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Endomannosidase is a Golgi-localized endoglycosidase, which provides an alternate glucosidase-independent pathway of glucose trimming. Using a protease protection assay we demonstrated that Golgi-endomannosidase is a type II membrane protein. The first 25 amino acids of this protein, containing the cytoplasmic tail and the transmembrane domain, were sufficient for Golgi retention of fused reporter proteins alpha1-antitrypsin or green fluorescent protein. However, shortening or deletion of the transmembrane domain prevented Golgi localization, while lengthening it partially reduced Golgi retention of the enzyme. Substitution of the highly conserved positively charged amino acids within the cytoplasmic tail had neither an effect on type II topology nor on the inherent Golgi localization of the enzyme. In contrast, cytoplasmic tail-deleted rat endomannosidase possessed an inverted topology resulting in endoplasmic reticulum mislocalization. Thus, proper topology rather than the presence of positively charged amino acids in the cytoplasmic tail is critical for Golgi localization of rat endomannosidase. 相似文献
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Endomannosidase processes oligosaccharides of α1-antitrypsin and its naturally occurring genetic variants in the Golgi apparatus 总被引:3,自引:0,他引:3
Torossi T Fan JY Sauter-Etter K Roth J Ziak M 《Cellular and molecular life sciences : CMLS》2006,63(16):1923-1932
Endomannosidase provides an alternate glucose-trimming pathway in the Golgi apparatus. However, it is unknown if the action
of endomannosidase is dependent on the conformation of the substrate. We have investigated the processing by endomannosidase
of the α1-antitrypsin oligosaccharides and its disease-causing misfolded Z and Hong Kong variants. Oligosaccharides of wild-type
and misfolded α1-antitrypsin expressed in castanospermine-treated hepatocytes or glucosidase II-deficient Phar 2.7 cells were
selectively processed by endomannosidase and subsequently converted to complex type oligosaccharides as indicated by Endo
H resistance and PNGase F sensitivity. Overexpression of endomannosidase in castanospermine-treated hepatocytes resulted in
processing of all oligosaccharides of wild-type and variants of α1-antitrypsin. Thus, endomannosidase does not discriminate
the folding state of the substrate and provides a back-up mechanism for completion of N-glycosylation of endoplasmic reticulum-escaped glucosylated glycoproteins. For exported misfolded glycoproteins, this would
provide a pathway for the formation of mature oligosaccharides important for their proper trafficking and correct functioning.
Received 18 April 2006; received after revision 12 June 2006; accepted 15 June 2006 相似文献
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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 相似文献
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