Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein.
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 for O-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 among O-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 analysis O-glycosylation was found to be sensitive to some particular changes but no unique sequence motif for O-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:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situ glycosylation 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.
de Haan
C A
CA
Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and the Institute of Biomembranes, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Roestenberg
P
P
de Wit
M
M
de Vries
A A
AA
Nilsson
T
T
Vennema
H
H
Rottier
P J
PJ
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D014763:Viral Matrix Proteins; D000116:Acetylgalactosamine