The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo

Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins, This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum, We have isolated two cDNA isoforms of the human a subunit (alpha 1 and alpha 2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit, The alpha 1 and alpha 2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2,7. Using COS7 cells, the coexpression of the beta subunit with the catalytic a subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex, Transfected cell extracts expressing either al or alpha 2 forms with the beta subunit showed similar activities, while mutating the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity, In order to compare the kinetic parameters of both alpha 1/beta and alpha 2/beta forms of human glucosidase II the protein was expressed,pith the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The KM1-2 values and apparent Ki1-2 for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics, The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.