Molecular cloning and primary structure of Man9‐mannosidase from human kidney

Abstract

Man₉-mannosidase, a processing enzyme found in the endoplasmic reticulum (ER), catalyses the removal of three distinct mannose residues from peptide-bound Man₉-GlcNAc₂ oligosaccharides producing a single Man₆ isomer [Bause, E., Breuer, W., Schweden, J., Roesser, R. & Geyer, R. (1992) Eur. J. Biochem. 208, 451–457]. We have isolated four Man₉-mannosidase-specific clones from a human kidney cDNA library and used these to construct a full-length cDNA of 3250 base pairs. A single open reading frame of 1875 nucleotides encodes a protein of approximately 71 kDa, consistent with data from immunological studies. Analysis of the coding sequence predicts that Man₉-mannosidase is a type II transmembrane protein consisting of a short cytoplasmic polypeptide tail, a single transmembrane domain acting as a non-cleavable signal sequence and a large luminal catalytic domain. This domain architecture closely resembles that of other ER and Golgi-located processing enzymes, pointing to common structural motifs involved in membrane insertion and topology. The protein sequence of the Man₉-mannosidase contains three potential N-glycosylation sites of which only one site is used. The amino acid sequence of several peptide regions, including a calcium-binding consensus sequence, bears striking similarities to an ER α-1,2-mannosidase from yeast, whereas, by contrast, no sequence similarity was detectable with rat liver ER α-mannosidase and Golgi α-mannosidase II. This finding may indicate that the mammalian α-mannosidases, which differ significantly in their substrate specificity, are coded for by evolutionarily unrelated genes, providing an attractive means of regulation and fine-tuning oligosaccharide processing, not only at the enzymic but also at the transcriptional level.