Title : Complete analysis of the glycosylation and disulfide bond pattern of human
beta-hexosaminidase B by MALDI-MS
Abstract :
- beta-hexosaminidase B is an enzyme that is involved in the degradation of glycolipids and glycans in the lysosome
- Mutation in the HEXB gene lead to Sandhoff disease, a glycolipid storage disorder characterized by severe neurodegeneration
- So far, little structural information on the protein is available
- Here, the complete analysis of the disulfide bond pattern of the protein is described for the first time
- Additionally, the structures of the N-glycans are analyzed for the native human protein and for recombinant protein expressed in SF21 cells
- For the analysis of the disulfide bond structure, the protein was proteolytically digested and the resulting peptides were analyzed by MALDI-MS
- The analysis revealed three disulfide bonds ( C91-C137 ; C309-C360 ; C534-C551 ) and a free cysteine (C487)
- The analysis of the N-glycosylation was performed by tryptic digestion of the protein , isolation of glycopeptides by lectin chromatography and mass measurement before and after enzymatic deglycosylation
- Carbohydrate structures were calculated from the mass difference between glycosylated and deglycosylated peptide
- For beta-hexosaminidase B from human placenta, four N-glycans were identified and analyzed, whereas the recombinant protein expressed in SF21 cells carried only three glycans
- In both cases the glycosylation belongs to the mannose-core- or high-mannose-type, and some carbohydrate structures are fucosylated