SUBSTRATE-SPECIFICITY OF ALPHA-1,6-MANNOSYLTRANSFERASE THAT INITIATESN-LINKED MANNOSE OUTER CHAIN ELONGATION IN SACCHAROMYCES-CEREVISIAE

Yeast Saccharomyces cerevisiae OCH1 gene encodes the mannosyltransfera se that is essential for the outer chain elongation of N-linked oligos accharides, Mannosyltransferase activity of OCH1 gene product (Och1p) was measured on HPLC by using pyridylaminated Man(8)GlcNAc(2) (Man(8)G lcNAc(2)-PA) as an acceptor and the reaction product was observed at t he retention time corresponding to Man(9)GlcNAc(2)-PA. H-1-NMR and fas t atom bombardment mass spectrometry (FAB-R;IS) fragmentation analysis of Man(9)GlcNAc(2)-PA showed that the additional mannose was attached with an alpha-1,6 linkage at the site where mannose outer chain elong ation initiates, Substrate specificity of Och1p was investigated by us ing various high mannose-type oligosaccharides as accepters, Man(8)Glc NAc(2) was the best acceptor for Och1p, The loss of one or two alpha-1 ,2-mannoses from Man(8)GlcNAc(2) reduced the mannosyltransferase activ ity and the Man(5)GlcNAc(2) completely lacking alpha-1,2-mannose resid ues did not serve as an acceptor, Man(8)GlcNAcOH that involves an open sugar ring by reduction of reducing terminal GlcNAc residue did not s erve as an acceptor for Och1p, The loss of three mannoses at the alpha -1,6-branch also reduced the Och1p activity, These results suggest tha t Och1p is an initiation specific alpha-1,6-mannosyltransferase that r equires the intact structure of Man(8)GlcNAc for efficient mannose out er chain initiation. (C) 1997 Federation of European Biochemical Socie ties.