Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major

A cell-free system based on washed Leishmania majormembranes was labelled with GDP-[3H]Man in the presence of synthetic glucosaminyl-phosphatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). In both cases, the major radiolabelled products were Manα1-4GlcNα1-6myo-inositol1-HPO4- (sn-1,2-dipalmitoylglycerol) and Manα1-4GlcNα1-6myo-inositol1-HPO4- (sn-1-palmitoyl-2-lyso-glycerol), to which an additional D-mannose residue was added when a chase with an excess of GDP-Man was performed. The L. majorcell-free system can therefore be used to observe the actions of four enzymes, namely GlcNAc-PI de-N-acetylase, Dol-P-Man–GlcN-PI α1-4-mannosyltransferase, a phospholipase A2-like activity and a second α-mannosyltransferase activity. The substrate specificities of the first two of these enzymes were studied using a series of substrate analogues. GlcNAc-PI de-N-acetylase was tested against a variety of N-acylated GlcN-PI substrates and was able to cleave N-acetyl and N-propyl groups but not larger groups such as N-butyl, N-isobutyl, N-pentyl and N-hexyl. The Dol-P-Man–GlcN-PI α1-4-mannosyltransferase activity required the amino group of the glucosamine residue and the d-configuration of the myo-inositol residue of the GlcN-PI acceptor substrate.