Abstract
A number of eukaryotic surface glycoproteins, including the variant surface glycoproteins of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosyl-phosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated precursor glycolipid that can be transferred en bloc to the polypeptide. We have reported the purification and partial characterization of a candidate precursor glycolipid (P2) and of a compositionally similar glycolipid (P3) from T. brucei (Menon, A. K., Mayor, S., Ferguson, M. A. J., Duszenko, M., and Cross, G. A. M. (1988) J. Biol. Chem. 263, 1970-1977). The primary structure of the glycan portions of P2 and P3 have now been analyzed by a combination of selective chemical fragmentation and enzymatic glycan sequencing at the subnanomolar level. The glycans were generated by deamination, NaB3H4 reduction, and dephosphorylation of glycolipids purified from different trypanosome variants. Glycan fragments derived from biosynthetically labeled glycolipids were also analyzed. The cumulative data strongly suggest that P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The structural similarities suggest that GPI membrane anchors are derived from common precursor glycolipids that become variably modified during or after addition to newly synthesized proteins.
Highlights
From the Oxford Glycobiology Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
Aqueous HF Dephosphorylation-The glycolipid anchor of membrane form variant surface glycoprotein (VSG) contains two phosphodiester linkages, one between the glycan core and diacylglycerol and the other connecting the glycan to the VSG polypeptide via ethanolamine
GlcN, identical to the core backbone structure found in the glycolipid anchors of VSG and Thy-l (Ferguson et al, 1988; Homans et al, 1988)
Summary
Chemical structures of the glycosylinositol phospholipid (GPI) membrane anchors of a trypanosome variant surface glycoprotein (VSG) (Ferguson et al, 1988), rat brain. ’ The abbreviations used are: PI-PLC, phosphatidylinositol-specific phospholipase C; VSG, variant surface glycoprotein; AHM, 2,5anhydromannitol; GPI, glycosylinositol phospholipid; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; GU, glucose units; ApcuM, A. phoenicis u-mannosidase I; JBaM, jack bean cu-mannosidase; Eh”-AChE, human erythrocyte acetylcholine esterase; Inos, inositol; TMS, trimethylsilyl; dansyl, 5-dimethylaminonaphthalene-lsulfonyl. The primary structure of the glycan portion of these glycolipids has been determined by a combination of selective chemical fragmentation and enzymatic glycan sequencing of subnanomolar amounts of material as well as by analyses of biosynthetically labeled material
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