CLONING AND EXPRESSION OF GLUCOSIDASE-I FROM HUMAN HIPPOCAMPUS

Glucosidase I, the first enzyme in the N-linked oligosaccharide proces sing pathway, cleaves the distal alpha 1,2-linked glucose residue from the Glc(3)-Man(9)-GlcNAc(2) oligosaccharide precursor highly specific ally. A human hippocampus cDNA library was screened against oligonucle otide probes, generated by PCR using primers derived from the amino ac id sequences of tryptic peptides of pig liver glucosidase I. Two indep endent lambda clones were isolated which allowed the construction of a full-length glucosidase I cDNA of 2881 bp. This cDNA construct encode s, in a single open reading frame, a polypeptide of 834 amino acids co rresponding 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 (a mino acids 38-58) which, most Likely, functions as a transmembrane anc hor. 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 clos e evolutionary relationship between the enzymes from human hippocampus and pig Liver. In contrast, the nucleotide and amino acid sequence re vealed no homology with other processing enzymes cloned so far. Transf ection of COS 1 cells with the glucosidase I cDNA construct resulted i n overexpression (about fourfold) of enzymic activity, which was inhib ited 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 i s in line with the localization of glucosidase I in the lumen of the e ndoplasmic reticulum, shown by immunofluorescence microscopy. The hydr ophobicity profile as well as the removal by trypsin of an approximate to kDa polypeptide from the membrane-associated glucosidase I in inta ct microsomal structures, supports the view that the enzyme is a type- II transmembrane glycoprotein, which contains a short cytosolic tail o f approximate to 37 amino acids, followed by a single transmembrane do main and a large C-terminal catalytic domain located on the luminal si de of the endoplasmic reticulum membrane.