This invention relates to nucleic acid and amino acid sequences of human galactosyltransferases and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, developmental disorders, reproductive disorders, and autoimmune/inflammatory disorders.
Epithelia, composed of sheets of highly differentiated epithelial cells, cover almost all internal and external body and organ surfaces, such as those of the intestine, kidney, pancreas, lung, mouth, and cervical tract. Epithelia regulate the exchange of substances between tissue compartments and with the outside environment. Regulated changes in embryonic epithelial cell arrangement and shape lead to the formation of internal organs. Secreted and membrane-bound proteins produced by the mesenchyme regulate these changes. It is hypothesized that regulation of cell/cell adhesion and cell motility plays an important role in epithelial morphogenesis. (Goode, S. et al. (1996) Development 122:3863-3879; Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, New York, N.Y. pp. 196-197, 623-624, 1167-1172; and Gumbiner, B. M. (1992) Cell 69:385-387.)
The follicular epithelium of the fruitfly Drosophila melanogaster has been used as a model system for epithelial morphogenesis. Drosophila is a useful system in which to study growth, differentiation, and tumor suppression as many of its genes have mammalian homologs. (Watson, K. L. et al. (1994) J. Cell Sci. Suppl. 18:19-33; and Lodish, supra., pp. 1167-1172.) The follicular epithelium, a monolayer of somatic cells that develops along with the germline during oogenesis, completely surrounds each developing egg chamber and eventually secretes components of the eggshell. Both the follicular epithelium and the oocyte have distinct dorsal-ventral asymmetry established by the interaction of at least 13 genes, some expressed in the follicle and some in the oocyte. Mutations in these genes lead to either dorsalization or ventralization of the eggshell and embryo. (Morisato, D. and Anderson, K. V. (1995) Annu. Rev. Genetics 29:371-399.)
Brainiac, a gene important for correct development of the follicular epithelium, may cooperate with the genes egghead and notch to mediate germline-follicle cell adhesion. Brainiac mutant females and their offspring have multiple defects including ventralization of the eggshell, gaps in the follicular epithelium, and multiple layers of follicle cells around oocytes. The described overproliferation of follicle cells is similar to adenoma tumors. Brainiac females lay fewer eggs than wild-type flies, an occurrence likely due to destruction of mutant egg chambers within the mother. The embryos produced have a cancer-like neurogenic phenotype due to the conversion of epidermal cells to neuroblasts, resulting in excess nervous tissue. The brainiac gene, present on the X chromosome, encodes a 325 to amino acid protein with a putative signal sequence. The brainiac gene is expressed constitutively in the germline during the first 12 hours of embryogenesis. (Morisato and Anderson, supra; Goode, S. et al. (1992) Development 116:177-192; Goode, S. et al. (1996) Developmental Biol. 178:35-50; and Goode, S. et al. (1996) Development, supra.)
