The alpha- and beta-subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver.
The alpha- and beta-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 alpha-subunit contained a 2835 bp ORF encoding a 107 kDa polypeptide. The 2095 bp cDNA for the beta-subunit encodes a approximately 60 kDa protein in a continuous 1605 bp ORF. The alpha- and beta-subunits each contain two potential Asn-Xaa-Thr/Ser acceptor sites, with only one site in the alpha-subunit (Asn97) being glycosylated. Additional lambda-clones were isolated for each subunit containing in-frame insertions/deletions within the coding region, indicating alternative splicing. Analysis of different human tissues revealed approximately 4.4 kb and approximately 2.4 kb transcripts for alpha- and beta-subunit, respectively, consistent with their full-length cDNA. Coexpression of the alpha- and beta-subunits in COS-1 cells resulted in >4-fold increase of glucosidase II activity. An inactive protein was obtained, however, after transfection with the alpha-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 beta-subunit, was catalytically active indicates that the beta-subunit is involved in alpha-subunit maturation rather than being required for enzymatic activity once the alpha-subunit has acquired its mature form. The alpha-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 alpha-subunit, when associated with the dimeric enzyme complex, is mediated by the C-terminal HDEL-signal in the beta-subunit, whereas the apparently incompletely folded form of the inactive alpha-subunit could be retained in the ER by the putative "glycoprotein-specific quality control machinery. "
Treml
K
K
Institut für Physiologische Chemie, Nussallee 11, 53115 Bonn, Germany.
Meimaroglou
D
D
Hentges
A
A
Bause
E
E
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D019281:Dimerization; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012322:RNA Precursors; D014162:Transfection; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D012322:RNA Precursors; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases