STRUCTURAL REQUIREMENTS FOR O-GLYCOSYLATION OF THE MOUSE HEPATITIS-VIRUS MEMBRANE-PROTEIN

The mouse hepatitis virus (MHV) membrane (M) protein contains only O-l inked oligosaccharides. We have used this protein as a model to study the structural requirements for O-glycosylation, We show that MHV M is modified by the addition of a single oligosaccharide side chain at th e cluster of 4 hydroxylamino acids present at its extreme amino termin us and identified Thr at position 5 as the functional acceptor site. T he hydroxylamino acid cluster, which is quite conserved among O-glycos ylated coronavirus M proteins, is not in itself sufficient for O-glyco sylation, Downstream amino acids are required to introduce a functiona l O-glycosylation site into a foreign protein, In a mutagenic analysis Oglycosylation was found to be sensitive to some particular changes b ut no unique sequence motif for O-glycosylation could be identified. E xpression of mutant M proteins in cells revealed that substitution of any 1 residue was tolerated, conceivably due to the occurrence of mult iple UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrans ferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situ glycosy lation assay based on the co-expression of endoplasmic reticulum-retai ned forms of the GalNAc transferases with endoplasmic reticulum-reside nt MHV M mutants. The GalNAc transferases were found to have largely o verlapping, but distinct substrate specificities. The requirement for a threonine as acceptor rather than a serine residue and the requireme nt for a proline residue three positions downstream of the acceptor si te were found to be distinctive features.