Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Abstract

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.

Figure 1

Comparison of the biophysical properties of hPDI and PDI-aba′c. (A) Refolding of GAPDH was analyzed in the presence of the indicated molar ratio of hPDI (○), PDIΔC1,2 (▾), and PDI-aba′c (▵), respectively. BSA (□) and carbamidomethylated PDI-aba′c (♦) served as control. The average of at least two independent experiments is given. (B) Redox equilibria of hPDI and PDI-aba′c with glutathione. hPDI (○) or PDI-aba′c (□) was incubated in refolding buffer with different molar ratios of GSH to GSSG (total concentration 2 mM). Fluorescence values as recorded at 332 nm (hPDI) and 335 nm (PDI-aba′c) are shown. The redox potential of hPDI and PDI-aba′c was calculated to be −0.15 V.

Figure 2

Influence of hPDI and PDI-aba′c on the kinetics and yield of hPI folding. Refolding of d/r-hPI was performed in refolding buffer with 1 mM GSH and 2 mM GSSG. Aliquots were removed at the indicated time points, folding stopped, and samples analyzed by RP-HPLC. (A) Spontaneous refolding of hPI (▵) and in the presence of a twofold molar excess of hPDI (○), PDI-aba′c (□), and PDIΔC1,2 (⋄). Data were fitted single exponentially. (B) Refolding of hPI with different molar ratios of hPDI (○), PDI-aba′c (□), and PDIΔC1,2 (⋄). Shown is the yield of native hPI analyzed after the renaturation was completed; given is the average and error of two to five independent experiments.

Figure 3

Formation of folding intermediates. hPI was refolded and analyzed as described in Figure 2 with a 1:5 molar ratio of hPDI to hPI. Refolding was stopped after 20 s (lower panel) or 90 s (upper panel). Early intermediates, late intermediates (numbered consecutively), and native hPI are indicated.

Figure 4

RP-HPLC chromatograms of hPI refolding with PDI-variants. hPI was refolded and analyzed as described in Figure 2. For all figures, the same scale was chosen. The peaks for early and late intermediates and native hPI (N) are indicated. Time courses similar to Figure 3 are shown for hPI folding noncatalyzed and with a 2:1 ratio of PDIΔC1,2 or a 1:5 ratio of hPDI or PDI-aba′c. The total recovery of hPI (sum of all peaks during folding) was comparable for all folding reactions.

Figure 5

Determination of the number of disulfide bonds in hPI intermediates on an 18% urea gel. Bands corresponding to fully IAM-modified or IAA-modified hPI, corresponding to the fully reduced or oxidized species, respectively, are indicated. d/r-hPI was completely IAM modified (lane 1) or mixed with partially IAA-modified hPI (lane 2, * indicates d/r-hPI) or competitively modified with IAM and IAA (lane 8). I₂, I₄, I₅, I₇, and I₈ (lanes 3–7) were first IAM modified, reduced with DTT, and subsequently IAA modified. Note that I₂ to I₈ are completely IAA modified indicating that they are completely oxidized.

Figure 6

Spontaneous refolding of I₂, I₄, I₅, I₇, and I₈. Intermediates were denatured and refolded as described in Figure 2. Peak areas were determined using peak fit. Note that intermediates convert into one another [I₂, ▿; I₄, □; I₅, ⋄; I₇, ▵; and I₈ (hexagon) and into native hPI (○)]. In the panel showing I₅, please note that the curve describing the disappearance of I₅ was fitted to better visualize this intermediate.

Figure 7

Refolding of I₄ spontaneously or with PDI variants. I₄ was denatured and refolded as described in Figure 2. The time courses of formation of native hPI (given as relative peak areas) during spontaneous folding (▵), and with PDIΔC1,2 (⋄; 2:1 molar ratio to hPI), hPDI (○, 1:5 ratio), or PDI-aba′c (□; 1:5 ratio), PDI-aba′c (▪; 2:1 ratio), and a combination of PDIΔC1,2 and PDI-aba′c (♦; 2:1 and 1:5 ratio) are shown.