = inosine):
 A+: 5' TWYTWYCTITWYGARCCICTITGGCAYST 3' (SEQ ID
 NO:03)
 B+: 5' CTIAAICTISTICWRCTISTIMGIRAYCC 3' (SEQ ID
 NO:04)
 B-: 5' GGRTYICKIASIAGYWGIASIAGITTIAG 3' (SEQ ID
 NO:05)
 C-: 5' AGRTCYTCRTAICKIAGIAGIAKRTA 3' (SEQ ID
 NO:06)
 In the first round PCR each reaction contained in a total volume of 50
 .mu.l 100 mM Tris-Cl (pH 8.3), 0.5 M KCl, 15 mM MgCl.sub.2, forward and
 reverse primer (0.5 .mu.M each), dATP, dCTP, dGTP, and dTTP (100 .mu.M
 each), 0.25 units Thermus aquaticus DNA polymerase (Boehringer Mannheim
 #1647679), and 0.5 .mu.l of HEV-message derived Cap-finder cDNA
 preparation (generated by Clontech Inc., cf. above). In "no template"
 control samples the cDNA was omitted.
 Each reaction was cycled as follows: hold 4 min@94.degree. C., then 35
 cycles of [30 sec@94.degree. C. followed by 30 sec@40.degree. C. followed
 by 1 min@72.degree. C.], then hold 6 min@72.degree. C. Following
 completion of PCR a 20 .mu.l aliqout of each reaction was analysed by
 standard horizontal agarose (1%) gel electrophoresis. No discernable band
 pattern was observed (data not shown)
 Therefore the unfractionated products of the first round PCR were used as
 template in a second round PCR. Here each reaction contained in a total
 volume of 50 .mu.l 100 mM Tris-Cl (pH 8.3), 0.5 M KCl, 15 mM MgCl.sub.2,
 forward and reverse primer (0.5 .mu.M each), dATP, dCTP, dGTP, and dTTP
 (100 .mu.M each), 0.25 units Thermus aquaticus DNA polymerase (Boehringer
 Mannheim #1647679), and 1 .mu.l of total PCR reaction from round 1 (cf.
 above).
 Each reaction was cycled as follows: hold 4 min@94.degree. C., then 35
 cycles of [30 sec@94.degree. C. followed by 30 sec@45.degree. C. followed
 by 1 min@72.degree. C.], then hold 6 min@72.degree. C. The entire
 reactions were then fractionated by standard horizontal agarose (1%) gel
 electrophoresis. Bands appearing at positions 2.1, 2.2 and 2.3, see FIG.
 4, were excised and DNA eluted from the gel using the QIAquick PCR
 purification kit (Qiagen Inc. #28104). Eluted DNA was then subcloned into
 the TA cloning vector pCR-II (stratagene) and E-coli transformed with
 recombinant plasmids. For each band eight colonies were expanded, and
 plasmid DNAs isolated and sequenced using standard dideoxynucleotide chain
 termination methodology with fluorimetric detection.
 In order to map the amplicons generated by the above homology PCR, public
 (dbest) and private (Incyte Inc.) EST databases were screened with by the
 ASTX algorithm (Karlin & Altschul, 1990 & 1993; cf. above) using the
 sequences of these amplicons as query sequences. Four sequences amplified
 from from HEV-cDNA with primers B+ and C- aligned with &gt;95% overall
 identity to Incyte EST #2620445 defined perviously as GST-3 (cf. above).
 All other query sequences did not pick up statistically significant
 matches in the specified databases.
 IV. GST-3 Is Expressed in HEC
 From the extended DNA sequence of Lifeseq clone #2620445=GST-3 we designed
 a nondegenerate primer pair located within the incomplete open reading
 frame encoded by this EST.
