Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus.
The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.
Wang
Zhi-Qiang
ZQ
Institute of Protein Research, Tongji University, Shanghai, China.
Han
Yu-Hong
YH
Shao
Xiao-Xia
XX
Chi
Cheng-Wu
CW
Guo
Zhan-Yun
ZY
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D020916:Conotoxins / Q000737:chemistry / Q000378:metabolism; D052078:Conus Snail / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D013379:Substrate Specificity
D020916:Conotoxins; D004220:Disulfides; D009113:Muramidase; D019704:Protein Disulfide-Isomerases