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A Mucor pusillus mutant defective in asparagine-linked glycosylation
1Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Abstract:
A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.
Insights
A Mucor pusillus mutant (1116) exhibits defective asparagine-linked glycosylation, secreting less-glycosylated aspartic proteinase (MPP). This suggests truncated oligosaccharide chains are transferred to MPP, with the mutation being recessive.
Area of Science:
- Mycology
- Biochemistry
- Molecular Biology
Background:
- Mucor pusillus secretes aspartic proteinase (MPP).
- Glycosylation is a critical post-translational modification affecting protein function.
- Defects in glycosylation pathways can alter protein structure and secretion.
Purpose of the Study:
- To characterize a Mucor pusillus mutant (1116) with defects in asparagine-linked glycosylation.
- To investigate the structure and glycosylation pattern of MPP secreted by the mutant.
- To determine the mechanism of glycosylation and the genetic nature of the mutation.
Main Methods:
- Enzyme susceptibility assays
- Lectin binding analysis
- Carbohydrate composition analysis
- Oligosaccharide processing inhibitor studies (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin)
- Genetic analysis using forced primary heterokaryons
Main Results:
- The mutant 1116 secreted MPP in a less-glycosylated form compared to the wild-type.
- Three MPP glycoforms were identified: one unglycosylated and two with truncated N-linked oligosaccharides (Man0-1GlcNAc2).
- MPPs in the mutant were glycosylated via transfer of truncated lipid-linked oligosaccharides, not aberrant processing.
- The mutation in strain 1116 was found to be recessive.
Conclusions:
- The Mucor pusillus mutant 1116 possesses a defect in asparagine-linked glycosylation affecting MPP secretion.
- The observed glycosylation pattern suggests a specific defect in oligosaccharide chain transfer or initial processing.
- The recessive nature of the mutation provides a basis for further genetic studies into the glycosylation pathway.