Effects of CaBP2, the rat analog of ERp72, and of CaBP1 on the refolding of denatured reduced proteins. Comparison with protein disulfide isomerase.
It has been shown previously that CaBP2, the rat analog of the murine protein ERp72, and CaBP1, the rat analogue of the hamster protein P5, represent members of the protein disulfide isomerase (PDI) family and are able to catalyze the reduction of insulin in the presence of various reductants (Nguyen Van et al., 1993). We have now examined the abilities of CaBP2 and CaBP1 to catalyze the renaturation of denatured reduced model proteins. Both CaBP2 and CaBP1 catalyzed the reappearance of the biological activity of the denatured reduced Fab fragment of a monoclonal anti-human creatine phosphokinase antibody. The reaction rate was positively correlated with the amount of CaBP2 or CaBP1 and dependent on the GSH/GSSG ratio (maximum at GSH/GSSG = 1). Peptide prolyl-cis,trans-isomerase (PPI), which catalyzed some renaturation on its own, showed synergistic effects with PDI, CaBP2, and CaBP1. No synergistic effects could be observed when the combinations CaBP2 + PDI, CaBP1 + PDI, or CaBP2 + CaBP1 were tested. Variation of [Ca2+] between 0 and 1 mM did not have any effect on the rate or amount of renaturation catalyzed by CaBP2, CaBP1, or PDI, nor were these parameters affected by the simultaneous presence of BiP or grp94. Both CaBP2 and CaBP1 catalyzed also the renaturation of denatured reduced ribonuclease AIII in a way that depended on the amounts of CaBP2 or CaBP1 and on the redox potential of the redox system used (GSH/GSSG or CSH/CSSC). PPI alone had no effect on the rate of RNase AIII renaturation and did not significantly affect renaturation catalyzed by PDI, CaBP2, or CaBP1. PDI showed a moderate but significant synergism with CaBP2, and a strong synergism with CaBP1. The results indicate that both CaBP2 and CaBP1 can catalyze the formation of disulfide bonds and protein disulfide isomerization and may thus be involved in the folding of nascent proteins in the secretory pathway. This does not exclude the possibility of additional functions of these proteins in the pre-Golgi compartments.
Rupp
K
K
Abteilung Klinische Biochemie, Universität Göttingen, Germany.
Birnbach
U
U
Lundström
J
J
Van
P N
PN
Söling
H D
HD
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D003402:Creatine Kinase / Q000276:immunology; D006224:Cricetinae; D003538:Cystamine / Q000494:pharmacology; D003543:Cysteamine / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D051381:Rats; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D019749:Sulfur-Sulfur Bond Isomerases
D002135:Calcium-Binding Proteins; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; C062477:endoplasmic reticulum glycoprotein p72; D003543:Cysteamine; D003402:Creatine Kinase; D012260:Ribonucleases; D007535:Isomerases; C085578:Ca-binding protein 2; C085577:Ca2+-binding protein-1; D019749:Sulfur-Sulfur Bond Isomerases; D019704:Protein Disulfide-Isomerases; D003538:Cystamine