ERp72, a member of the PDI family, has been identified as a luminal ER protein and is believed to participate in the formation and isomerization of disulfide bonds in cellular secretory proteins (Mazzarella et al 1990
; Van et al 1993
; Rupp et al 1994
; Feng et al 1996
). Although ERp72 shares the sequence (WCGHCK) responsible for redox activity with other PDI family proteins, the functional roles of the 3 thioreoxin homology domains of ERp72 have not been elucidated. In this study, we characterized the functional properties of the 3 individual thioredoxin homology domains of ERp72 by focusing on the reduction of disulfide bonds in insulin. Presumably, this reflected one of the steps involved in the oxidative folding process in which “nonnative” bonds are reduced by ERp72 before rearrangement.
Kinetic analysis of ERp72 mutants with Cys to Ser mutations at 2 cysteine residues in the individual thioredoxin homology domains revealed that triple mutants with substitutions in domains 1, 2, and 3 had no insulin reduction activity, indicating that at least one of these thioredoxin homology domains of ERp72 is involved in catalyzing insulin reduction.
Single mutation in domain 1 (intact domains 2 and 3) reduced kcat compared with that of wild type, but hardly altered Km values, indicating that the active site of domain 1 chiefly contributed to catalytic activity. Single mutation in domain 2 (intact domains 1 and 3) reduced kcat and increased Km, indicating that the domain 2 active site contributed to both binding affinity for substrate and catalytic activity. Single mutation in domain 3 (intact domains 1 and 2) doubled Km without altering kcat, indicating that the principal role of the domain 3 active site was apparently to enhance the recognition and binding of substrate in the steady state.
Preservation of intact domain 2 represented by double mutations in domains 1 plus 3 (mut-1+3) and of intact domain 1 represented by double mutations in domains 2 plus 3 (mut-2+3) had 50–60% activity of wild type but showed about 2-fold elevation in Km, indicating that the active sites of domains 1 and 2 chiefly contributed to catalytic activity but not binding affinity for substrate. Whereas, preservation of intact domain 3 by double mutations in domains 1 plus 2 (mut-1+2) had similar Km to that of wild type, indicating that domain 3 made the most significant contribution to binding affinity for substrates among domains 1, 2, and 3. These results of assays using single mutation and double mutations indicated in a consistent manner that the active site of domain 1 contributed to catalytic activity and the active site of domain 3 contributed to binding affinity for substrates. However, the results on domain 2 appeared inconsistent between single mutation study and double mutation study.
We interpret the results on domain 2 as follows. Compared with mut-1+2 (intact domain 3), mut-1 (intact domains 2 and 3) elevated Km, indicating that domain 2 was involved in binding affinity for substrate. However, compared with mut-2+3 (intact domain 1), mut-3 (intact domains 1 and 2) did not elevate Km, indicating that domain 2 did not contribute to binding affinity for substrate. Therefore, domain 2 appeared to be involved in binding affinity in a somewhat indirect manner, that is, only in combination with domain 3. Altogether, all 3 domains contributed to catalyzing insulin reduction, and individual domains acted synergistically rather than having simply additive effects.
The PDI has 5 μM of Km
for insulin, and loss of C-terminal cysteines of PDI affects the apparent steady-state binding of substrate as evidenced by a 4-fold increase in Km
). Compared with this, elevation in Km
caused by double mutations in domains 1 plus 3 or domains 2 plus 3 of ERp72 was relatively small (approximately 2-fold), which could be because of the innate low binding affinity for insulin (36.7 μM for wild-type ERp72). Therefore, it is natural to raise a question whether the differences in function among domains of ERp72 have biological significance. We claim that they do, on the basis of the following discussion. First, a decrease in kcat
caused by double mutations in domains 1 plus 2 of ERp72 is comparable with that caused by loss of N-terminal cysteins of PDI (30–40% residual activity for N-terminal mutated PDI). Second, there is no information regarding the accurate concentration of ribonuclease (Rnase) or insulin in the ER, whereas the major folding enzymes and chaperones, PDI, BiP/GRP78, GRP94, and ERp72, are known to be present in millimolar concentrations in the ER (Hillson et al 1984
; Lyles and Gilbert 1991
). Therefore, we consider that if functional differences between thioredoxin domains of PDI have biological significance in disulfide bond formation and isomerizaton of RNase or insulin in vivo, functional differences among individual domains of ERp72 must also be biologically significant in vivo. We propose in this study that there may be a transfer of substrate to a low affinity site to allow catalysis and that binding of substrate to the higher affinity site may cause a decrease in the folding rate.
The manner of action of the thioredoxin homology domains of ERp72 was similar to that of the 2 thioredoxin homology domains of PDI (Lyles and Gilbert 1994
). Both thioredoxin homology domains of PDI have been reported to contribute to some extent to Km
. However, the method of contribution differs in that the C-terminal domain contributes more to Km
and the N-terminal domain contributes more to kcat
(Lyles and Gilbert 1994
). shows the alignment of homologies between the PDI N- and C-terminal thioredoxin homology domains and the ERp72 thioredoxin homology domains 1, 2, and 3. There is complete conservation of the 11 amino acid– sequence EFYAPWCGHCK in the PDI and ERp72 thioredoxin homology domains, but the homologies of the amino acid sequence preceding or following the 11 amino acid–sequence are very different among the thioredoxin homology domains. ERp72 domains 1 and 2 are more homologous with the PDI N-terminal domain (48% and 51% identical, respectively) when compared with the homology between the ERp72 domain 3 and the PDI N-terminal domain (24% identical). In contrast, ERp72 domain 3 is more homologous with the PDI C-terminal domain (46% identical) when compared with the homologies of ERp72 domains 1 and 2 with the PDI N-terminal domain (26% and 23% identical, respectively). Domains 1 and 2 contributed more to catalyzing efficacy, and domain 3 contributed more to binding affinity. This assignment of function to individual domains is similar to that observed in PDI domains, which is consistent with the high homology between the ERp domains 1 and 2 and the PDI N-terminal domain, and that between the ERp72 domain 3 and the PDI N-terminal domain.
Fig 4. Alignment of homologies between protein disulfide isomerase (PDI) N-terminal thioredoxin homology domain and ERp72 thioredoxin domains 1, 2, and 3 (top), as well as homologies between PDI C-terminus and ERp72 thioredoxin domains 1, 2, and 3 (bottom). (more ...)
Given the general principle that essentially important sequences that exert fundamental biological functions are strictly conserved through evolution, the amino acid sequences flanking the active site cysteines, which are well conserved between ERp72 and PDI regardless of the very different sequences preceding and following this site, must represent essentially important roles, such as enzymatic catalysis of disulfide bond formation or isomerization and formation of secondary or tertiary domain structures. Further characterization of the thioredoxin homology domains of ERp72 requires future studies on oxidative folding and thiol-dependent isomerization of substrates such as reduced or scrambled RNase.