Man9-mannosidase from human kidney is expressed in COS cells as a Golgi-resident type II transmembrane N-glycoprotein.
Man9-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Völker, C. & Schmidt, B. (1993) Eur. J. Biochem. 217, 533-540]. Transient expression in COS 1 cells of the enzyme resulted in a more than 20-fold increase of a catalytic activity cleaving specifically alpha 1,2-mannosidic linkages in [14C]Man9-GlcNAc2 or [14C]Man5-GlcNAc2. Man9-mannosidase is expressed as a N-glycoprotein with a molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and inhibited strongly by 1-deoxymannojirimycin (50% at 100 microM). Proteolytic studies with the membrane-associated form of Man9-mannosidase support the view that the enzyme is a type II transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man9-mannosidase, as expressed, is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites. Approximately 50% of the N-linked oligosaccharide chains are removed by endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is observed, when transfected cells were cultured in the presence of the Golgi mannosidase II inhibitor swainsonine, indicating that the sugar moiety of Man9-mannosidase is processed partially by Golgi-resident enzymes. This observation is consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man9-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man9-mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles.
Bieberich
E
E
Institut für Physiologische Chemie, Bonn, Germany.
Bause
E
E
D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008361:Mannosidases / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000032:analysis*; D043323:alpha-Mannosidase
D008562:Membrane Glycoproteins; D008361:Mannosidases; D043323:alpha-Mannosidase