MAN(9)-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

Man(9)-mannosidase, one of three different alpha 1,2-exo-mannosidases known to be involved in N-linked oligosaccharide processing, has been cloned in lambda gt10, using a mixed-primed pig liver cDNA library. Th ree 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 approximate to 73 kDa. The Man(9)-mannosidase specificity of t he cDNA construct was verified by the observation that all peptide seq uences derived from a previously purified, catalytically active 49-kDa fragment were found within the coding region. The N-terminus of the 4 9-kDa fragment aligns with amino acid 175 of the translated cDNA, indi cating that the catalytic activity is associated with the C-terminus. Transfection of COS 1 cells with the Man(9)-mannosidase cDNA gave rise to a > 30-fold over-expression of a 73-kDa protein whose catalytic pr operties, including substrate specificity, susceptibility towards alph a-mannosidase inhibitors and metal ion requirements, were similar to t hose of the 49-kDa enzyme fragment. Thus deletion of 174 N-terminal am ino acids in the 73-kDa protein appears to have only marginal influenc e 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 Man(9)-mannosidase being a non-glycosylate d type-II transmembrane protein. This protein contains a 48-residue cy tosolic tail followed by a 22-residue membrane anchor (which probably functions as internal and non-cleavable signal sequence), a lumenal ap proximate to 100-residue-stem region and a large 49-kDa C-terminal cat alytic domain. As shown by immune-fluorescence microscopy, the pig liv er enzyme expressed in COS 1 cells, is resident in the endoplasmic ret iculum, in contrast to COS 1 Man(9)-mannosidase from human kidney whic h is Golgi-located [Bieberich, E. & Bause, E. (1995) Eur: J. Biochem. 233, 644-649]. Localization of the porcine enzyme in the endoplasmic r eticulum is consistent with immuno-electron-microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man(9)-mannosidase is, therefore, enzyme-specific ra ther than a COS-1-cell-typical phenomenon. Since we oberserve approxim ate to 81% sequence similarity between the two alpha-mannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence-dependent.