The effects of phenylmethylsulfonyl fluoride on inositol-acylation and fatty acid remodeling in African trypanosomes.
Phenylmethylsulfonyl fluoride (PMSF) has been shown to inhibit the addition of ethanolamine phosphate to glycosylphosphatidylinositol (GPI) intermediates in Trypanosoma brucei (Masterson, W. J., and Ferguson, M. A. J. (1991) EMBO J. 10, 2041-2045). Here we show that the Man3-GlcN-PI intermediate that accumulates in the presence of PMSF can undergo fatty acid remodeling, suggesting that the fatty acid remodeling enzymes are not specific for ethanolamine phosphate-containing GPI intermediates. We also show that PMSF inhibits the acylation of the inositol residue of GPI intermediates in bloodstream form T. brucei. Pulse-chase experiments demonstrate that glycolipid C (ethanolamine-PO4-Man3-GlcN-(acyl)PI) is not an obligatory precursor of glycolipid A (ethanolamine-PO4-Man3-GlcN-PI) and that glycolipid C can be converted to glycolipid A. These data suggest a model where glycolipid C is the terminal product of the GPI biosynthetic pathway, in dynamic equilibrium with glycolipid A. The inhibition of ethanolamine phosphate addition and inositol acylation by PMSF was also observed for procyclic forms of T. brucei but not for mammalian HeLa cells. These results suggest differences between the relevant parasite and mammalian enzymes.
Güther
M L
ML
Department of Biochemistry, University of Dundee, Scotland, United Kingdom.
Masterson
W J
WJ
Ferguson
M A
MA
D000215:Acylation; D000818:Animals; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005227:Fatty Acids / Q000378:metabolism*; D006017:Glycolipids / Q000096:biosynthesis* / Q000302:isolation & purification; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000302:isolation & purification; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007294:Inositol / Q000378:metabolism*; D007700:Kinetics; D008361:Mannosidases / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D051381:Rats; D013997:Time Factors; D014316:Tritium; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism; D043323:alpha-Mannosidase
D005227:Fatty Acids; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D014316:Tritium; D006155:Guanosine Diphosphate Mannose; D007294:Inositol; D014537:Uridine Diphosphate N-Acetylglucosamine; D010664:Phenylmethylsulfonyl Fluoride; D010727:Phosphoric Diester Hydrolases; D008361:Mannosidases; D043323:alpha-Mannosidase; D043265:Phosphatidylinositol Diacylglycerol-Lyase