Glycobiology, Vol 8, 585-595, Copyright © 1998 by Society for Glycobiology
LO Tremblay, N Campbell Dyke and A Herscovics
Class I alpha1,2-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 alpha1, 2-mannosidase IA whereas the other was very
similar to murine alpha1, 2-mannosidase IB. Northern blot analysis of human
tissues with these two alpha1,2-mannosidase probes revealed very different
patterns of tissue-specific expression. Similar tissue-specific expression
of alpha1,2-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 alpha1,2- 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 alpha1,2- mannosidase IB (Herscovics et al., 1994, J. Biol. Chem.,
269, 9864- 9871). A truncated soluble form of the human
alpha1,2-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 alpha1,2-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 alpha1,2- mannosidases in the human and
murine genomes with very distinct transcriptional regulation in different
tissues.
ORIGINAL ARTICLES
Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human alpha1,2-mannosidase gene involved in N-glycan maturation
McGill Cancer Centre, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6.
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