 Forward: 5'AAACTCAAGAAGGAGGACCAACCCTACTATGTGATGC 3' (SEQ ID NO:07)
 Reverse:5'ATAAAGCTTGTGGATTTGTTCAGGGACATTCCAGGTAGACAGAAGAT 3'(SEQ ID NO:08)
 Using RT-PCR, a PCR product of appropriate length (500 bp) was amplified
 from HEC cDNA with this primer pair. This product could not be amplified
 from cDNAs prepared from tonsillar lymphocytes or primary cultured human
 umbilical vein endothelial cells (HUVEC). See FIG. 5 Control primers for
 hypoxanthine phosphoribosyl transferase (HPRT, a ubiquitously expressed
 cellular "housekeeping enzyme") were used in parallel to establish that
 similar amounts of template were used in each set of PCR reactions and
 that none of the template DNAs were substantially degraded. These RT-PCR
 results confirm that the gene corresponding to the PCR product is
 expressed in HEC but not in lymphocytes or HUVEC. Northern analysis has
 failed to detect mRNA for the new gene in a variety of human tissues and
 organs, establishing that the expression of this gene is is highly
 restricted.
 V. GST-3 Cloning
 A full length cDNA from the HEC library described in the previous section
 was cloned as follows. The pool selection procedure described in Bakker et
 al., J. Biol. Chem. (1997) 272:29942-6) was used to quickly isolate the
 cDNA. It was first established that the elevant template was contained
 within the library by successfully amplifying the above described PCR
 product from the library stock comprising the entire library. An aliquot
 of this bacterial stock was then divided into 200 pools of 2000-3000
 colonies each. Each pool was plated out on LB plates and the colonies were
 allowed to grow to a healthy size. The colonies were harvested in LB and
 allowed to grow further at 37.degree. C., at which time glycerol stocks
 were prepared from each pool. By PCR analysis of the pools, nine positives
 were identified in this first round of screening. The corresponding
 bacterial stock for one of these pools was then titered and plated at 100
 colony forming units (cfu) per plate in 40 plates. Plates were grown,
 harvested, preserved and analyzed as in the first round, resulting in the
 identification of three positive subpools. At this stage, one of the three
 positive pools was plated at a density (300 cfu) so that individual
 colonies could be analyzed by PCR. One cDNA clone was obtained by this
 approach. It contains a complete open reading frame which which encodes a
 novel 386 amino acid protein, termed GST-3. This full length cDNA sequence
 was now used as template in a BLASTN search of the public (dbest) and
 Lifeseq EST databases. In this manner, two so far unrecognized ESTs
 #2617407 (from Lifeseq; derived from a human gall bladder cDNA library)
 and g2262929 (from the mouse EST collection included in the dbest
 database, derived from a murine mammary gland cDNA library) were
 identified. The former EST included the 5' end of GST-3 open reading
 frame. Since this EST was generated with an oligo dT-primer, it contains
 therefore the entire open reading frame plus all 3' untranslated sequence
 of the human GST-3 cDNA. This EST was retrieved from Incyte in the form of
 a plasmid-transformed E.coli culture, expanded into Luria Bertoni Medium
 (with 0.1 mg/ml Ampicillin), plasmid isolated from a 500 ml culture, and
 sequenced using standard dideoxynucleotide chain termination methodology
 with fluorimetric detection. Since, in contrast to the Cap-finder
 methodology employed in generation of our HEV-library, no PCR-step was
 used in generating the full length GST-3 Lifeseq EST Incyte #2167407, the
 GST-3 sequence obtained from Incyte #2617407 is free of PCR errors. The
 sequence is provided in SEQ ID NO:01 and shown in FIG. 1.
 VI. Characterization of GST-3
 Three cDNA clones which encode three different human homologs for C6ST/KSST
 have been obtained. The predicted GST proteins are type 2 membrane
 proteins 411, 477, and 386 amino acids in length, respectively. Each has a
 relatively short transmembrane domain and a short amino-terminal
 cytoplasmic tail. Table 2 demontrates the high homologies among the 3
 human proteins and the chick CS6T/KSST. Overall homologies at the amino
 acid level ranged from 28% to 40% identity. Strikingly, there are three
 regions of 16 to 29 amino acids in which identity among the three GSTs
 ranged from 50-59% and similarity ranged from 65-94%. See FIG. 6. In FIG.
