Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-12-10
2002-07-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C536S023200, C435S320100, C435S325000, C435S252300
Reexamination Certificate
active
06416988
ABSTRACT:
BACKGROUND OF THE INVENTION
Beta-1,3-galactosyltransferase molecules are classified in the family of glycosyltransferases. In addition to transferring carbohydrate molecules to glycoproteins during biosynthesis, members of this family have also been detected on the cell surface where they are thought to be involved in varying aspects of cell-cell interactions. This family includes carbohydrate transferring enzymes, such as sialyltransferases and fucosyltransferases, and galactosyltransferases. During the formation of O-linked glycoproteins and the modification of N-linked ones, each sugar transfer is catalyzed by a different type of glycosyltransferase. Each glycosyltransferase enzyme is specific for both the donor sugar nucleotide and the acceptor molecule.
Galactosyltransferases promote the transfer of an activated galactose residue in UDP-galactose to the monosaccharide N-acetylglucosamine. This transfer is a step in the biosynthesis of the carbohydrate portion of galactose-containing glycoproteins, such as oligosaccharides and glycolipids, in animal tissues. The Beta-1,3-galactosyl-transferases are characterized by the elongation of type I oligosaccharide chains, and the Beta-1,4-galactosyl-transferases are characterized by the elongation of type II oligosaccharide chains. Both types of carbohydrate structures are present in soluble oligosaccharides of human milk, and are also found on glycoproteins and glycolipids, and are important precursors of blood group antigens. Both galactosyltransferases require a divalent cation (Mn
2+
) to function. Beta-1,4-galactosyltransferases are expressed in various cell types and tissues, while the Beta-1,3-galactosyltransferases seem to have more restricted tissue distributions.
Some galactosyltransferases are found in the Golgi apparatus. These Golgi-localized enzymes have structure similarity: a short N-terminal domain that faces the cytosol, a single transmembrane &agr; helix, and a large C-terminal domain that faces the Golgi lumen and that contains the catalytic site. The transmembrane &agr; helix is necessary and sufficient to restrict the enzyme to the Golgi. Of the Beta-1,3-galactosyltransferase family two members (See Amado, M. et al.,
J. Biol. Chem.
273, 21: 12770-12778, 1998) have been predicted to have two potentially different initiation codons, resulting in two different N-terminal cytoplasmic domains.
Additionally, galactosyltransferases have been shown to be expressed on the cell surface, where their function is theorized to participate in cellular interactions, perhaps as receptors, or receptor-like complementary molecules. As a cell surface carbohydrate, galactosyltransferases have been implicated in varied biology such as cell migration, contact inhibition, tissue interactions, neuronal specificity, fertilization, embryonic cell adhesions, limb bud morphogenesis, mesenchyme development, immune recognition, growth control, and tumor metastasis. See, for example, Shur, B. D.,
Mol Cell Bioc.
61:143-158, 1984.
The failure of tumor cell-tumor cell adhesion is believed to be a contributing factor to tumor metastases. See, for example, Zetter,
Cancer Biology,
4: 219-29, 1993. Metastases, in turn, are generally associated with poor prognosis for cancer treatment. The metastatic process involves a variety of cellular events, including angiogenesis, tumor cell invasion of the vascular or lymphatic circulation, tumor cell arrest at a secondary site; tumor cell passage across the vessel wall into the parenchymal tissue, and tumor cell proliferation at the secondary site. Thus, both positive and negative regulation of adhesion are necessary for metastasis. That is, tumor cells must break away from the primary tumor mass, travel in circulation and adhere to cellular and/or extracellular matrix elements at a secondary site. Molecules capable of modulating cell-cell and cell-matrix adhesion are therefore sought for the study, diagnosis, prevention or treatment of metastases.
&bgr;1→3 Galactosyltransferases have limited homology to each other. In contrast to other glycosyltransferases, they do not appear to be localized to the same chromosomes. Additionally, a member of this family has recently been identified in Drosophila. This molecule, Brainiac, is involved in contact and adhesion between germ-line and follicle cells (Amado, M. et al.,
J. Biol. Chem.
273, 21: 12770-12778, 1998).
A deficiency of Beta-1,3-galactosyltransferase enzymes has been noticed in the Tn-syndrome. This syndrome is a rarely acquired disorder affecting all hemopoietic lineages, and is characterized by the expression of the Tn and the sialosyl-Tn antigens on the cell surface. The Tn is &agr;N-acetylgalactosamine linked O-glycosidically to threonine or serine residues of membrane proteins. These antigens bind naturally occurring serum antibodies thereby leading to mild hemolytic anemia and pronounced thrombopenia. Thus, the blood cells in the Tn-syndrome are expected to carry less sialic acid if galactose can not be transferred to N-Acetylgalactosamine. The expression of Tn and sialosyl-Tn antigens as a consequence of imcomplete or disordered gylcan biosynthesis has been recognized as a cancer-associated phenomenon. Tn and sialosyl-Tn antigens are among the most investigated cancer-associated carbohydrates antigens.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
Within one aspect, the present invention provides an isolated polypeptide comprising residues 148 to 397 of SEQ ID NO:2. Within an embodiment, the isolated polypeptide comprises residues 19 to 397 of SEQ ID NO:2. Within another embodiment, the isolated polypeptide comprises residues 1 to 397 of SEQ ID NO:2.
Within another aspect, the present invention provides an isolated polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 18 of SEQ ID NO:2; a polypeptide comprising residues 19 to 147 of SEQ ID NO:2; a polypeptide comprising residues 148 to 397 of SEQ ID NO:2; a polypeptide comprising residues 19 to 397 of SEQ ID NO:2; and a polypeptide comprising residues 1 to 397 of SEQ ID NO:2.
Within another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide wherein the polypeptide comprises residues 148 to 397 of SEQ ID NO:2. Within an embodiment, the polypeptide molecule comprises residues 19 to 397 of SEQ ID NO:2. Within another embodiment, the polypeptide molecule comprises residues 1 to 397 of SEQ ID NO:2.
Within another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide molecule wherein the polypeptide is selected from the group consisting of: a polypeptide comprising residues 1 to 18 of SEQ ID NO:2; a polypeptide comprising residues 19 to 147 of SEQ ID NO:2; a polypeptide comprising residues 148 to 397 of SEQ ID NO:2; a polypeptide comprising residues 19 to 397 of SEQ ID NO:2; and a polypeptide comprising residues 1 to 397 of SEQ ID NO:2. Within an embodiment is provided an expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment wherein the DNA segment is a polynucleotide encoding the polypeptide of claim 1; and a transcription terminator. Within another embodiment the DNA segment contains an affinity tag. Within another embodiment, the invention provides a cultured cell into which has been introduced the expression vector, wherein said cell expresses the polypeptide encoded by the DNA segment. Within another embodiment the invention provides a method of producing a polypeptide comprising culturing the cell, whereby said cell expresses the polypeptide encoded by the DNA segment; and recovering the polypeptide.
Within another aspect is provided a method of producing an antibody comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of: a polypeptide comprising residues 1 to 18 of SEQ ID NO:2; a polypeptide comprising residues 19 to 147
Conklin Darrell C.
Gao Zeren
Jaspers Stephen R.
Yamamoto Gayle
Adams Robyn
Prouty Rebecca E.
Steadman David J.
Zymogenetics Inc.
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