GLYCOPROTEIN-BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE - NGD29, AN N-GLYCOSYLATION MUTANT ALLELIC TO OCH1 HAVING A DEFECT IN THE INITIATION OF OUTER CHAIN FORMATION

Outer drain glycosylation in Saccharomyces cerevisiae leads to heterog eneous and immunogenic asparagine-linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [H-3] mannose suicide selection procedure a collection of N-glycosylation de fective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displa yed a temperature growth sensitivity at 37 degrees C allowing the isol ation of the corresponding gene by complementation. Cloning, sequencin g and disruption of NGD29 showed that it is a non lethal gene and iden tical to OCH1, It complemented both the glycosylation and growth defec t, Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from mild type ce lls, to transfer mannose from GDP mannose to Man(8)GlcNAc(2), the in v ivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutan t produced a secreted uniform glycoprotein with exclusively Man(8)GlcN Ac(2) structure that in mild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the e xpression of recombinant glycoproteins from different origin in S. cer evisiae to obtain mammalian oligomannosidic type N-linked carbohydrate chains.