Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation.
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 [3H]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 degrees 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 Man8GlcNAc2, 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 Man8GlcNAc2 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.
Lehle
L
L
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Germany.
Eiden
A
A
Lehnert
K
K
Haselbeck
A
A
Kopetzki
E
E
D000483:Alleles; D000818:Animals; D001216:Asparagine; D002240:Carbohydrate Sequence; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D005949:Glucose Oxidase / Q000096:biosynthesis; D006026:Glycoside Hydrolases / Q000032:analysis / Q000096:biosynthesis; D006031:Glycosylation; D008322:Mammals; D008364:Mannosyltransferases / Q000096:biosynthesis / Q000235:genetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D016366:Open Reading Frames; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D043324:beta-Fructofuranosidase
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; C075636:OCH1 protein, S cerevisiae; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D005949:Glucose Oxidase; D008364:Mannosyltransferases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase