A single tryptophan residue of endomannosidase is crucial for Golgi localization and in vivo activity.
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 alpha1-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 endomannosidase is of critical importance for correct subcellular localization and in vivo activity of the enzyme.
Torossi
T
T
Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland.
Roth
J
J
Ziak
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000201:enzymology*; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D014364:Tryptophan / Q000737:chemistry / Q000502:physiology*
D018076:DNA, Complementary; D014364:Tryptophan; D008361:Mannosidases