Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Abstract

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-.beta.-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-.beta.-glucosaminyl) asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man.alpha.1 .fwdarw. 6(Man.alpha.1 .fwdarw. 3) Man.alpha.1 .fwdarw. 6(Man.alpha.1 .fwdarw. 3)Man.beta.1 .fwdarw. 4GlycNAc.beta.1 .fwdarw. GlcNAc.beta.1 .fwdarw., were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man.alpha.1 .fwdarw. 6(Man.alpha.1 .fwdarw. 3)Man.alpha.1 .fwdarw. 6 and Man.alpha. 1 .fwdarw. 3Man.alpha.1 .fwdarw. 6(Gal.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw. 2Man.alpha.1 .fwdarw. 3)Man.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw., were found at Asn12, as previously published [Schluter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. and Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc.alpha.2 .fwdarw. 3 or Gal.alpha.1 .fwdarw. 3Gal.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw. 2Man.alpha.1 .fwdarw. 6(NeuAc.alpha.2 .fwdarw. 6 or NeuAc.alpha.2 .fwdarw. 3Gal-.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw. 2Man.alpha.1 .fwdarw. 3)Man.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw. 4GlcNAc.beta.1 .fwdarw. with some bifurcation at .fwdarw. 6Man.alpha.1 .fwdarw. 6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal.beta.1 .fwdarw. 3GalNAc.alpha.1 .fwdarw., monosialylated or disialylated at position 3 of Gal or/and position 6 of GalNAc.