Molecular cloning and primary structure of Man9-mannosidase from human kidney.
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.
Bause
E
E
Institut für Physiologische Chemie, Universität Bonn, Germany.
Bieberich
E
E
Rolfs
A
A
Völker
C
C
Schmidt
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D016415:Sequence Alignment; D013552:Swine
D018076:DNA, Complementary; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase