Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human 1,2-mannosidase gene involved in N-glycan maturation

Abstract

Class I α1,2-mannosidases play an essential role in the elaboration of complex and hybrid N-glycans in mammalian cells. Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and lymphocyte cDNAs. One of these was related to the previously cloned human and murine α1,2-mannosidase IA whereas the other was very similar to murine α1,2-mannosidase IB. Northern blot analysis of human tissues with these two α1,2-mannosidase probes revealed very different patterns of tissue-specific expression. Similar tissue-specific expression of α1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues. A human placenta cDNA library was screened and PCR of brain, placenta, and lymphocyte cDNAs was performed in order to isolate the human α1,2-mannosidase IB cDNA. This cDNA encodes a type II membrane protein of 73 kDa that is 94% identical in amino acid sequence to the murine α1,2-mannosidase IB (Herscovics et al., 1994, J. Biol. Chem., 269, 9864-9871). A truncated soluble form of the human α1,2-mannosidase IB lacking its N-terminal transmembrane domain was expressed as a secreted protein in Pichia pastoris. The recombinant enzyme was incubated with [³H]Man₉GlcNAc and [³H]Man₈GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that [³H]Man₉GlcNAc was readily converted to [³H]Man₆GlcNAc and much more slowly to [³H]Man₅GlcNAc, whereas [³H]Man₈GlcNAc was rapidly trimmed to [³H]Man₅GlcNAc. The human α1,2-mannosidase IB gene was isolated from a P1 human genomic library and shown to be at least 60 kb in size and to contain at least 13 exons. The gene was localized by fluorescence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers. These results show that there are at least two Class I α1,2-mannosidases in the human and murine genomes with very distinct transcriptional regulation in different tissues.