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Glycobiology, 2001, Vol. 11, No. 7 549-556
© 2001 Oxford University Press

Complete analysis of the glycosylation and disulfide bond pattern of human ß-hexosaminidase B by MALDI-MS

Christina Gabriele Schuette1, Judith Weisgerber and Konrad Sandhoff2

Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn,Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany

ß-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 analyized 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 ß-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.

1 Present address: Max-Planck-Institut for Biophysical Chemistry, Dept. 190 Am Fassberg 11, D-37077 Göttingen, Germany.

2 To whom correspondence should be addressed


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