Expression cloning of human globoside synthase cDNAs - Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine: Globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase

By using a eukaryocytic cell expression cloning system, we have isolated cD NAs of the globoside synthase (beta1,3-N-acetylgalactosaminyltransferase) g ene. Mouse fibroblast L cells transfected with SV40 large T antigen and pre viously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA libr ary prepared from mRNA from human kidney together with Forssman synthase cD NA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading fra me predicting a type II membrane protein with 351 amino acids. Surprisingly , the cDNA clones turned out to be identical with previously reported beta 3Gal-T3, which had been cloned by sequence homology with other galactosyltr ansferases. Substrate specificity analysis with extracts from cDNA-transfec ted L cells confirmed that the gene product was actually pl,3-N-acetylgalac tosaminyltransferase that specifically catalyzes the transfer of N-acetylga lactosamine onto globotriaosylceramide. Results of TLC immunostaining of ne utral glycolipids from the cDNA-transfected cells also supported the identi ty of the newly synthesized component as globoside. The results show that g lycosyltransferases apparently belonging to a single glycosyltransferase fa mily do not necessarily catalyze reactions utilizing the same acceptor or e ven the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation bet a 3GalNAc-T1 for the cloned globoside synthase gene.