 6 shows that all four of the sulfotransferases are type II transmembrane
 proteins with short cytoplasmic tails (TM). There are three regions
 (region A, B and C) in which identities among the human GSTs range from
 50-59% and similarities range from 65 to 94%. The amino acid sequence for
 the regions are:
 A: (T/S)XRSGSSF(V/F)G(Q/E)LFXQX(P/L)(D/E)VF(F/Y)L(F/Y/M)EP(L/V/A)(W/Y)HV
 B: L(N/D)L(K/H)(V/I)(I/V)XLVRDPR(A/G)(V/I)(LAF)
 C: PXXL(Q/K)XXY(L/M)(L/V)VRYEDL(A/V)XXP
 TABLE 2
 Percent amino acid identities for the predicted coding sequences
 GST 1 GST 2 GST 3 CS6T/KSST
 GST 1 -- 31 32 40
 GST 2 -- -- 35 28
 GST 3 -- -- -- 31
 VII. GST-3 Sulfates GlyCAM-1
 In expression experiments, the sulfotransferase activity of the GST 3
 protein by transient expression of its cDNA into COS cells has been
 investigated. Since the HEC library yielding the GST 3 cDNA was in the
 pcDNA1.1 expression vector, there was no need subclone the GST 3 insert
 prior to transfection. Co-transfection of the GST 3 cDNA with a cDNA
 encoding a GlyCAM-1/human IgG1 Fc chimera resulted in a &gt;10 fold enhanced
 incorporation of .sup.35 S-SO.sub.4 relative to transfection with the
 GlyCAM-1 chimera alone. Co-transfection with vector cDNA had no effect. By
 SDS-PAGE analysis, incorporation of .sup.35 S-SO.sub.4 counts into the
 GlyCAM-1 chimera was confirmed. The results are shown in FIG. 7. The
 results indicate that GST 3 encodes a sulfotransferase that can utilize
 GlyCAM-1 as an acceptor.
 It is apparent from the above results and discussion that a novel human
 glycosyl sulfotransferase, as well as polypeptides related thereto and
 nucleic acid compositions encoding the same are provided by the subject
 invention. These polypeptide and nucleic acid compositions find use in a
 variety of diverse applications, including research, diagnostic, screening
 and therapeutic applications. Also provided are improved methods of
 treating diseases associated with selectin-sulfated ligand mediated
 binding events, since agents that selectively reduce or inhibit the
 activity of the subject enzyme are employed, so that other
 sulfotransferases whose activity is beneficial are not adversely affected.
 All publications and patent applications cited in this specification are
 herein incorporated by reference as if each individual publication or
 patent application were specifically and individually indicated to be
 incorporated by reference. The citation of any publication is for its
 disclosure prior to the filing date and should not be construed as an
 admission that the present invention is not entitled to antedate such
 publication by virtue of prior invention.
