ER-60 domains responsible for interaction with calnexin and calreticulin.
ER-60 is a thiol oxidoreductase family protein of the endoplasmic reticulum that facilitates the oxidative folding of glycoproteins via interaction with calnexin (CNX) and calreticulin (CRT). In this study, we tried to identify the site of interaction with CNX and CRT in the ER-60 molecule. ER-60 was shown to be composed of at least four domains, named a, b, b', and a', by limited proteolysis. Recombinant fragments of ER-60, a, b', and a'c, were each expressed in Escherichia coli as an individual soluble folded protein that underwent a cooperative unfolding transition along a urea gradient. These fragments each gave the circular dichroism (CD) spectrum of the folded protein. On the other hand, fragment b, which did not undergo the cooperative unfolding transition along a urea gradient gel, did not show any sign of the folded structure on the CD measurement. However, subtraction of the spectra showed that the b domain was folded in wild-type ER-60 or abb'. Both a and a'c, which have a catalytic center CGHC motif, showed activity almost equivalent to half of that of wild-type ER-60. Extension from a or a'c to ab and abb' or b'a'c had little effect on their isomerase activity, suggesting that the b and b' domains hardly contribute to the catalytic activity of ER-60. The contribution of both the b and b' domains to the binding with CNX and CRT was revealed by surface plasmon resonance analysis and oxidative-refolding experiments of monoglucosylated RNase B with addition of the luminal domain of CNX.
Urade
Reiko
R
Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. urade@soya.food.kyoto-u.ac.jp
Okudo
Hirokazu
H
Kato
Hiroyuki
H
Moriyama
Tatsuya
T
Arakaki
Yukino
Y
D001665:Binding Sites; D037281:Calnexin / Q000378:metabolism*; D037282:Calreticulin / Q000378:metabolism*; D002942:Circular Dichroism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014508:Urea / Q000494:pharmacology
D037282:Calreticulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D037281:Calnexin; D014508:Urea; D011490:Protein Disulfide Reductase (Glutathione)