Complete analysis of the glycosylation and disulfide bond pattern of human beta-hexosaminidase B by MALDI-MS.
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.
Schuette
C G
CG
Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany.
Weisgerber
J
J
Sandhoff
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004220:Disulfides / Q000378:metabolism*; D006031:Glycosylation; D054819:Hexosaminidase B; D006801:Humans; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D018411:Spodoptera; D001619:beta-N-Acetylhexosaminidases / Q000097:blood* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004220:Disulfides; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases