Mf. Pelletier et al., The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo, GLYCOBIOLOG, 10(8), 2000, pp. 815-827
Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the
two innermost alpha-1,3-linked glucose residues from N-linked oligosacchari
des on nascent glycoproteins, This processing allows the binding and releas
e of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin a
nd calreticulin, the lectin-like chaperones of the endoplasmic reticulum, W
e 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 sub
unit, The alpha 1 and alpha 2 forms have distinct mobilities on SDS-PAGE an
d are expressed in most of the cell lines we have tested, but were absent f
rom the glucosidase II-deficient cell line PHA(R) 2,7. Using COS7 cells, th
e coexpression of the beta subunit with the catalytic a subunit was found t
o be essential for enzymatic activity, solubilization, and/or stability, an
d ER retention of the alpha/beta complex, Transfected cell extracts express
ing either al or alpha 2 forms with the beta subunit showed similar activit
ies, while mutating the nucleophile (D542N) predicted from the glycoside hy
drolase Family 31 active site consensus sequence abolished enzymatic activi
ty, In order to compare the kinetic parameters of both alpha 1/beta and alp
ha 2/beta forms of human glucosidase II the protein was expressed,pith the
baculovirus expression system. Expression of the human alpha or beta subuni
t alone led to the formation of active human/insect heteroenzymes, demonstr
ating functional complementation by the endogenous insect glucosidase II su
bunits. 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 a
nd apparent Ki1-2 for deoxynojirimycin and castanospermine were determined
and found to be identical for both isoforms suggesting they have similar ca
talysis and inhibition characteristics, The substrate specificities of both
isoforms using the physiological oligosaccharides were assessed and found
to be similar.