32] Enzymatic N-deacylation of sphingolipids

Abstract

Lysosphingolipids, sphingolipids with an N-deacylated ceramide moiety, are present at low levels in normal tissues, but accumulate abnormally in cells in various lysosomal storage diseases. For example, in Krabbe's disease, caused by a deficiency of β -galactosylceramidase, abnormal accumulation of galactosylceramide as well as its lyso form is observed. Lyso-GM2 and lysosphingomyelin have been detected in the brain of patients with Tay-Sachs and Niemann-Pick type-A disease, respectively, whereas they are barely detectable in the normal brain. Lysosphingolipids inhibit protein kinase C, which could be responsible for the pathogenesis of sphingolipidoses. Several lines of evidence have suggested the biological significance of lysosphingolipids in various cell activities. Lysosphingolipids are useful for preparing sphingolipid derivatives containing appropriately labeled fatty acids and can be coupled with either appropriate proteins or gel matrix for affinity columns utilizing the amino groups newly generated in lysosphingolipids. This chapter describes a novel enzyme, tentatively designated sphingolipid ceramide N-deacylase (SCDase), which is capable of cleaving the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. To date, the preparation of lysosphingolipids has been performed using purely chemical procedures, which are somewhat troublesome, time-consuming, and give a low yield. Using the SCDase we were able to obtain easily the lyso forms of all species of glycosphingolipids and sphingomyelin without any alternation of their polar portions and sphingoid moieties.