STRUCTURE OF THE N-LINKED OLIGOSACCHARIDES THAT SHOW THE COMPLETE LOSS OF ALPHA-1,6-POLYMANNOSE OUTER CHAIN FROM OCH1, OCH1 MNN1, AND OCH1 MNN1 ALG3 MUTANTS OF SACCHAROMYCES-CEREVISIAE

The periplasmic invertase was purified from Saccharomyces cerevisiae o ch1=LEU2 disruptant cells (DELTAoch1), which have a defect in elongati on of the outer chain attached to the N-linked core oligosaccharides ( Nakayama, K., Nagasu, T., Shimma, Y., Kuromatsu, J., and Jigami, Y. (1 992) EMBO J. 11, 2511-2519). Structural analysis of the pyridylaminate d (PA) neutral oligosaccharides released by hydrazinolysis and N-acety lation confirmed that the ochl mutation causes a complete loss of the alpha-1,6-polymannose outer chain, although the PA oligosaccharides (M an9GlcNAc2-PA and Man10GlcNAC2-PA), in which one or two alpha-1,3-link ed mannose(s) attached to the endoplasmic reticulumn (ER)-form core ol igosaccharide (Man8GlcNAc2) were also detected. Analysis of the DELTAo ch1 mnn1 strain oligosaccharides released from total cell mannoprotein revealed that the DELTAoch1 mnn1 mutant eliminates the alpha-1,3-mann ose attached to the core and accumulates predominantly a single ER-for m oligosaccharide species (Man8GlcNAc2), suggesting a potential use of this strain as a host cell to produce glycoproteins containing mammal ian high mannose type oligosaccharides. The DELTAoch1 mnn1 alg3 mutant s accumulated Man5GlcNAC2 and Man8GlcNAC2 in total cell Tnannoprotein, confirming the lack of outer chain addition to the incomplete corelik e oligosaccharide and the leaky phenotype of the alg3 mutation. All th e results suggest that the OCH1 gene encodes an alpha-1,6-mannosyltran sferase that is functional in the initiation of alpha-1,6-polymannose outer chain addition to the N-linked core oligosaccharide (Man5GlcNAC2 and Man8GlcNAC2) in yeast.