 Although the foregoing invention has been described in some detail by way
 of illustration and example for purposes of clarity of understanding, it
 is readily apparent to those of ordinary skill in the art in light of the
 teachings of this invention that certain changes and modifications may be
 made thereto without departing from the spirit or scope of the appended
 claims.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 9
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 2032
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 1
 ggctcgaggc caggatgcct ccagtctggg ggaaaatgct tcctcatttg cttctcccag 60
 cccacctcaa gcagtctccc caccccttga gtctcagcag tgttaaagct gttactttca 120
 cagcttcctg ggagcgagtg ctttctcaag cccgtcttgc aaggtcttcc acttcagcac 180
 aatgctactg cctaaaaaaa tgaagctcct gctgtttctg gtttcccaga tggccatctt 240
 ggctctattc ttccacatgt acagccacaa catcagctcc ctgtctatga aggcacagcc 300
 cgagcgcatg cacgtgctgg ttctgtcttc ctggcgctct ggctcttctt ttgtggggca 360
 gctttttggg cagcacccag atgttttcta cctgatggag cccgcctggc acgtgtggat 420
 gaccttcaag cagagcaccg cctggatgct gcacatggct gtgcgggatc tgatacgggc 480
 cgtcttcttg tgcgacatga gcgtctttga tgcctacatg gaacctggtc cccggagaca 540
 gtccagcctc tttcagtggg agaacagccg ggccctgtgt tctgcacctg cctgtgacat 600
 catcccacaa gatgaaatca tcccccgggc tcactgcagg ctcctgtgca gtcaacagcc 660
 ctttgaggtg gtggagaagg cctgccgctc ctacagccac gtggtgctca aggaggtgcg 720
 cttcttcaac ctgcagtccc tctacccgct gctgaaagac ccctccctca acctgcatat 780
 cgtgcacctg gtccgggacc cccgggccgt gttccgttcc cgagaacgca caaagggaga 840
 tctcatgatt gacagtcgca ttgtgatggg gcagcatgag cagaaactca agaaggagga 900
 ccaaccctac tatgtgatgc aggtcatctg ccaaagccag ctggagatct acaagaccat 960
 ccagtccttg cccaaggccc tgcaggaacg ctacctgctt gtgcgctatg aggacctggc 1020
 tcgagcccct gtggcccaga cttcccgaat gtatgaattc gtgggattgg aattcttgcc 1080
 ccatcttcag acctgggtgc ataacatcac ccgaggcaag ggcatgggtg accacgcttt 1140
 ccacacaaat gccagggatg cccttaatgt ctcccaggct tggcgctggt ctttgcccta 1200
 tgaaaaggtt tctcgacttc agaaagcctg tggcgatgcc atgaatttgc tgggctaccg 1260
 ccacgtcaga tctgaacaag aacagagaaa cctgttgctg gatcttctgt ctacctggac 1320
 tgtccctgag caaatccact aagagggttg agaaggcttt gctgccacct ggtgtcagcc 1380
 tcagtcactt tctctgaatg cttctgagcc ttgcctacat ctctgagcct taactacatg 1440
 tctgtgggta tcacactgag tgtgagttgt gtccacacgt gctcaagcag aaggactttt 1500
 gtgtccatgc ttgtgtctag aaaacagact ggggaacctt atgtgagcag cacatcccac 1560
 cagtgaaaca gggtattgct cttcttcttt tcttgatctt cctgtctggg cagacttcag 1620
 agactttgtg gcctggaggc ctattaagca cgacacagta tcagtggaat tgatccataa 1680
 acctccctgt ccacatcttg cccaatgggg aatggatctt tcaccaaaga gctcaccagc 1740
 attttccaca gagatgcaaa ttctgagccc ttggagttcc cagtggattc aaggaaggaa 1800
 gtgggaacaa ggttggatgc ctacttatga gcttgaccat cacagctatc ggtaatcaga 1860
 aatatgaaac aaaatctctg cacaaaagag caagctctta agttcacagg gtgcctgggc 1920
 tgcatttgaa tatcacttcc cctctgcatt ttcccatcac atagaagact ttgacctgtg 1980
 aagctgccat ctgttaatac taaaattccc aaataagaaa aaaaaaaaaa aa 2032
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 386
