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

Abstract

Man,-mannosidase, an crl,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) EUE J. Biochein. 217, 533-5401. Transient expression in COS 1 cells of the enzyme resulted in a more than 20-fold increase of a catalytic activity cleaving specifically al,2-mannosidic linkages in [ "C]Man,-GlcNAcZ or [ "'C]Man,-GlcNAc2. Man,-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 I-deoxymannojirimycin (50% at 100 pM). Proteolytic studies with the mernbrane-associated form of Man,-mannosidase support the view that the enzyme is a type I1 transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man,-mannosidase, as expressed, is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites. Approximately SO c/o 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 I1 inhibitor swainsonine, indicating that the sugar moiety of Man,-mannosidase is processed partially by Golgi-resident enzymes. This observation is consistent with the rewlts of indirect immunofluorescence studies, pointing to a localization of the Man,-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man,,-mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles.Keywords: Man,-mannosidase; human kidney; expression in COS 1 cells; type TI transmembrane Nglycoprotein subcellular location.The maturation of N-linked oligosaccharides begins with the removal by glucosidases I and I1 of three glucose residues from the Gk-Man,-GlcNAc, precursor oligosaccharide, followed by cleavage of up to four al,2-linked mannose residues by at least three different al,2-mannosidases (endoplasmic reticulum mannosidase, Man,-mannosidase, and Golgi mannosidase I) [I -71. The resulting Man5-GlcNAc2-structure is then modified by GlcNAc transferase I, Golgi mannosidase IT, and other glycosyltransferases, finally yielding complex glycan chains [I l. The biological significance of al,2-mannosidases, all of which act on Man,-GlcNAcZ, is still unknown. These enzymes may, however, be responsible for generating distinct oligosaccharides with specific functions [8].Man,-mannosidase was purified recently from pig liver to homogeneity [3]. A catalytically active 49-kDa fragment cleaves three distinct mannose residues from the Man,-GlcNAc2 substrate. two from the previously glucosylated crl,3-branch, and one residue from the outer al,b-branch [8]. The enzyme is strongly inhibited by 1 -deoxymannojirimycin and requires divalent cations for activity 131. Based on sequence information obtained from the 49-kDa pig liver enzyme, a cDNA probe was ...

Man9‐Mannosidase from Pig Liver is a Type‐II Membrane Protein That Resides in the Endoplasmic Reticulum

“…8) [20]. This is in line with the observation that an antibody against the 49-kDa pig liver enzyme fragment cross-reacts specifically with the COS-expressed human kidney enzyme [21]. The two Man,-mannosidases differ substantially, however, in their cytosolic N-termini, which contain 12 amino acids in the human kidney and 48 in the pig liver species.…”

“…By contrast, the pattern is clearly different from that of the human kidney Man,-mannosidase, which is characteristic of the Golgi complex (Fig. 7D) [21]. We conclude from this data that pig liver Man,-mannosidase is expressed as an ER-resident protein in COS 1 cells, in contrast to the human kidney enzyme which is associated with the Golgi.…”

“…3). This indicates that pig liver Man,-mannosidase is, similar to the human kidney enzyme [21], a type-I1 transmembrane protein with the bulk of its amino acid sequence, including the catalytic side, located in the lumen. Type-I1 topology is consistent with the positive-inside rule proposed by van Heijne which predicts a net positive charge on the cytosolic side of the membrane-flanking peptide regions [34,351.…”

“…This view is supported by the observation that the degree of sequence similarity approaches 90% when only the lumenal part of their polypeptides is considered. There are a number of structural differences, however, which may be relevant for a distinct and specific interaction of the three enzymes in the processing pathway, both in terms of time and space: (a) pig liver Man,-mannosidase is not N-glycosylated, whereas the human kidney enzyme carries a heterogenous, hybridthigh-mannose-type sugar chain at Asn485 [21]; (b) the cytosolic N-terminus of the porcine enzyme is 37 amino acids larger than for the human kidney enzyme but is similar in murine a-mannosidase (Fig. 8) [17]; (c) pig liver and human kidney Man,-mannosidase were found to exhibit different subcellular localization, with the pig liver enzyme being ER-resident and the kidney enzyme Golgi-located (Fig.…”

“…We have shown that the Man,-mannosidase from human kidney can be expressed in COS 1 cells as a 73-kDa N-glycoprotein [21]. The catalytic properties of the expressed enzyme greatly resemble those of a stable 49-kDa Man,-mannosidase fragment purified from pig liver, indicating that the two enzymes are closely related.…”