A single tryptophan residue of endomannosidase is crucial for Golgi localization and <Emphasis Type="Italic">in vivo</Emphasis> activity |
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Authors: | T Torossi J Roth M Ziak |
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Institution: | (1) Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland |
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Abstract: | Golgi-endomannosidase provides an alternate glucosidase-independent pathway of glucose trimming. Activity for endomannosidase
is detectable in various tissues and cell lines but not in CHO cells. Cloning of CHO cell endomannosidase revealed that the
highly conserved Trp188 and Arg177 of vertebrate endomannosidase were both substituted by Cys. The Trp188Cys substitution
was functionally important since it alone resulted in endoplasmic reticulum (ER) mislocalization of endomannosidase and caused
the greatly reduced in vivo activity. These effects could be reversed in cells with a back-engineered Cys188Trp CHO cell endomannosidase, in particular
N-glycans of α1-antitrypsin became fully processed. The intramolecular disulfide bridge of CHO cell endomannosidase formed
with the additional Cys188 was not solely responsible for the reduced enzyme activity since endomannosidase with engineered
Cys188Ala or Ser substitutions did not restore enzyme activity and was ER mislocalized. Thus, the conserved Trp188 residue
in endomannosidases is of critical importance for correct subcellular localization and in vivo activity of the enzyme.
Received 7 May 2007; received after revision 31 May 2007; accepted 11 June 2007 |
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Keywords: | Endomannosidase Golgi apparatus endoplasmic reticulum N-glycosylation α 1-antitrypsin CHO Lec 23 cells |
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