Man₉‐Mannosidase from Human Kidney is Expressed in COS Cells as a Golgi‐Resident Type II Transmembrane N‐Glycoprotein

Man₉‐mannosidase, an α1,2–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., Volker, 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 α1,2–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 μM). 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.