Cloning, expression and characterization of a cDNA (6A8) encoding a novel human α‐mannosidase

A 3300-bp cDNA (6A8) has been isolated from a human tonsil cell λgt11 cDNA library (GenBank accession number: AF044414). The 6A8 gene is localized on human chromosome 13q31–32. Its cDNA has an open reading frame from position 57 bp to 3243 bp, encoding a 1062 amino-acid polypeptide. The sequence of the polypeptide has 89% identity to rat liver ER α-mannosidase. Homogenates of COS-7 cells transfected with 6A8 cDNA showed an enhanced enzymatic activity with p-nitro-phenyl-α-D-mannopyranoside, which was not inhibited by swainsonine. These data suggest that 6A8 α-mannosidase belongs to the class II α-mannosidase. Western blot analysis showed a band for 6A8 cDNA encoded protein of ≈120 kDa. Northern blot analysis revealed two 6A8 mRNA transcripts with different tissue distribution. Enhanced concanavalin A (ConA) binding to CNE-2L2 cells transfected with a reverse 6A8 DNA was observed, indicating that the 6A8 protein is an important cellular α-mannosidase.