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Biochemical Society Transactions ( 1 995) 23 The in v i m catalysis of protein folding by endoplasrnic reticulum luminal peptidyl prolyl cis-frans isomerase SUCHIRA BOSE’.’, HAUKE LILIE’, JOHANNES BUCHNER’ and ROBERT B. FREEDMAN’ ‘Research School of Biosciences, Biological Laboratory, University of Kent, Canterbury, Kent, CTZ 7NJ, U.K. ’Institut fur Biophysik und Physikalische Biochemie, Universitat Regensburg. D-93040 Regensburg, Germany. Protein folding in the cell is assisted by niany auxiliary proteins that catalyse covalent isomerisation steps, or ‘chaperone’ the folding of nascent chains and prevent them from entering non-productive pathways (1.2). Several catalysts and chaperones have now been identified which assist in such cellular protein folding processes. Molecular chaperones, some of which are induced under conditions of heat shock or stress and are also referred to as ‘heat shock proteins’ or ‘stress proteins’, have been implicated in the formation and maintenance of protein structure under physiological conditions (2). Protein disulphide isomerase (PDI)’ catalyses disulphide bond isomerisation associated with protein folding and was the first example of a protein folding catalyst (3). Peptidyl prolyl cb-truns-isomerase (PPI), a second protein folding catalyst, is an enzyme that catalyses the cis-trans interconversion of prolyl peptide bonds (4). Isomerisations of Xaa-Pro peptide bonds have been identified as slow steps in in v i m folding of some proteins ( 5 ) . Nascent proteins are presumably all fruns polypeptide chains and cis Xaa-Pro peptide bonds are common under native conditions (6); this suggests a role for PPI under in vivo conditions to act as folding catalyst by introducing native cis prolyl peptide bonds or reconverting to truns any non-native cis prolyl peptide bonds that may form in the period between protein synthesis and folding. Cytosolic PPI (cyclophilin A) has been detected from many sources and purified from porcine kidney (4). This enzyme catalyses some, but not all, proline-limited slow protein folding steps, in vitro (5,7). The lumen of the endoplasmic reticulum (ER) has a specialist role in the folding of newly synthesised membrane-bound and secretory proteins. In our previous studies we detected a latent PPI activity, distinct from the abundant cytosolic PPI, within mammalian microsomal vesicles (8). This activity has been purified and identified as a cyclophilin-type PPI present within the secretory pathway (9). We report here that this ER PPI (cyclophilin B) is able to catalyse the in v i m refolding of oxidised ribonuclease Ti (RNase T I ) and an antibody Fab fragment. Furthermore. ER PPI and PDI together catalyse the in ritm refolding of reduced and denatured Fab fragments. RNase T I , in the native conformation, contains two cis prolylpeptides (Tyr38-Pro39 and SerS4-ProS5) and two truns prolylpeptides (Trp59-Pro60 and Ser72-Pro73). In the denatured state, the rruns form predominates and the isomerisation of the truns to the cis prolyl-peptide bonds for Pro39 and Pro55 account for the two slow steps observed in the refolding kinetics (10). Here, the refolding of RNase Ti in the absence and presence of 24nM ER PPl was followed by measuring the increase in tryptophan fluorescence at 320nm after excitation at 778nm. Both slow folding steps were catalysed by ER PPI and catalysis of the intermediate phase was more efficient than the very slow phase. The two refolding phases differ in rates and in catalysis by PPls because they differ in the accessibility of the prolylpeptide bond to the enzyme (11). The presence of 24nM bovine serum albumin (BSA) did not affect the kinetics of refolding of the protein. IpM cyclosporin A (CsA) blocked the catalysis by ER PPI of RNase T i refolding. These results are comparable to those found for cytosolic PPI ( 1 1 ) . ’ Ahhrcviations used: PDI. protein disulphide isomerase; PPI, peptidyl prolyl ci.!