| Abstract | Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost α-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of mono-glucosylated (Glc₁Man₉GlcNAc₂) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endo-plasmic reticulum. We have isolated two cDNA isoforms of the human α subunit (α1 and α2) differing by a 66 bp stretch, and a cDNA for the corresponding β subunit. The α1 and α2 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 PHAR 2.7. Using COS7 cells, the coexpression of the β subunit with the catalytic α subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the α/β complex. Transfected cell extracts expressing either α1 or α2 forms with the β 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 α1/β and α2/β forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human α or β 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 α-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. |
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