Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major

Terry K. Smith, Fiona C. Milne, Deepak K. Sharma, Arthur Crossman, John S. Brimacombe, Michael A. J. Ferguson

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    37 Citations (Scopus)

    Abstract

    A cell-free system based on washed Leishmania major membranes 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 Mana1-4GlcNa1-6myo-inositoll-HPO4-(sn-1,2-dipalmitoylglycerol) and Mana1-4GlcNa1-6myo-inositoll-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. major cell-free system can therefore be used to observe the actions of four enzymes, namely GlcNAc-PI de-N-acetylase, Dol-P-Man-GlcN-PI a1-4-mannosyltransferase, a phospholipase A2-like activity and a seconda-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 a1-4-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.

    Original languageEnglish
    Pages (from-to)393-400
    Number of pages8
    JournalBiochemical Journal
    Volume326
    Issue number2
    Publication statusPublished - 1 Sep 1997

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