Cloning of a mouse beta 1,3 N-acetylglucosaminyltransferase GlcNAc(beta 1,3)Gal(beta 1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-ser ies glycolipids is catalyzed by the beta1,3 N-acetylglucosaminyltransferase GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 syn thase activity has been shown to be regulated during development, especiall y during brain morphogenesis. Here, we report the molecular cloning of a mo use gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open r eading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the beta1,3 glycosyltransferase superfamily. The Lc3 syn thase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor . The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using anti bodies TE-8 and 1B2 that recognize Lc3 and Gal(beta1,4)GlcNAc(beta1,3)Gal(b eta1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the ini tiating activity for lacto-chain synthesis, the Lc3 synthase could extend t he terminal N-acetyllactosamine unit of nLc4 and also had a broad specifici ty for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryon ic development. In situ hybridization analysis revealed that that the Lc3 s ynthase was expressed in most tissues at embryonic day 11 with elevated exp ression in the developing central nervous system. Postnatally, the expressi on was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthes is during embryonic development.