Functional Analysis of Sphingolipids Using a Specific Enzyme and Animal Cells

Abstract

Glycosphingolipids (GSLs) and sphingomyelin (SM) are amphipathic compounds composed of a hydrophilic head group and a common hydrophobic structure, ceramide. Recently, they were found to be enriched with cholesterol and GPI-anchor proteins, and to form microdomains on the plasma membranes of vertebrates [1]. GSLs, SM and their metabolites have emerged as a new class of lipid biomodulators, which regulate cell-cell interaction and recognition, cellular proliferation, differentiation and apoptosis [2]. Some of GSLs function as a receptor for microbial infections that occur on the cell surface [3]. It was suggested that these functions of GSLs could be determined by not only the structure of the carbohydrate but also that of the ceramide moiety, especially the fatty acid composition [4]. We have found sphingolipid ceramide N-deacylase (SCDase) which releases fatty acids from the ceramide moieties of GSLs and SM to generate the respective lyso-sphingolipids [5]. SCDase is completely different from ceramidase (EC 3.5.1.23), because SCDase hardly hydrolyzes free ceramide, while ceramidases are specific to free ceramide and do not hydrolyze GSLs or SM. Recently, SCDase was found to condense fatty acids to lysosphingolipids (sphingolipids without fatty acids) to generate sphingolipids [6]. This condensation reaction was successfully applied to the preparation of radioisotope- [7, 8] and fluorescence-labeled sphingolipids [9, 10]. SCDase can be used as ‘scissors’ and as ’paste’ for remodeling of the fatty acid moieties of sphingolipids, and thus the enzyme will facilitate the further development of sphingolipid research. This paper describes the characterization of the reversible nature of SCDase, remodeling of fatty acid moieties of GSLs by the enzyme, and functional analysis of the remodeled GSLs using mouse neuroblastoma Neuro2a cells.