Molecular cloning and expression of cDNAs encoding human α-mannosidase II and a previously unrecognized α-mannosidase IIX isozyme

Abstract

Golgi α-mannosidase II (α-MII) 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 α-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding α-MII were isolated. One such gene was found to encode an isozyme, designated α-MIIx. A 5-kb cDNA clone encoding α-MIIx was then isolated from a human melanoma cDNA library. However, comparison between α-MIIx and α-MII cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while α-MII consists of 1144 amino acid residues. To reevaluate the sequence of α-MIIx cDNA, polymerase chain reaction (PCR) was performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the α-MIIx genomic sequence revealed that alternative splicing of the α-MIIx transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of α-MIIx in various tissues, suggesting that the α-MIIx gene is a housekeeping gene. COS cells transfected with α-MIIx cDNA containing the full-length open reading frame showed an increase of α-mannosidase activity. The α-MIIx gene was mapped to human chromosome 15q25, whereas the α-MII gene was mapped to 5q21-22.