Toyohisa Kurita

A Novel Enzyme That Cleaves the N-Acyl Linkage of Ceramides in Various Glycosphingolipids as Well as Sphingomyelin to Produce Their Lyso Forms

We describe a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids. The enzyme was purified about 300-fold with 5% recovery from the culture filtrate of a newly isolated bacterium (Pseudomonas sp. TK4) by ammonium sulfate precipitation followed by several steps of high performance liquid chromatography. The purified enzyme preparation was completely free of exoglycosidases, sphingomyelinase, and proteases, and showed a single protein band corresponding to a molecular mass of 52 kDa on SDS-polyacrylamide slab gel electrophoresis after staining with Coomassie Brilliant Blue. The enzyme shows quite wide specificity, i.e. it hydrolyzes both neutral and acidic glycosphingolipids, and simple glycosphingolipid cerebrosides to polysialogangliosides such as GQ1b. Furthermore the enzyme also hydrolyzes sphingomyelin to produce the respective lyso form. However, the enzyme shows hardly any activity on ceramides, indicating that it is completely different from the ceramidase (EC 3.5.1.23) reported previously. This enzyme, which is tentatively named sphingolipid ceramide N-deacylase, should greatly facilitate the further study of sphingolipids as well as lysosphingolipids.

Enzymatic N-deacylation of sphingolipids

Publisher Summary 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 Krabbes 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.