MAN(9)-MANNOSIDASE FROM PIG-LIVER IS A TYPE-II MEMBRANE-PROTEIN THAT RESIDES IN THE ENDOPLASMIC-RETICULUM - CDNA CLONING AND EXPRESSION OF THE ENZYME IN COS-1 CELLS
E. Bieberich et al., MAN(9)-MANNOSIDASE FROM PIG-LIVER IS A TYPE-II MEMBRANE-PROTEIN THAT RESIDES IN THE ENDOPLASMIC-RETICULUM - CDNA CLONING AND EXPRESSION OF THE ENZYME IN COS-1 CELLS, European journal of biochemistry, 246(3), 1997, pp. 681-689
Man(9)-mannosidase, one of three different alpha 1,2-exo-mannosidases
known to be involved in N-linked oligosaccharide processing, has been
cloned in lambda gt10, using a mixed-primed pig liver cDNA library. Th
ree clones were isolated which allowed the reconstruction of a 2731-bp
full-length cDNA. The cDNA construct contained a single open reading
frame of 1977 bp, encoding a 659-residue polypeptide with a molecular
mass of approximate to 73 kDa. The Man(9)-mannosidase specificity of t
he cDNA construct was verified by the observation that all peptide seq
uences derived from a previously purified, catalytically active 49-kDa
fragment were found within the coding region. The N-terminus of the 4
9-kDa fragment aligns with amino acid 175 of the translated cDNA, indi
cating that the catalytic activity is associated with the C-terminus.
Transfection of COS 1 cells with the Man(9)-mannosidase cDNA gave rise
to a > 30-fold over-expression of a 73-kDa protein whose catalytic pr
operties, including substrate specificity, susceptibility towards alph
a-mannosidase inhibitors and metal ion requirements, were similar to t
hose of the 49-kDa enzyme fragment. Thus deletion of 174 N-terminal am
ino acids in the 73-kDa protein appears to have only marginal influenc
e on the catalytic properties. Structural and hydrophobicity analysis
of the coding region, as well as the results from tryptic degradation
studies, point to pig liver Man(9)-mannosidase being a non-glycosylate
d type-II transmembrane protein. This protein contains a 48-residue cy
tosolic tail followed by a 22-residue membrane anchor (which probably
functions as internal and non-cleavable signal sequence), a lumenal ap
proximate to 100-residue-stem region and a large 49-kDa C-terminal cat
alytic domain. As shown by immune-fluorescence microscopy, the pig liv
er enzyme expressed in COS 1 cells, is resident in the endoplasmic ret
iculum, in contrast to COS 1 Man(9)-mannosidase from human kidney whic
h is Golgi-located [Bieberich, E. & Bause, E. (1995) Eur: J. Biochem.
233, 644-649]. Localization of the porcine enzyme in the endoplasmic r
eticulum is consistent with immuno-electron-microscopic studies using
pig hepatocytes. The different intracellular distribution of pig liver
and human kidney Man(9)-mannosidase is, therefore, enzyme-specific ra
ther than a COS-1-cell-typical phenomenon. Since we oberserve approxim
ate to 81% sequence similarity between the two alpha-mannosidases, we
deduce that the localization in either endoplasmic reticulum or Golgi
is likely to be sequence-dependent.