Man₉-mannosidase from pig liver is a type-II membrane protein that resides in the endoplasmic reticulum. cDNA cloning and expression of the enzyme in COS 1 cells

Man₉-mannosidase, one of three different α1,2-exo-mannosidases known to be involved in N-linked oligosaccharide processing, has been cloned in λgt10, using a mixed-primed pig liver cDNA library. Three clones were isolated which allowed the reconstruction of a 2731-bp full-length cDNA. The cDNA construct contained a single open reading frame of 1977 bp, encoding a 659-residue polypeptide with a molecular mass of ~73 kDa. The Man₉-mannosidase specificity of the cDNA construct was verified by the observation that all peptide sequences derived from a previously purified, catalytically active 49-kDa fragment were found within the coding region. The N-terminus of the 49-kDa fragment aligns with amino acid 175 of the translated cDNA, indicating that the catalytic activity is associated with the C-terminus. Transfection of COS 1 cells with the Man₉-mannosidase cDNA gave rise to a >30-fold over-expression of a 73-kDa protein whose catalytic properties, including substrate specificity, susceptibility towards α-mannosidase inhibitors and metal ion requirements, were similar to those of the 49-kDa enzyme fragment. Thus deletion of 174 N-terminal amino acids in the 73-kDa protein appears to have only marginal influence on the catalytic properties. Structural and hydrophobicity analysis of the coding region, as well as the results from tryptic degradation studies, point to pig liver Man₉-mannosidase being a non-glycosylated type-II transmembrane protein. This protein contains a 48-residue cytosolic tail followed by a 22-residue membrane anchor (which probably functions as internal and non-cleavable signal sequence), a lumenal ~100-residue-stem region and a large 49-kDa C-terminal catalytic domain. As shown by immune-fluorescence microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the endoplasmic reticulum, in contrast to COS 1 Man₉-mannosidase from human kidney which is Golgi-located [Bieberich, E. and Bause, E. (1995) Eur. J. Biochem. 233, 644-649]. Localization of the porcine enzyme in the endoplasmic reticulum is consistent with immuno-electron-microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man₉-mannosidase is, therefore, enzyme-specific rather than a COS-1-cell-typical phenomenon. Since we oberserve ~81% sequence similarity between the two α-mannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence-dependent.