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 2
 Met Leu Leu Pro Lys Lys Met Lys Leu Leu Leu Phe Leu Val Ser Gln
 1 5 10 15
 Met Ala Ile Leu Ala Leu Phe Phe His Met Tyr Ser His Asn Ile Ser
 20 25 30
 Ser Leu Ser Met Lys Ala Gln Pro Glu Arg Met His Val Leu Val Leu
 35 40 45
 Ser Ser Trp Arg Ser Gly Ser Ser Phe Val Gly Gln Leu Phe Gly Gln
 50 55 60
 His Pro Asp Val Phe Tyr Leu Met Glu Pro Ala Trp His Val Trp Met
 65 70 75 80
 Thr Phe Lys Gln Ser Thr Ala Trp Met Leu His Met Ala Val Arg Asp
 85 90 95
 Leu Ile Arg Ala Val Phe Leu Cys Asp Met Ser Val Phe Asp Ala Tyr
 100 105 110
 Met Glu Pro Gly Pro Arg Arg Gln Ser Ser Leu Phe Gln Trp Glu Asn
 115 120 125
 Ser Arg Ala Leu Cys Ser Ala Pro Ala Cys Asp Ile Ile Pro Gln Asp
 130 135 140
 Glu Ile Ile Pro Arg Ala His Cys Arg Leu Leu Cys Ser Gln Gln Pro
 145 150 155 160
 Phe Glu Val Val Glu Lys Ala Cys Arg Ser Tyr Ser His Val Val Leu
 165 170 175
 Lys Glu Val Arg Phe Phe Asn Leu Gln Ser Leu Tyr Pro Leu Leu Lys
 180 185 190
 Asp Pro Ser Leu Asn Leu His Ile Val His Leu Val Arg Asp Pro Arg
 195 200 205
 Ala Val Phe Arg Ser Arg Glu Arg Thr Lys Gly Asp Leu Met Ile Asp
 210 215 220
 Ser Arg Ile Val Met Gly Gln His Glu Gln Lys Leu Lys Lys Glu Asp
 225 230 235 240
 Gln Pro Tyr Tyr Val Met Gln Val Ile Cys Gln Ser Gln Leu Glu Ile
 245 250 255
 Tyr Lys Thr Ile Gln Ser Leu Pro Lys Ala Leu Gln Glu Arg Tyr Leu
 260 265 270
 Leu Val Arg Tyr Glu Asp Leu Ala Arg Ala Pro Val Ala Gln Thr Ser
 275 280 285
 Arg Met Tyr Glu Phe Val Gly Leu Glu Phe Leu Pro His Leu Gln Thr
 290 295 300
 Trp Val His Asn Ile Thr Arg Gly Lys Gly Met Gly Asp His Ala Phe
 305 310 315 320
 His Thr Asn Ala Arg Asp Ala Leu Asn Val Ser Gln Ala Trp Arg Trp
 325 330 335
 Ser Leu Pro Tyr Glu Lys Val Ser Arg Leu Gln Lys Ala Cys Gly Asp
 340 345 350
 Ala Met Asn Leu Leu Gly Tyr Arg His Val Arg Ser Glu Gln Glu Gln
 355 360 365
 Arg Asn Leu Leu Leu Asp Leu Leu Ser Thr Trp Thr Val Pro Glu Gln
 370 375 380
 Ile His
 385
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: primer
 &lt;400&gt; SEQUENCE: 3
 twytwyctnt wygarccnct ntggcayst 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: primer
 &lt;400&gt; SEQUENCE: 4
 ctnaanctns tncwrctnst nmgnraycc 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 29
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: primer
 &lt;400&gt; SEQUENCE: 5
 ggrtynckna snagywgnas nagnttnag 29
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 26
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: primer
 &lt;400&gt; SEQUENCE: 6
 agrtcytcrt ancknagnag nakrta 26
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 37
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 7
 aaactcaaga aggaggacca accctactat gtgatgc 37
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 8
 &lt;211&gt; LENGTH: 47
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 8
 ataaagcttg tggatttgtt cagggacatt ccaggtagac agaagat 47
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 9
 &lt;211&gt; LENGTH: 6
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 9
 Val Arg Tyr Glu Asp Leu
 1 5