Recent work suggests that brainiac protein is a xcex21,3-galactosyltransferase. (Yuan, Y. P. et al. (1997) Cell 88:9-11; and Hennet, T. et al. (1998) J. Biol. Chem. 273:58-65.) Galactosyltransferases are enzymes that transfer galactose to N-acetylglucosamine (GlcNAc)-terminating oligosaccharide chains that are part of glycoproteins or glycolipids or are free in solution. (Kolbinger, F. et al. (1998) J. Biol. Chem. 273:433-440.) xcex21,3-galactosyltransferases form Type I carbohydrate chains with Gal (xcex21-3)GlcNAc linkages. Known human and mouse xcex21,3-galactosyltransferases appear to have a short cytosolic domain, a single transmembrane domain, and a catalytic domain with eight conserved regions. (Kolbinger, supra; and Hennet, supra.) In mouse UDP-galactose:xcex2-N-acetylglucosamine xcex21,3-galactosyltransferase-I region 1 is located at amino acid residues 78-83, region 2 is located at amino acid residues 93-102, region 3 is located at amino acid residues 116-119, region 4 is located at amino acid residues 147-158, region 5 is located at amino acid residues 172-183, region 6 is located at amino acid residues 203-206, region 7 is located at amino acid residues 236-246, and region 8 is located at amino acid residues 264-275. (Hennet, supra.) A variant of a sequence found within mouse UDP-galactose:xcex2-N-acetylglucosamine xcex21,3-galactosyltransferase-I region 8 is also found in bacterial galactosyltransferases, suggesting that this sequence defines a galactosyltransferase sequence motif. (Hennet, supra.) xcex21,4-galactosyltransferases, which form Type II carbohydrate chains with Gal (xcex21-4)GlcNAc linkages, are localized to both the Golgi and the cell surface. These enzymes have a short cytosolic domain, a transmembrane domain, and stem and catalytic domains which face the Golgi lumen or cell surface. A soluble xcex21,4-galactosyltransferase is formed by cleaving the membrane-bound form. Amino acids conserved among xcex21,4-galactosyltransferases include two disulfide-bonded cysteines and a putative UDP-galactose-binding site in the catalytic domain. (Yadav, S. and Brew, K. (1990) J. Biol. Chem. 265:14163-14169; Yadav, S. P. and Brew, K. (1991) J. Biol. Chem. 266:698-703; and Shaper, N. L. et al. (1997) J. Biol. Chem. 272:31389-31399.) xcex21,4-galactosyltransferases have several specialized roles in addition to synthesizing carbohydrate chains on glycoproteins or glycolipids. In mammals, a xcex21,4-galactosyltransferase, as part of a heterodimer with xcex1-lactalbumin, functions in lactating mammary gland lactose production. A xcex21,4-galactosyltransferase on the surface of sperm functions as a receptor that specifically recognizes the egg. Cell surface xcex21,4-galactosyltransferases also function in cell adhesion, cell/basal lamina interaction, and normal and metastatic cell migration. (Shur, B. D. (1993) Curr. Opin. Cell Biol. 5:854-863; and Shaper, supra.) An aberrantly cleaved soluble xcex21,4-galactosyltransferase is secreted by a human ovarian cancer cell line. (Uejima, T. et al. (1992) Cancer Res. 52:6158-6163.)
Galactosyltransferases are part of a larger class of enzymes, the glycosyltransferases, which are implicated in the regulation of cellular growth, development, and differentiation. Many glycosyltransferases are localized to the Golgi while others are present on the cell surface and as soluble extracellular proteins. Cell surface membrane-bound glycosyltransferases may function in cell adhesion by binding carbohydrate substrates on adjacent cell surfaces or in the extracellular matrix. Secreted glycosyltransferases, derived in some cases from proteolytic cleavage of membrane-bound forms, may trigger cell surface receptors by binding their bound carbohydrates or may modify carbohydrates on cell surface molecules in a regulated fashion. Extracellular carbohydrate moieties are developmentally regulated and may be involved in the regulation of cell migration. (Yuan, supra; Shur, supra; and Paulson, J. C. and Colley, K. J. (1989) J. Biol. Chem. 264:17615-17618.) Glycosyltransferases may be involved in autoimmune/inflammatory disorders as many humans with autoimmune thyroid disorders have high levels of circulating antibodies directed against the enzymatic product of xcex11,3galactosyltransferase. (Etienne-Decerf, J. et al. (1987) Acta Endocrinol. 115:67-74.)
The discovery of new human galactosyltransferases and the polynucleotides encoding them satisfies a need in the art by providing new compositions useful in the diagnosis, treatment, and prevention of cancer, developmental disorders, reproductive disorders, and autoimmune/inflammatory disorders.
The invention features substantially purified polypeptides, human galactosyltransferases, referred to collectively as xe2x80x9cHUGAxe2x80x9d and individually as xe2x80x9cHUGA-1xe2x80x9d and xe2x80x9cHUGA-2.xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:3, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as a purified agonist and a purified antagonist to the polypeptide. The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a developmental disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing an autoimmune/inflammatory disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.