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

Abstract

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...

Recent progress in lysosomal α-mannosidase and its deficiency

“…Northern blotting detects one main transcript of approximate 3.0 kb in most tissues investigated, but one commercial Northern blot of different human tissues shows smear bands of 2.0 to 3.6 kb, indicating that human pre-mRNA is alternatively spliced to generate different transcripts (Liao et al, 1996). This is consistent with the fact that a 3.6 kb alternatively spliced cDNA is isolated from human spleen (Liao et al, 1996).…”

“…Among the reported cDNAs for human MANB, the retina/muscle composite cDNA (Nebes and Schmidt, 1994) is relatively distinct from the others (Liao et al, 1996;Nilssen et al, 1997;Sun et al, 1999a), with an identity of only 90% in terms of predicted amino acid sequence. The differences include a 69 bp deletion as well as a number of other smaller deletions/insertions, which cause several frame shifts and significant difference in the predicted amino acid sequences.…”

“…Northern blotting detects one main transcript of approximate 3.0 kb in most tissues investigated, but one commercial Northern blot of different human tissues shows smear bands of 2.0 to 3.6 kb, indicating that human pre-mRNA is alternatively spliced to generate different transcripts (Liao et al, 1996). This is consistent with the fact that a 3.6 kb alternatively spliced cDNA is isolated from human spleen (Liao et al, 1996). RT-PCR analysis of HeLa and HepG2 cells also suggests that alternative splicing is involved in human MANB pre-mRNA processing, in which at least three minor bands ranging from 2.3 to 2.8 kb, as well as the 3.0 kb main message, are detected (Sun et al, 2000).…”

“…The second cDNA for human MANB was isolated by RT-PCR of human spleen, which encodes a polypeptide of 988 amino acids (Liao et al, 1996). The cDNA expresses detectable MANB activity in methylotropic yeast (Pichia).…”

“…The coding region is nearly identical to that of the spleen cDNA (Liao et al, 1996), but more 5'-sequence encoding the putative signal peptide and 3'-untranslated sequence were obtained. As with the spleen cDNA, whether the chimeric cDNA could express the active enzyme in α-mannosidosis is not known.…”