Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major.
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 Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1, 2-dipalmitoylglycerol) and Man alpha 1-4GlcN alpha 1-6myo-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. 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 alpha 1-4-mannosyltransferase, a phospholipase A2-like activity and a second alpha-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 alpha1-4-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.
Smith
T K
TK
Department of Biochemistry, University of Dundee, Scotland, U.K.
Milne
F C
FC
Sharma
D K
DK
Crossman
A
A
Brimacombe
J S
JS
Ferguson
M A
MA
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D018320:Leishmania major / Q000201:enzymology* / Q000378:metabolism*; D008364:Mannosyltransferases / Q000378:metabolism; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity
D017261:Glycosylphosphatidylinositols; D011104:Polyisoprenyl Phosphate Oligosaccharides; C038433:dolichol pyrophosphate Man(1-4)-GlcNAc(1-4)GlcNAc; D008364:Mannosyltransferases; D000581:Amidohydrolases; C087696:N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase