Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-21
2001-11-27
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S183000, C536S023100
Reexamination Certificate
active
06323332
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to enzymes and more specifically to sulfotransferases that synthesize the HNK-1 carbohydrate epitope.
2. Background Information
Neural and immune cells express certain characteristic carbohydrate glycans on their cell surfaces (Jessell et al. (1985) Ann. Rev. Neurosci. 13, 227-255; Schachner and Martini (1995) Trends Neurosci. 18, 183-191; all references cited herein are incorporated by reference). One such glycan is the HNK-1 carbohydrate epitope, originally discovered by a monoclonal antibody raised against Human Natural Killer cells (Abo and Balch (1981) J. Immunol. 127, 1024-1029). The functional significance of the HNK-1 carbohydrate was first recognized as an autoantigen involved in peripheral demyelinative neuropathy. The structural analysis of glycolipids reacting with these autoantibodies led to the discovery that the HNK-1 epitope is sulfo→3GlcA&bgr;1→3Gal&bgr;1→4GlcNAc&bgr;1→R (Chou et al., (1986) J. Biol. Chem. 261, 11717-11725; Ariga et al., (1987) J. Biol. Chem. 262, 848-853).
By using HNK-1-specific antibodies and carbohydrate structural studies, the HNK-1 glycan has been found in a number of neural cell adhesion molecules including N-CAM, myelin-associated glycoprotein, L1, contactin and P0 (Schachner and Martini (1995) Trends Neurosci. 18, 183-191; McGarry et al., (1983) Nature (London) 306, 376-378; Kruse et al., (1984) Nature (London) 311, 153-155; Gennarini et al., (1989) J. Neurosci. Res. 22, 1-12; Voshol et al., (1996) J. Biol. Chem. 271, 22957-22960). The studies, using either monoclonal antibodies or isolated carbohydrates, demonstrated that the HNK-1 glycan is involved in cell-cell and cell-substratum interactions (Keilhauer et al. (1985) Nature (London) 316, 728-730; Mohan et al., (1990) J. Neurochem. 54, 2024-2031).
Expression of the HNK-1 epitope is spatially and developmentally regulated, and is found on migrating neural crest cells, cerebellum and myelinating Schwann cells in motor neurons, but not on those in the sensory neurons (Bronner-Fraser (1986) Dev. Biol. 115, 44-55; Eisenman and Hawkes, (1993) J. Comp. Neurol. 335, 586-605; Martini et al., (1992) Eur. J. Neurosci. 4, 628-639). In addition, the HNK-1 carbohydrate binds to P-selectin and L-selectin (Needham and Schnaar, (1993) Proc. Natl. Acad. Sci., U.S.A. 90, 1359-1363), suggesting that interactions between immune cells and the nervous system may be mediated through binding of the HNK-1 glycan in neural cells.
The HNK-1 glycan is synthesized in a stepwise manner by adding a &bgr;1,3-linked glucuronic acid to a precursor N-acetyllactosamine, followed by adding a sulfate group to GlcA&bgr;1→3Gal&bgr;1→4GlcNAc&bgr;1→R (Jungalwala, (1994) Neurochem. Res. 19, 945-957; Chou and Jungalwala, (1993) J. Biol. Chem. 268, 330-336). A &bgr;-1,3-glucuronyl transferase GlcAT-P (glycoprotein-specific glucuronyltransferase)—which forms an HNK-1 precursor carbohydrate, GlcA&bgr;1→3Gal&bgr;1→4GlcNAc&bgr;1→R, in glycoproteins—has been cloned (Terayama et al., (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 6093-6098).
Desulfation experiments have shown that the sulfate group plays a critical role (Mohan et al., (1990) J. Neurochem. 54, 2024-2031). A sulfotransferase involved in the synthesis of HNK-1 has been cloned from rat cells (“raHNK-1ST”) (Bakker et al., (1997) J. Biol. Chem. 272, 29942-29946.), although it is believed that the sequence of this sulfotransferase was not publicly known or available until after the date of conception or reduction to practice of the present invention. For the sake of comparison, however, the rat sequence is described herein.
