In previous work (Herscovics et al., J. Biol. Chem., 269, 9864-9871),
a novel mouse alpha-mannosidase cDNA was isolated by homology, taking
advantage of identical regions between the amino acid sequences of the
yeast and rabbit liver processing alpha 1,2-mannosidases of different
specificities to design degenerate oligonucleotides for reverse trans
cription/polymerase chain reaction. The cDNA isolated from a mouse 3T3
cDNA library encodes a 73 kDa type II membrane protein with a cytopla
smic region of similar to 35 amino acids and a large C-terminal region
that contains a consensus Ca2+-binding sequence. To study the propert
ies of this enzyme, the C-terminal part lacking the transmembrane regi
on (beginning at either amino acid 106 or 171) was transiently express
ed in COS cells as a secreted protein A fusion protein, and the enzyma
tic properties of the fusion protein bound to IgG-Sepharose were inves
tigated. The enzyme is an alpha 1,2-mannosidase that trims Man(9)GlcNA
c to Man(5)GlcNAc (where Man is mannose and GlcNAc is N-acetyl glucosa
mine). The activity requires divalent cations since it is greatly inhi
bited by ethylene diamine tetraacetic acid (EDTA). Although Ca2+ is th
e most effective, the enzyme may also use Mg2+, Mn2+ or Co2+, but not
Zn2+, which is inhibitory. The enzyme is inhibited by 1-deoxymannojiri
mycin, but not by swainsonine. We propose that this novel alpha 1,2-ma
nnosidase cDNA encodes mouse Golgi alpha-mannosidase IB.