Glycobiology, 2000, Vol. 10, No. 5 493-502
© 2000 Oxford University Press
The 
- and ß-subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver
Institut für Physiologische Chemie, Nussallee 11, 53115 Bonn, Germany
The 
- and ß-subunits of the hetero-dimeric glucosidase II complex from human liver were cloned and expressed in COS-1 cells. The 4106 bp full-length cDNA for the -subunit contained a 2835 bp ORF encoding a 107 kDa polypeptide. The 2095 bp cDNA for the ß-subunit encodes a ~60 kDa protein in a continuous 1605 bp ORF. The - and ß-subunits each contain two potential Asn-Xaa-Thr/Ser acceptor sites, with only one site in the -subunit (Asn97) being glycosylated. Additional 
-clones were isolated for each subunit containing in-frame insertions/deletions within the coding region, indicating alternative splicing. Analysis of different human tissues revealed ~4.4 kb and ~2.4 kb transcripts for - and ß-subunit, respectively, consistent with their full-length cDNA. Coexpression of the - and ß-subunits in COS-1 cells resulted in >4-fold increase of glucosidase II activity. An inactive protein was obtained, however, after transfection with the -subunit alone, showing that both subunits are essential for expression of active glucosidase II. The observation that the enzyme, previously purified from pig liver and lacking the ß-subunit, was catalytically active indicates that the ß-subunit is involved in -subunit maturation rather than being required for enzymatic activity once the -subunit has acquired its mature form. The -subunit is expressed in COS-1 cells as an ER-located protein, whether inactive or part of a catalytically active complex. This suggests that ER-localization of the -subunit, when associated with the dimeric enzyme complex, is mediated by the C-terminal HDEL-signal in the ß-subunit, whereas the apparently incompletely folded form of the inactive -subunit could be retained in the ER by the putative "glycoprotein-specific quality control machinery."
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