Download The in vitro catalysis of protein folding by endoplasmic reticulum

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.
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