Thus, HNK-1 sulfotransferases have not yet been described as of the present invention. Thus, a need exists for a HNK-1 sulfotransferase involved in synthesizing the HNK-1 glycan. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides an HNK-1 sulfotransferase that synthesizes the HNK-1 carbohydrate epitope in cells of the nervous system. The sequences for human and mouse HNK-1 sulfotransferases are provided. The invention also provides peptide portions of HNK-1ST that catalytically active. Also provided are antibodies that bind to human HNK-1ST, as well as cells producing such antibodies.
The present invention also provides a nucleic acid encoding a HNK-1ST polypeptide. In addition, the invention provides polynucleotide sequences that hybridize under stringent conditions to a nucleic acid molecule encoding human HNK-1ST, but not to a nucleic acid molecule encoding rat HNK-1ST. Also provided are vectors containing a nucleic acid molecule of the invention.
The invention further provides methods of synthesizing an HNK-1 carbohydrate epitope on a glycoprotein or glycolipid precursor, by contacting the precursor with human HNK-1ST. Such contacting can occur in vitro or in vivo and, when performed in vitro, can be performed in a cell-free system using a substantially purified precursor or using cells, which express the precursor, in culture. The invention also provides a method of functional expression cloning.
REFERENCES:
Adams et al. GenBank Accession No. AA338789, Apr. 21, 1997.*
Hillier et al. Genbank Accession No. R18331, Apr. 14, 1995.*
Aigner et al., “CD24, a Mucin-Type Glycoprotein, Is a Ligand for P-Selectin on Human Tumor Cells,”Blood, 89:3385-3395 (1997).
Bakker et al., “Expression Cloning of a cDNA Encoding a Sulfotransferase Involved in the Biosynthesis of the HNK-1 Carbohydrate Epitope,”J. Biol. Chem., 272(47):29942-29946 (1997).
Chou et al., “Characterization and Developmental Expression of a Novel Sulfotransferase for the Biosynthesis of Sulfoglucuronyl Glycolipids in the Nervous System,”J. Biol. Chem., 268:330-336 (1993).
Fukuta et al., “Molecular Cloning and Expression of Chick Chondrocyte Chondroitin 6-Sulfotransferase,”J. Biol. Chem., 270:18575-18580 (1995).
Honke et al., “Molecular Cloning and Expression of cDNA Encoding Human 3′-Phosphoadenylylsulfate:galactosylceramide 3′-Sulfotransferase,”J. Biol. Chem., 272:4864-4868 (1997).
Jungalwala, F.B., “Expression and Biological Functions of Sulfoglucuronyl Glycolipids (SGGLs) in the Nervous System—A Review,”Neurochem. Res., 19:945-957 (1994).
Jackson et al., “CD24, a Signal-transducing Molecule Expressed on Human B Cells, Is a Major Surface Antigen on Small Cell Lung Carcinomas,”Cancer Res., 52:5264-5270 (1992).
Kobayashi et al., “Molecular Cloning and Expression of Chinese Hamster Ovary Cell Heparan-sulfate 2-Sulfotransferase,”J. Biol. Chem., 272:13980-13985 (1997).
Mohan et al., “Sulfoglucuronyl Glycolipids Bind Laminin,”J. Neurochem., 54:2024-2031 (1990).
Oka et al., “A Novel Glucuronyltransferase in Nervous System Presumably Associated with the Biosynthesis of HNK-1 Carbohydrate Epitope on Glycoproteins,”J. Biol. Chem., 267:22711-22714 (1992).
Schachner et al., “Glycans and the modulation of neural-recognition molecule function,”Trends Neurosci., 18:183-191 (1995).
Shworak et al., “Molecular Cloning and Expression of Mouse and Human cDNAs Encoding Heparan Sulfate D-Glycosaminyl 3-O-Sulfotransferase,”J. Biol. Chem., 272:28008-28019 (1997).
Terayama et al., “Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope HNK-1,”Proc. Natl. Acad. Sci. USA, 94:6093-6098 (1997).
Fukuda Minoru
Ong Edgar
Campbell & Flores LLP
Prouty Rebecca E.
Rao Manjunath
The Burnham Institute
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