Analysis of galactolipids and UDP-galactose: Ceramide galactosyltransferase

Glycosphingolipids are enriched in the outer leaflet of the plasma membrane of most eukaryotic cells where they are thought to be involved in cell recognition and signaling. Although glycosphingolipids constitute only a few mol% of the lipids in most membranes, they are major components of the myelin sheath, where GalCer and SGalCer are involved in axonal insulation, myelin function, and stability. The apical plasma membrane of epithelial cells in the gastrointestinal and urinary tracts is enriched in glycosphingolipids. In rodents, these are typically glucolipidsy whereas in humans most are galactolipids. Glycosphingolipids play a structural role in rigidifying and protecting the apical cell surface. Their role in sorting lipids and proteins to various membranes along the exocytotic and endocytotic transport routes is not fully understood. The foremost enzyme involved in the biosynthesis of galactosphingolipids is the UDP-galactose:ceramide galactosyltransferase, CGalT or GalT-1. CGalT catalyzes the transfer of galactose from UDP-galactose to Cer yielding GalCer and has relatively promiscuous substrate specificity. Whether there are one or more CGalT enzymes with distinct specificity and cellular localization has been a controversial issue. The localization of CGalT has long been enigmatic. The enzyme is exclusively localized to the endoplasmic reticulum (ER) by immunogold electron microscopy on ultrathin cryosections. CGalT is a high mannose-type glycoprotein that is N-glycosylated at Asn-78 and Asn-333 and contains a putative carboxy terminal Lys-Lys-Val-Lys ER-retrieval signal. The conceptual translation product exhibits no amino acid sequence similarity with other glycosyltransferases. Instead, CGalT is related to the superfamily of UDP-glucuronosyltransferases.