MAN(9)-MANNOSIDASE FROM HUMAN KIDNEY IS EXPRESSED IN COS CELLS AS A GOLGI-RESIDENT TYPE-II TRANSMEMBRANE N-GLYCOPROTEIN

Citation
E. Bieberich et E. Bause, MAN(9)-MANNOSIDASE FROM HUMAN KIDNEY IS EXPRESSED IN COS CELLS AS A GOLGI-RESIDENT TYPE-II TRANSMEMBRANE N-GLYCOPROTEIN, European journal of biochemistry, 233(2), 1995, pp. 644-649
Citations number
25
Categorie Soggetti
Biology
ISSN journal
00142956
Volume
233
Issue
2
Year of publication
1995
Pages
644 - 649
Database
ISI
SICI code
0014-2956(1995)233:2<644:MFHKIE>2.0.ZU;2-I
Abstract
Man(9)-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-lin ked 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 expressi on in COS 1 cells of the enzyme resulted in a more than 20-fold increa se of a catalytic activity cleaving specifically alpha 1,2-mannosidic linkages in [C-14]Man(9)-GlcNAc(2) or [C-14]Man(5)-GlcNAc(2). Man(9)-m annosidase is expressed as a N-glycoprotein with a molecular mass of 7 3 kDa. Its enzymic activity is metal ion dependent and inhibited stron gly by 1-deoxymannojirimycin (50% at 100 mu M). Proteolytic studies wi th the membrane-associated form of Man(9)-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 Man(9)-manno sidase, as expressed, is N-glycosylated at one of three potential Asn- Xaa-Thr/Ser/Cys acceptor sites. Approximately 50% of the N-linked olig osaccharide 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 inhibi tor swainsonine, indicating that the sugar moiety of Man(9)-mannosidas e is processed partially by Golgi-resident enzymes. This observation i s consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man(9)-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man(9)-mannosidase which appears to be located in th e endoplasmic reticulum and transient vesicles.