IN VIVO SPECIFICITY OF OLIGOSACCHARYLTRANSFERASE FOR TRUNCATED GLYCANS

GARCIA, MICAELA DAIANA; PARODI ARMANDO; D'ALESSIO CECILIA

Mendoza

Congreso; XLVIII Reunión Anual 48th Annual Meeting; 2012

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

The endoplasmic reticulum membrane oligosaccharytransferase complex (OST) transfers glycan Glc3Man9GlcNAc2 from a dolichol-PP derivative upon protein N-glycosylation. Glycan transfer enhances glycoprotein folding efficiency. Defects in the transfer reaction due either to OST mutations or to truncated glycan structures may result in protein hypoglycosylation thus causing diseases known as congenital disorders of glycosylation type I. The OST has been shown to have strict preference for the complete glycan but no systematic studies have been performed concerning the in vivo transfer rate of glycans not bearing the full complement of 3 glucoses or 9 mannoses. We constructed four series of mutants of the fission yeast Schizosaccharomyces pombe that synthesize dolichol-PP-linked glycans bearing from 0 to 3 glucoses and 5, 6, 7 or 9 mannoses and expressed Saccharomyces cerevisiae carboxypeptidase Y (CPY), a protein bearing four N-glycosylation sites. The degree of CPY hypoglycosylation provides a good indication of OST transfer efficiency. Our results indicate that N-glycan mannose content does not influence glycan transfer by the OST in vivo and that in S. pombe, unlike what happens in other known organisms, three glucoses may be added to glycans in the absence of the full complement of mannoses.