Related Experiment Videos
Mod A: a post-translational mutation affecting phosphorylated and sulfated glycopeptides in Dictyostelium discoideum
Abstract:
The Mod A mutation in Dictyostelium discoideum results in a post-translational modification which reduces the activity and electrophoretic mobility of a group of lysosomal glycoproteins. To determine whether this mutation might affect protein bound oligosaccharides, metabolically labeled [2] 3H-mannose glycopeptides were isolated from wild-type (AX3) and mutant cells (M31) of Dictyostelium discoideum. A group of large, negatively charged glycopeptides are significantly depleted in strain M31 compared to AX3. Cells of each strain double labeled with 3H-mannose and 35SO4 or 32PO4 showed that the large, negatively charged glycopeptides of AX3 contain both sulfate and phosphate while those of M31 are depleted in these groups. The kinetics of 35SO4 release from the glycopeptides of each strain suggested that both contained similar sulfated sugar(s), but that M31 glycopeptides contained three-fold less than those of AX3. Acid hydrolysis of 32PO4 containing 3H-mannose glycopeptides showed the presence of 3H-mannose-6-32-P-phosphate in the AX3 hydrolysates while the glycopeptides of M31 contain only 15% as much mannose-6-phosphate as those of AX3.
Insights
The Mod A mutation in Dictyostelium discoideum impacts lysosomal glycoproteins, significantly reducing sulfated and phosphorylated oligosaccharides in mutant cells. This study reveals key differences in glycopeptide composition due to the mutation.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Genetics
Background:
- The Mod A mutation in Dictyostelium discoideum affects lysosomal glycoproteins.
- This mutation leads to altered activity and electrophoretic mobility of these glycoproteins.
Purpose of the Study:
- To investigate the impact of the Mod A mutation on protein-bound oligosaccharides.
- To analyze the composition of glycopeptides in wild-type and mutant Dictyostelium discoideum strains.
Main Methods:
- Metabolic labeling of glycopeptides with 3H-mannose.
- Double labeling with 35SO4 or 32PO4 to assess sulfate and phosphate content.
- Analysis of glycopeptide composition and hydrolysis products.
Main Results:
- A significant depletion of large, negatively charged glycopeptides was observed in the M31 mutant strain compared to wild-type AX3.
- These glycopeptides in AX3 cells contained both sulfate and phosphate, while M31 cells showed depletion of these groups.
- M31 glycopeptides had three-fold less sulfate and 15% less mannose-6-phosphate compared to AX3.
Conclusions:
- The Mod A mutation in Dictyostelium discoideum alters the post-translational modification of lysosomal glycoproteins.
- This alteration results in a significant reduction in sulfated and phosphorylated oligosaccharides, particularly mannose-6-phosphate, on these glycoproteins.