Control of O-Glycan Branch Formation

Abstract

Core 2 O-glycan branching catalyzed by UDP-N-acetyl-D-glucosamine: acceptor 1,6-N-acetylglucosa-minyltransferases (6GlcNAc-Ts) is an important step in mucin-type biosynthesis. Core 2 complex-type O-glycans are involved in selectin-mediated adhesion events, and O-glycan branching appears to be highly regulated. Two homologous 6GlcNAc-Ts functioning in O-glycan branching have previously been characterized, and here we report a third homologous 6GlcNAc-T designated C2GnT3. C2GnT3 was identified by BLAST analysis of human genome survey sequences. The catalytic activity of C2GnT3 was evaluated by in vitro analysis of a secreted form of the protein expressed in insect cells. The results revealed exclusive core 2 6GlcNAc-T activity. The product formed with core 1-para-nitrophenyl was confirmed by 1 H NMR to be core 2-para-nitrophenyl. In vivo analysis of the function of C2GnT3 by coexpression of leukosialin (CD43) and a full coding construct of C2GnT3 in Chinese hamster ovary cells confirmed the core 2 activity and failed to reveal I activity. The C2GnT3 gene was located to 5q12, and the coding region was contained in a single exon. Northern analysis revealed selectively high levels of a 5.5-kilobase C2GnT3 transcript in thymus with only low levels in other organs. The unique expression pattern of C2GnT3 suggests that this enzyme serves a specific function different from other members of the 6GlcNAc-T gene family.