MOLECULAR-CLONING, CHROMOSOMAL MAPPING AND TISSUE-SPECIFIC EXPRESSIONOF A NOVEL HUMAN ALPHA-1,2-MANNOSIDASE GENE INVOLVED IN N-GLYCAN MATURATION

Class I alpha 1,2-mannosidases play an essential role in the elaborati on of complex and hybrid N-glycans in mammalian cells. Using degenerat e primers based on amino acid sequences conserved in all members of th is enzyme family for RT-PCR, two distinct PCR products were obtained f rom placenta and lymphocyte cDNAs. One of these was related to the pre viously cloned human and murine alpha 1,2-mannosidase IA whereas the o ther was very similar to murine alpha 1,2-mannosidase IB. Northern blo t analysis of human tissues with these two alpha 1,2-mannosidase probe s revealed very different patterns of tissue-specific expression. Simi lar tissue-specific expression of alpha 1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues. A human placen ta cDNA library was screened and PCR of brain, placenta, and lymphocyt e cDNAs was performed in order to isolate the human alpha 1,2-mannosid ase IB cDNA. This cDNA encodes a type II. membrane protein of 73 kDa t hat is 94% identical in amino acid sequence to the murine alpha 1,2-ma nnosidase IB (Herscovics et al., 1994, J. Biol. Chem., 269, 9864-9871) . A truncated soluble form of the human alpha 1,2-mannosidase IB lacki ng its N-terminal transmembrane domain was expressed as a secreted pro tein in Pichia pastoris. The recombinant enzyme was incubated with [H- 3]Man(9)GlcNAc and [H-3]Man(8)GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that [H-3]Man(9) GlcNAc was readily converted to [H-3]Man(6)GlcNAc and much more slowly to [H-3]Man(5)GlcNAc, whereas [H-3]Man(8)GlcNAc was rapidly trimmed t o [H-3]Man(5)GlcNAc. The human alpha 1,2-mannosidase IB gene was isola ted 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 fluor escence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers. These re sults show that there are at least two Class I alpha 1,2-mannosidases in the human and murine genomes with very distinct transcriptional reg ulation in different tissues.