Abstract
While expression-cloning enzymes involved in heparan sulfate biosynthesis, we isolated a cDNA that encodes a protein 65% identical to the UDP-GlcUA:glycoprotein beta1, 3-glucuronosyltransferase (GlcUAT-P) involved in forming HNK-1 carbohydrate epitopes (3OSO3GlcUAbeta1,3Gal-) on glycoproteins. The cDNA contains an open reading frame coding for a protein of 335 amino acids with a predicted type II transmembrane protein orientation. Cotransfection of the cDNA with HNK-1 3-O-sulfotransferase produced HNK-1 carbohydrate epitopes in Chinese hamster ovary (CHO) cells and COS-7 cells. In vitro, a soluble recombinant form of the enzyme transferred GlcUA in beta-linkage to Galbeta1,3/4GlcNAcbeta-O-naphthalenemethanol, which resembles the core oligosaccharide on which the HNK-1 epitope is assembled. However, the enzyme greatly preferred Galbeta1, 3Galbeta-O-naphthalenemethanol, a disaccharide component found in the linkage region tetrasaccharide in chondroitin sulfate and heparan sulfate. During the course of this study, a human cDNA clone was described that was thought to encode UDP-GlcUA:Galbeta1,3Gal-R glucuronosyltransferase (GlcUAT-I), involved in the formation of the linkage region of glycosaminoglycans (Kitagawa, H., Tone, Y., Tamura, J., Neumann, K. W., Ogawa, T., Oka, S., Kawasaki, T., and Sugahara, K. (1998) J. Biol. Chem. 273, 6615-6618). The deduced amino acid sequences of the CHO and human cDNAs are 95% identical, suggesting that they are in fact homologues of the same gene. Transfection of a CHO cell mutant defective in GlcUAT-I with the hamster cDNA restored glycosaminoglycan assembly in vivo, confirming its identity. Interestingly, transfection of the mutant with GlcUAT-P also restored glycosaminoglycan synthesis. Thus, both GlcUAT-P and GlcUAT-I have overlapping substrate specificities. However, the expression of the two genes was entirely different, with GlcUAT-I expressed in all tissues tested and GlcUAT-P expressed only in brain. These findings suggest that, in neural tissues, GlcUAT-P may participate in both HNK-1 and glycosaminoglycan production.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF113703
By partially purifying the enzymes and noting differences in phospholipid activation and pH dependence, Oka et al [9] concluded that the glucuronosyltransferase involved in the synthesis of HNK-1 epitopes on glycoproteins (GlcUAT-P)1 differs from the one that acts on glycolipids (GlcUAT-L)
Isolation of a cDNA Clone for a Glucuronosyltransferase from Chinese Hamster Ovary Cells—During a series of experiments to identify genes involved in glycosaminoglycan synthesis, we found a cDNA clone that showed high homology to the recently cloned rat glycoprotein glucuronosyltransferase involved in the assembly of HNK-1 epitopes (GlcUAT-P) [10]
Summary
Cell Cultures—Chinese hamster ovary (CHO-K1, ATCC CCL-61), COS-7 (ATCC CRL-1651), and Lec (ATCC CRL-1736) cells were obtained from the American Type Culture Collection (Manassas, VA). The cDNA fragment encoding amino acids 39 –347 of rat GlcUAT-P (GenBankTM/EBI accession number D88035) [10] was prepared by PCR using pcDNA3-GlcUAT-P as a template Cells were washed twice with PBS, blocked at room temperature for 10 min with 2% (w/v) bovine serum albumin in PBS, and incubated at room temperature for 30 – 40 min with mouse monoclonal anti-HNK-1 antibody (Becton Dickinson Advanced Cellular Biology) diluted 1:100 in buffer. The cells were incubated at room temperature for 20 –30 min with fluorescein isothiocyanate-conjugated goat antimouse IgM antibody (Sigma) diluted 1:100 with buffer. Material that comigrated with the authentic UDP-GlcUA was eluted from the paper with 10% (v/v) ethanol/water, lyophilized, and dissolved in 100 l of buffer containing 10 units of calf intestinal phosphatase (Life Technologies, Inc.).
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