Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine.
We studied the post-translational modification of NS26, the protein product of rotavirus gene 11 segment. Based on the presence of a putative N-glycosylation site and the high content of serine and threonine residues in gene 11 amino acid sequence we investigated whether NS26 is modified by carbohydrate addition. Specific antibodies raised against the gene 11 product expressed in Escherichia coli recognized in infected cells two polypeptides with apparent molecular weight of 26,000 (26-kDa polypeptide) and 28,000 (28-kDa polypeptide). Pulse-chase experiments demonstrated that the 26-kDa product was processed to the 28-kDa polypeptide. Both polypeptides were metabolically labeled with [3H]glucosamine, indicating the presence of a carbohydrate moiety on the protein. NS26 was found to be resistant to endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F/peptide:N-glycosidase F treatment, but sensitive to removal by alkali-induced beta-elimination, suggesting that the saccharide chain was attached to the protein via an O-glycosidic linkage. Chromatographic analysis of total acid hydrolysates of [3H]glucosamine-labeled NS26-bound carbohydrate indicated the presence of N-acetylglucosamine. In addition, mild alkaline treatment of NS26 in the presence of NaB3H4 identified the O-linked carbohydrate moiety as N-acetylglucosamine. Taken together, these data demonstrate that NS26 is processed to a 28-kDa polypeptide by addition of O-linked monosaccharide residues of N-acetylglucosamine.
González
S A
SA
Instituto de Investigaciones Bioquímicas, Fundación Campomar-CONICET, Buenos Aires, Argentina.
Burrone
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OR
D000117:Acetylglucosamine / Q000378:metabolism*; D002213:Capsid / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D008970:Molecular Weight; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D012401:Rotavirus / Q000378:metabolism*; D014758:Viral Core Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D011994:Recombinant Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D006026:Glycoside Hydrolases; D000117:Acetylglucosamine