Glucosidase I, the first enzyme in the N‐linked oligosaccharide processing pathway, cleaves the distal α1,2‐linked glucose residue from the Glc3‐Man9‐GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent λ clones were isolated which allowed the construction of a full‐length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92‐kDa protein contains a single N‐glycosylation site of the Asn‐Xaa‐Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N‐terminus (amino acids 38–58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far. Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1‐deoxynojirimycin or N,N‐dimethyl‐deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93‐kDa form, indicating that it carries a high‐mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an ≈4‐kDa polypeptide from the membrane‐associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type‐II transmembrane glycoprotein, which contains a short cytosolic tail of ≈37 amino acids, followed by a single transmembrane domain and a large C‐terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.
- Human hippocampus glucosidase I
- cDNA cloning
- glucosidase I expression in COS 1 cell
- oligosaccharide processing
- subcellular location
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