-rr.irrrsisomerdse; ER. endoplasmic reliculum; BSA, hovine serum albumin; CsA, cyclosporin A; RNase T , . rihonuclease T , . 63s The second substrate studied was the Fab tragnient of the murine monoclonal antibody MAK 33. The slow folding phases of oxidised antibody domains are known to be limited in rate by the cis-rruns isomerisation of Xaa-Pro bonds (12) and are effectively catdysed by cyclophilins (7). In contrast to the isolated light chain, denaturation of the Fab molecule is not completely reversible and depends largely on the conditions of the experiment. Here, renaturation of I-ab,, was examined under conditions previously employed by others (13, 14). Under these conditions, approximately 20-30% of the molecules gained the native conformation which is comparable to previous findings (13). In the presence of 10-fold molar excess of ER PPI, the rate of reactivation of Fab was found to be accelerated three-fold. ER PPI was found to influence not only the rate of reactivation but also the yield. This increase in yield is probably due to competition between correct and non-productive folding pathways that are influenced by the rate of proline isonierisations. Again, theje results are comparable to that with the abundant cytosolic PPI (cyclophilin) (14). In most studies, the role of PPI in protein folding has concentrated on the refolding of proteins where the disulphide bonds remained intact in the unfolded protein. We have studied the effects of folding catalysts on the renaturation of reduced and denatured antibody molecules. Experiments performed in the presence of excess PDI and ER PPI resulted in an increase in the rate of formation and yield of functional antibody molecules Excess PDI (or PDI plus BSA) only increased the yield of functional antibody molecules. as reported previously (15). These results imply the existence of at least two rate limiting steps during the renaturation of the reduced and denatured protein. The first is the formation of the intradomain disulphide bonds, which is catalysed by PDI, and the second is limitttl in rate by cis-fruns isomerisation of Xaa-Pro bonds. which is catdysed by ER PPI. Studies on the oxidative refolding of reduced RNase TI have also shown a synergistic effect of PDI and cytosolic PPI (16). We have now demonstrated that ER PPI and PDI together catalyse the in virro refolding of reduced and denatured Fab fragments, suggesting that the two enzymes work synergistically in the cell. We acknowledge the financial support of the British Council and a SERC studentship to S.B. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 I5 16 Gething, M-I. & Samhrook, J. (1992) Nature, 335, 33-45 Jaenicke. R. & Buchner, I. (1993) Chemtracts Biochein. Mol. B i o l . , 4, 1-30 Freedman, R. B. (1992) in Protein Folding (T. E. Crcighton, d), Freeman, N e w York, 455-539 Fischer, G . , Bang, H. Ck Mcch. C. (1984) Bionied. Iiixhiin Acta, 43. 1101-11I I Fischer, G . , Wittnian-Lichold. B.. Lang, K.,Kielhahcr, ‘I. K Schmid, F. X . (1989) Nature, 337. 476-478 Stewart. D. E.. Sarkar, A , . 62 Warnpler, J . E. (1990) J . hl(11. B ~ o l . , 214, 253-260 Lang, K., Schmid. F. X . Ck Fischer, G . (1987) Nature. 329. 268-270 Bose, S . ck Freedinan. R . B. (1904) Biochem. J . , 300, 86.1-870 Bose, S . . Miickc, M . CG Freedman. R. B. (1994) Blocham. J . , 300. 871-875 Schmid, F. X . (1992) in Prolam Folding (T. E. Creighton, ed.). Freeman, N e w York, 191.242 SchBnhrunner, E . R . , Mayer, S . , Trapschug, M.. Fi,cher. G.. Takahaahi, N . ck Schinid, F. X . (1991) I . B i d . Chem.. 266, 36303635 Goto, Y. Ck Hainaguchi, K . (1982) J . Mol. Biol.. 156. 891-910 Schmidt, M . ck Buchncr, J. (1992.) J . Biol. Chem., 267. 16829-16833 L i l i e , 13.. Lang, K . , Rudolph, R. Ck Buchnrr. J . (1993) [’rot. Sci., 2. 1490- I496 Lilie, H., McLaughlin, S. H . . Frcr*lmim. R. B. Ck Buchncr. J . , (1994) J. Biol. Chem., 269, 14290-14296 Schonhrunner, E. R. & Schinid, F. X . (1992) Prot. Natl. Acad. Sci.. 89. 4 5 10-4513