Abstract
In humans and rodents, the lysosomal catabolism of core Man(3)GlcNAc(2) N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal alpha-mannosidase (LysMan), core-specific alpha1,6-mannosidase, beta-mannosidase, and cleavage at the reducing terminus by a di-N-acetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 alpha-mannosidase with catalytic characteristics similar to those established previously for the core-specific alpha1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol, and stimulation by Co(2+) and Zn(2+)). Substrate specificity studies comparing the novel human alpha-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the alpha1-6mannose residue from Man(3)GlcNAc but not Man(3)GlcNAc(2) or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before alpha1,6-mannosidase activity. In contrast, LysMan cleaved all of the alpha-linked mannose residues from high mannose oligosaccharides except the core alpha1-6mannose residue. alpha1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine alpha1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5'-flanking sequences for the bovine alpha1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and alpha1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.
Highlights
An enzyme specific for the hydrolysis of the core ␣1– 6Man linkage was initially identified from human ␣-mannosidosis fibroblasts [24] and partially purified from human spleen [25] and rat liver [26], but the cDNA encoding this enzyme activity has not been identified
A second clade contains the broad specificity lysosomal ␣-mannosidase involved in glycoprotein catabolism (LysMan from humans and mice [12, 53, 54] as well as multiple orthologs in D. melanogaster, C. elegans, and A. thaliana)
The third clade is a heterogeneous collection of enzymes represented by the mammalian ER/cytosolic ␣-mannosidase involved in dolichol-oligosaccharide turnover and catabolism of glycans on glycoproteins that failed ER quality control and were translocated into the cytosol in mammalian species [2, 55]
Summary
Cloning and Expression of a Novel Human ␣-Mannosidase—The human cDNA homolog of the pig/mouse 135-kDa ␣-mannosidase was identified by sequence searching of a cloned cDNA library (GenBankTM accession number AL553663, human placenta; Invitrogen), and the cDNA in the pCMV script vector was fully sequenced to confirm that it matched the corresponding GenBankTM reference sequence (accession number NM_015274). The concentrated enzyme preparations were further purified by loading onto a Superdex 200 gel filtration column (16 ϫ 700 mm; Amersham Biosciences) pre-equilibrated with 50 mM HEPES (pH 7.5) and 200 mM NaCl. Fractions containing ␣-mannosidase activity were pooled and used for substrate specificity studies and kinetic analysis. The blots were prehybridized, hybridized, and washed as described previously [38] using radiolabeled probes comprised of the 740-bp or 1.3-kb amplimers from human LysMan or novel ␣-mannosidase coding regions, respectively, generated as described above. Mannosidase Activity Assays with High Mannose Substrate—Various pyridylamine (PA)-tagged glycans, including Man9GlcNAc2PA, Man8GlcNAc2-PA, Man7GlcNAc2-PA, Man6GlcNAc2-PA, Man5GlcNAc2-PA, and GlcNAcMan5GlcNAc2-PA, were isolated as described previously [43,44,45] and incubated with the novel human ␣-mannosidase or human LysMan in 100 mM sodium acetate buffer (pH 4.0) for 24 h at 37 °C. Comparative pairwise analysis of DNA sequences was accomplished using the Compare and DotPlot subroutines [50] of the University of Wisconsin Genetics Computer Group (GCG software, version 10.2) with a window of 21 residues and a stringency of 14
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