Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins.
Trypanosoma brucei variant surface glycoproteins are apparently synthesized with a hydrophobic carboxyl-terminal peptide that is cleaved and replaced by a complex glycosylphosphatidylinositol membrane anchor within 1 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated core glycolipid that would be transferred en bloc to the variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent with a role as a variant surface glycoprotein glycolipid donor. This candidate glycolipid precursor has been defined by thin-layer chromatography of extracts of trypanosomes metabolically labeled with radioactive myristic acid, ethanolamine, glucosamine, mannose, and phosphate and by enzymatic, chemical, and gas chromatographic-mass spectrometric analysis. Mild alkali released 100% of the myristic acid, and reaction with phospholipase A2 released 50%. Nitrous acid deamination generated dimyristylphosphatidylinositol, and periodate oxidation released phosphatidic acid. Treatment of purified glycolipid with phosphatidylinositol-specific phospholipase C released dimyristylglycerol and a water-soluble glycan that was sized on Bio-Gel P-4 columns. The candidate precursor contained mannose, myristic acid, phosphate, and ethanolamine with an unsubstituted amino group, but not galactose.
Menon
A K
AK
Rockefeller University, New York, New York 10021.
Mayor
S
S
Ferguson
M A
MA
Duszenko
M
M
Cross
G A
GA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis; D005690:Galactose / Q000032:analysis; D006017:Glycolipids / Q000302:isolation & purification*; D008358:Mannose / Q000032:analysis; D008565:Membrane Proteins / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011498:Protein Precursors / Q000302:isolation & purification*; D014346:Trypanosoma brucei brucei; D010738:Type C Phospholipases / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000096:biosynthesis*
D004983:Ethanolamines; D005227:Fatty Acids; D006017:Glycolipids; D008565:Membrane Proteins; D009227:Myristic Acids; D011498:Protein Precursors; D014643:Variant Surface Glycoproteins, Trypanosoma; D019814:Myristic Acid; D019856:Ethanolamine; D010738:Type C Phospholipases; D008358:Mannose; D005690:Galactose