Molecular cloning and primary structure of Man<sub>9</sub>‐mannosidase from human kidney

Bause, Ernst; Bieberich, Erhard; Rolfs, Andreas; Völker, Christof; Schmidt, Bernhard

doi:10.1111/j.1432-1033.1993.tb18274.x

Cited by 68 publications

(25 citation statements)

References 33 publications

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“…The gene encoding lysosomal ␣-mannosidase has been isolated from D. discoideum (5) and sequence comparisons (4) with other mammalian ␣-mannosidases identified regions similar to the previously cloned Golgi processing enzyme, ␣-mannosidase II (21), and the endoplasmic reticulum/cytosolic ␣-mannosidase (22). No sequence similarities were detected with the ␣1,2-mannosidases involved in early steps of glycoprotein biosynthesis (23)(24)(25)(26). Thus, a classification of the ␣-mannosidase sequences was proposed whereby the early processing ␣1,2-mannosidases were categorized as Class I ␣-mannosidases, and the Class II ␣-mannosidases were subdivided into Golgi processing enzymes (i.e.…”

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Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Liao

Lal

Moremen

1996

Journal of Biological Chemistry

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We have cloned and expressed two cDNAs encoding the human lysosomal ␣-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from ␣-mannosidosis fibroblasts, and p-nitrophenyl-␣-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, K m , and V max characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No ␣-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb ␣-mannosidase cDNA immunoprecipitated lysosomal ␣-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including ␣-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal ␣-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239 -245) and several differences were found relative to the functional lysosomal ␣-mannosidase encoded by the 3-kb spleen cDNA.The lysosomal catabolism of N-glycans on mammalian glycoproteins occurs through the sequential exoglycosidase digestion of oligosaccharides from the non-reducing terminus down to the carbohydrate-peptide core region (1). Included among the hydrolytic activities responsible for this oligosaccharide degradation is a broad specificity exo-␣-mannosidase activity (EC 3.2.1.24) that catalyzes the hydrolysis of ␣1,2-, ␣1,3-, and ␣1,6-mannoside linkages present in complex, hybrid, and high mannose Asn-linked glycans (2). This enzyme is distinguished from other ␣-mannosidase activities by a combination of a low pH optimum (pH 4.5), a broad natural substrate specificity, activity toward the synthetic substrate p-nitrophenyl-␣-mannoside, and sensitivity to inhibition by swainsonine (3, 4). An enzyme activity with these characteristics has been identified and purified from several species sources including Dictyostelium discoideum (5), and a variety of mammalian tissues (6 -11). Metabolic radiolabeling studies have indicated that the human enzyme is initially synthesized as a polypeptide of ϳ110 kDa that is sub...

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Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Liao

Lal

Moremen

1996

Journal of Biological Chemistry

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“…Several a-Ms have been cloned to date. These include Golgi a-MIT (3,4), ER/cytosolic a-MI (5), two isozymes of Golgi a-MI (6)(7)(8), lysosomal a-M (9), Dictyostelium a-M (10), and yeast a-M (11) (for a recent review, see ref. 2).…”

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Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

Misago

Liao

Kudo

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Golgi a-mannosidase II (a-Mll) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human a-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding a-MIl were isolated. One such gene was found to encode an isozyme, designated acMIIx. A 5-kb cDNA clone encoding a-MIIX was then isolated from a human melanoma cDNA library. However, comparison between a-MIIX and a-MIl cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while a-MI consists of 1144 amino acid residues. To reevaluate the sequence of a-MIIx cDNA, polymerase chain reaction (PCR) was-performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the a-MIIX genomic sequence revealed that alternative splicing of the a-MIIX transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of a-MIIX in various tissues, suggesting that the a-MIIX gene is a housekeeping gene. COS cells transfected with c-MIIX cDNA containing the full-length open reading frame showed an increase of a-mannosidase activity. The ca-MIIX gene was mapped to human chromosome 15q25, whereas the a-MIl gene was mapped to 5q21-22.a-Mannosidase (a-M) activities are involved in both biosynthesis and catabolism of N-linked glycans (1, 2). These enzyme activities are present in cells ranging from yeast to human. There are different forms of a-Ms: lysosomal a-Ms are soluble and involved in degradation of N-glycans, endoplasmic reticulum (ER) and Golgi a-Ms are involved in processing of newly synthesized N-glycans, and cytoplasmic a-Ms may be involved in degradation of dolichol intermediates that are not needed for protein glycosylation or oligosaccharides derived from glycoprotein turnover in the ER (1).. Substrate specificities of these a-Ms differ from each other, and Golgi a-MIl specifically hydrolyzes two peripheral mannosyl residues from Manal1-6(Manal --3)Manal ->6(GlcNAcf31--2Mana1 ->3)[Man/31 ->4GlcNAc,lB --4GlcNAc31 ->]asparagine structure. Several a-Ms have been cloned to date. These include Golgi a-MIT (3, 4), ER/cytosolic a-MI (5), two isozymes of Golgi a-MI (6-8), lysosomal a-M (9), Dictyostelium a-M (10), and yeast a-M (11) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…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 synthesized by PCR and used to screen a human kidney cDNA library [4]. Several Man,,-mannosidase-specific clones allowed the construction of a cDNA that encoded the complete amino acid sequence (625 amino acids) of the human kidney enzyme.…”

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

Bieberich¹,

Bause²

1995

European Journal of Biochemistry

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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 ...

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Molecular cloning and primary structure of Man₉‐mannosidase from human kidney

Cited by 68 publications

References 33 publications

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

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

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Molecular cloning and primary structure of Man9‐mannosidase from human kidney

Cited by 68 publications

References 33 publications

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Molecular cloning and expression of cDNAs encoding human alpha-mannosidase II and a previously unrecognized alpha-mannosidase IIx isozyme.

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

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Molecular cloning and primary structure of Man₉‐mannosidase from human kidney

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