Chapter 19 In Situ Glycosylation of Flagellar Lipids

Abstract

Publisher Summary The glycosyltransferases involve in functions including glycosylation of proteins for sorting, modification of the proteins and lipids that determine antigenic properties of cell surfaces, and mediation of a variety of cell–cell interactions. Glycosyltransferases can covalently bind sugars to a variety of lipids. In Euglena, steryl glucosides have been identified in lipid extracts of flagella, and some of these appear to be distinct from the steryl glucosides present in the adjacent cell surface membrane. Some of these lipids are glycosylated in situ as isolated flagella, and detergent extracts of flagella can glycosylate endogenous and exogenous lipid substrates, indicating that functional glycosyltransferases are residents of the flagellar membrane. Flagellar-specific glycosyltransferases that use UDP-glucose may provide unique markers for identifying flagellar membranes and for following the ontogeny and development of flagellar membranes. This chapter summarizes procedures for the detection and characterization of lipid substrates and lipid glucosyltransferases in Euglena flagella. The chapter details the protocol for demonstrating glucosyltransferase activity—the solutions and materials, the donor, the acceptor, and the product. There are notes on the cofactors and products. One requisite for sugar transfer is a nucleotide diphosphate-activated sugar donor. Controls include the addition of unlabeled nucleotide sugars and unlabeled sugars to assess their competitive effects on the incorporation of UDP-[ 3 H] glucose. Various assays indicate that the native acceptors have properties in common with sterols, and that one of these acceptors is unique to the flagellum, whereas the other major acceptor can also be extracted from the cell surface membrane. The use of an exogenous sterol acceptor can greatly expedite the assay for, and facilitate the identification of, the flagellar glycosyltransferase(s). For the Glucosyltransferase(s)—their enrichment and identification on polyacrylamide gels—the chapter details the solutions and materials, flagellar preparation, methods for enrichment of enzyme activity, and 3-[(3-cholamidopropyI) dimethylammonio]-1-propanesulfonate extraction of flagellar vesicles.