Section 6
Chapter 5,532

Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin

Polonoff, E.; Machida, C.A.; Kabat, D.

Journal of Biological Chemistry 257(23): 14023-14028


ISSN/ISBN: 0021-9258
PMID: 6292217
Accession: 005531793

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Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analog .beta.-hydroxynorvaline. In conditions of partial analog substitution, a series of subglycosylated components is formed which are related by a constant apparent MW difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent MW of 1 oligosaccharide into the total apparent MW difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with a known number of oligosaccharides. The gp70 membrane envelope glycoproteins of certain ecotropic MuLV contain 7 oligosaccharides; the GIX+ antigen-containing variant gp70 contains 1 fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only 1 asparagine-linked oligosaccharide. The gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains 4 asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogs or by loss of oligosaccharides. Evidently, loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis. [Rat kidney NRK cells were used in this study.].

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