The characterization of a novel human core-specific lysosomal [alpha]1-6mannosidase involved in N-glycan catabolism
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In humans and rodents lysosomal catabolism of Man3GlcNAc2 core N-glycan structures results from the concerted actions of exoglycosidases including the broad specificity lysosomal ±-mannosidase (LysMan), a core-specific ±1-6mannosidase, and ²-mannosidase, as well as the core chitobiose cleavage by an di-N-acetylchitobiase. In ungulates and carnivora, both the chitobiase and the ±1-6mannosidase enzyme activities are absent suggesting a co-regulation of the two enzymes. We describe here the cloning, expression, purification and characterization of the human core-specific ±1-6mannosidase with similarity to members of the glycosylhydrolase family 38. The recombinant enzyme had a pH optimum of 4.0, was potently inhibited by +2swainsonine and 1,4-dideoxy1,4-imino-D-mannitol, and was stimulated by Co. NMR-based time course substrate specificity studies comparing the ±1-6mannosidase with human LysMan revealed that the former enzyme selectively cleaved the ±1-6mannose residue from Man3GlcNAc, but not Man3GlcNAc2 or other larger high mannose structures, indicating the requirement for chitobiase action prior to ±1-6mannosidase cleavage. In contrast, LysMan cleaved all of the ±-linked mannose residues from Man9-5GlcNAc2, Man3GlcNAc2, or Man3GlcNAc structures except the core ±1-6mannose residue. Transcripts encoding the ±1-6mannosidase were ubiquitously expressed in human tissues and expressed sequence tag searches in various mammalian species demonstrated a similar distribution in species-specific expression as the chitobiase. No expressed sequence tags were identified for bovine ±1-6mannosidase despite the identification of two homologs in the bovine genome. The lack of conserved 5’ flanking sequences for the bovine gene relative to the human ±1-6mannosidase gene suggests that transcriptional gene silencing may account for the undetectable enzyme activity in bovine tissues similar to the transcriptional silencing previously identified for the bovine chitobiase gene.