Disulfide bond formation is a determinant of glycosylation site usage in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus

L W McGinnes, T G Morrison
1997 Journal of Virology  
Determinants of glycosylation site usage were explored by using the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus Newcastle disease virus. The amino acid sequence of the HN protein, a type II glycoprotein, has six N-linked glycosylation addition sites, G1 to G6, two of which, G5 and G6, are not used for the addition of carbohydrate (L. McGinnes and T. Morrison, Virology 212:398-410, 1995). The sequence of this protein also has 13 cysteine residues in the ectodomain (C2 to
more » ... ectodomain (C2 to C14). Mutation of either cysteine 13 or cysteine 14 resulted in the addition of another oligosaccharide chain to the protein. These cysteine residues flank the normally unused G6 glycosylation addition site, and mutation of the G6 site eliminated the extra glycosylation found in the cysteine mutants. These results suggested that failure to form an intramolecular disulfide bond resulted in the usage of a normally unused glycosylation site. This conclusion was confirmed by preventing cotranslational disulfide bond formation in cells by using dithiothreitol. Under these conditions, the wild-type protein acquired extra glycosylation, which was eliminated by mutation of the G6 site. These results suggest that localized folding events on the nascent chain, such as disulfide bond formation, which block access to the oligosaccharyl transferase are a determinant of glycosylation site usage. MATERIALS AND METHODS Cells, vectors, and viruses. Cos-7 cells, obtained from the American Type Culture Collection, were maintained in Dulbecco's modified Eagle medium supplemented with nonessential amino acids, vitamins, glutamine, penicillin, streptomycin, and 10% fetal calf serum. The NDV HN gene (25, 26) was expressed in Cos cells by using pSVL obtained from Pharmacia (32). NDV strain AV was purified after growth in embryonated chicken eggs by standard protocols. Antibodies. The antibody used to detect antigenically mature HN protein was an anti-NDV antibody raised in rabbits against UV-inactivated NDV virions (strain AV) by standard protocols (32). This antibody reacts with mature HN
doi:10.1128/jvi.71.4.3083-3089.1997 fatcat:wrscrqcjejaozltdluf2dktjne