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Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29 , an N ‐glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation
Author(s) -
Lehle Ludwig,
Eiden Antje,
Lehnert Klaus,
Haselbeck Anton,
Kopetzki Erhard
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00789-c
Subject(s) - complementation , glycosylation , mutant , mannose , saccharomyces cerevisiae , glycoprotein , biochemistry , biology , mutagenesis , microbiology and biotechnology , yeast , gene
Outer chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine‐linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [ 3 H]mannose suicide selection procedure a collection of N ‐glycosylation defective mutants (designated ngd ) was isolated. One mutant, ngd29 , was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37°C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1 . It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man 8 GlcNAc 2 , the in vivo acceptor for building up the outer chain . Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man 8 GlcNAc 2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N ‐linked carbohydrate chains.