Cloning and expression of a novel galactoside beta1, 3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope.
We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.
Shimoda
Y
Y
Department of Biochemical Cell Research, Tokyo Metropolitan Institute of Medical Science (RINSHOKEN), Tokyo 113-8613, Japan.
Tajima
Y
Y
Nagase
T
T
Harii
K
K
Osumi
N
N
Sanai
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000196:embryology / Q000378:metabolism; D018959:CD57 Antigens / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000096:biosynthesis*; D005915:Globosides / Q000378:metabolism; D005965:Glucuronates / Q000378:metabolism; D020723:Glucuronic Acid; D014453:Glucuronosyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009844:Oligosaccharides / Q000096:biosynthesis*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D014018:Tissue Distribution
D018959:CD57 Antigens; D018076:DNA, Complementary; D000939:Epitopes; D005915:Globosides; D005965:Glucuronates; D006023:Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D020723:Glucuronic Acid; C119714:galactoside beta-1,3-glucuronyltransferase; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase