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Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein
C A de Haan1, P Roestenberg, M de Wit
1Institute 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.
Abstract:
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.
Insights
Mouse hepatitis virus (MHV) membrane protein O-glycosylation requires specific downstream amino acids, not just the hydroxylamino acid cluster. Multiple GalNAc transferases can modify the protein, indicating distinct substrate specificities.
Area of Science:
- Virology
- Glycobiology
- Molecular Biology
Background:
- Mouse hepatitis virus (MHV) membrane (M) protein is characterized by O-linked oligosaccharides.
- Understanding O-glycosylation structural requirements is crucial for viral protein modification.
Purpose of the Study:
- To investigate the structural determinants for O-glycosylation using the MHV M protein as a model.
- To identify the specific amino acid residues and sequence motifs essential for O-glycosylation.
Main Methods:
- Mutagenic analysis of the MHV M protein to identify functional acceptor sites.
- Co-expression of endoplasmic reticulum-resident MHV M mutants with GalNAc transferases in an in situ glycosylation assay.
Main Results:
- O-glycosylation occurs at Threonine (Thr) at position 5 of the MHV M protein.
- A conserved hydroxylamino acid cluster is necessary but not sufficient; downstream amino acids are required.
- MHV M protein is a substrate for GalNAc transferases 1, 2, and 3, which exhibit overlapping specificities.
- Distinctive features include a preference for threonine over serine and a proline residue three positions downstream.
Conclusions:
- The MHV M protein's O-glycosylation is a complex process influenced by specific amino acid sequences.
- Multiple GalNAc transferases can catalyze MHV M O-glycosylation, suggesting functional redundancy and distinct substrate preferences.
- These findings provide insights into the structural basis of O-glycosylation in viral proteins.