33] Genetic approaches for studies of glycolipid synthetic enzymes

Abstract

Publisher Summary The synthesis of sphingolipids starts from the condensation of L -serine and palmitoyl-CoA. After several reactions, ceramide, a pivotal sphingolipid, is synthesized as the core structure of all sphingolipids, including sphingomyelin (SM) and over 300 species of glycosphingolipids (GSLs). The synthesis of most GSLs begins with the glucosylation of ceramide catalyzed by GlcCer synthase (ceramide glucosyltransferase, GlcT-1, EC 2.4.1.80) and its product glucosylceramide (GlcCer). These GSLs are believed to play important roles in a variety of cellular processes such as cell recognition, growth, development, and differentiation. Ceramide glucosylation is also important as a regulatory factor for the intracellular level of ceramide, which is now regarded as a second messenger. Enzymes involved in the initial steps of sphingomyelin and glycosphingolipid syntheses are usually difficult to isolate because they are tightly attached to membranes. The development of expression cloning by Seed and Aruffo offered an excellent system for the isolation of sphingolipid-metabolizing enzymes. Theoretically, the method can be applied for the isolation of most of the cDNAs encoding sphingolipid synthetic enzymes. This chapter describes the methods used for the cloning of GlcCer synthase and discusses the possible application of this approach to the isolation of other sphingolipid synthetic enzymes.