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 alpha-1,2-mannosidase from yeast, whereas, by contrast, no sequence similarity was detectable with rat liver ER alpha-mannosidase and Golgi alpha-mannosidase II. This finding may indicate that the mammalian alpha-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.