Glycobiology, 2000, Vol. 10, No. 8 815-827
© 2000 Oxford University Press
The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo
5Genetics Group and 6Enzymology Group, Biotechnology Research Institute, National Research Council of Canada, Montreal H4P 2R2, Canada, and 7Department of Biology and 8Department Anatomy and Cell Biology, McGill University, Montreal H3A 2B2, Canada
Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost 
–1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc1Man9GlcNAc2) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human subunit (1 and 2) differing by a 66 bp stretch, and a cDNA for the corresponding ß subunit. The 1 and 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 PHAR 2.7. Using COS7 cells, the coexpression of the ß subunit with the catalytic subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the /ß complex. Transfected cell extracts expressing either 1 or 2 forms with the ß 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 1/ß and 2/ß forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human or ß 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 -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.
1 To whom correspondence should be addressed at: Genetics Group, National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada 2Present address: Pharmacor Inc., 535 Boulevard Cartier Est CP-320, Succ. Laval-des-Rapides, Canada H7M 4Z9 3Present address: Mikrobiologisches Institut, ETH Zürich, LFV E20, CH-8092 Zürich, Switzerland 4Contributed equally to this work
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