| Autor: |
de Haan, Cornelis A.M., Roestenberg, Peggy, de Wit, Marèl, de Vries, Antoine A.F., Nilsson, Tommy, Vennema, Harry, Rottier, Peter J.M. |
| Zdroj: |
Journal of Biological Chemistry; November 1998, Vol. 273 Issue: 45 p29905-29914, 10p |
| Abstrakt: |
The mouse hepatitis virus (MHV) membrane (M) protein contains only O-linked oligosaccharides. We have used this protein as a model to study the structural requirements forO-glycosylation. We show that MHV M is modified by the addition of a single oligosaccharide side chain at the cluster of 4 hydroxylamino acids present at its extreme amino terminus and identified Thr at position 5 as the functional acceptor site. The hydroxylamino acid cluster, which is quite conserved amongO-glycosylated coronavirus M proteins, is not in itself sufficient for O-glycosylation. Downstream amino acids are required to introduce a functional O-glycosylation site into a foreign protein. In a mutagenic analysisO-glycosylation was found to be sensitive to some particular changes but no unique sequence motif forO-glycosylation could be identified. Expression of mutant M proteins in cells revealed that substitution of any 1 residue was tolerated, conceivably due to the occurrence of multiple UDP-N-acetylgalactosamine:polypeptideN-acetylgalactosaminyltransferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situglycosylation assay based on the co-expression of endoplasmic reticulum-retained forms of the GalNAc transferases with endoplasmic reticulum-resident MHV M mutants. The GalNAc transferases were found to have largely overlapping, but distinct substrate specificities. The requirement for a threonine as acceptor rather than a serine residue and the requirement for a proline residue three positions downstream of the acceptor site were found to be distinctive features. |
| Databáze: |
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