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. 2018 Jun 22;7:e34843. doi: 10.7554/eLife.34843

Figure 3. ERp5 cleaves the βI-domain Cys177-Cys184 disulfide bond.

(A) One or more disulfide bonds are cleaved in platelet β3 integrin upon platelet activation. Washed platelets were untreated or activated with ADP and unpaired cysteine thiols in platelet surface proteins labelled with the biotin-linked maleimide, MPB. The β3 integrin was immunoprecipitated from the platelet lysate and blotted for MPB. (B) Differential cysteine alkylation and mass spectrometry method of measuring the redox state of the β3 integrin cysteines. Unpaired cysteine thiols in purified β3 integrin are alkylated with 12C-IPA and the disulfide bonded cysteine thiols with 13C-IPA following reduction with DTT. Sixty-eight peptides (Supplementary file 1) encompassing cysteines representing 24 of the 28 β3 integrin disulfide bonds were analyzed. (C) Positions of the β3 integrin disulfide bonds (yellow and grey spheres) in a modelled open structure of the complete αIIbβ3 integrin ectodomain (blue ribbon). We were able to map 24 of the 28 β3 disulfide bonds. The mapped disulfides are in yellow and the unmapped bonds are in grey. (D–E) Redox state of 24 of the 28 β3 integrin disulfide bonds in the absence or presence of 2- or 10-fold molar excess of ERp5 or redox inactive ERp5 (D), or PDI or redox inactive PDI (E). (F–G) Redox state of 24 of the 28 β3 integrin disulfide bonds in the absence or presence of 2- or 10-fold molar excess of ERp5 or redox inactive ERp5 (D), or PDI or redox inactive PDI (E), and 1 mM RGD peptide. The bars and errors (1 SD) are for 5–15 measurements from three different integrin preparations. The β3 Cys177-Cys184 disulfide bond (indicated by red arrow) is significantly cleaved by 10-fold molar excess of ERp5 (p<0.05) (H) The βI-domain Cys177-Cys184 disulfide bond is cleaved on the platelet surface by platelet ERp5. Washed platelets were incubated with function-blocking anti-ERp5 antibodies or isotype control antibodies and the redox state of the βI-domain disulfide determined in the integrin immunoprecipitated from lysate. The bars and errors (1 SD) are from three different platelet preparations from healthy donors. ***p<0.005; ****p<0.001; assessed by unpaired, two-tailed Student’s t-test.

Figure 3.

Figure 3—figure supplement 1. Differential cysteine alkylation and mass spectrometry analysis of the β3 Cys177-Cys184 disulfide bond.

Figure 3—figure supplement 1.

(A) HPLC resolution of the β3 ISPPEALENPCY peptide containing Cys177 labelled with either 12C-IPA or 13C-IPA. (B) Representative tandem mass spectra of the β3 ISPPEALENPCY peptide. At left is an example of 12C-IPA-alkylation of Cys177, and at right is an example of 13C-IPA-alkylation of Cys177. The accurate mass spectrum of the peptide is shown in the inset (left, observed [M + 2 hr]2+ = 733.3370 m/z and expected [M + 2 hr]2+ = 687.3207 m/z; right, observed [M + 2 hr]3+ = 736.3471 m/z and expected [M + 2 hr]2+ = 690.331).
Figure 3—figure supplement 2. Redox state of the β3 Cys177-Cys184 disulfide bond in the absence or presence of 10-fold molar excess of ERp5 and 1 mM RGD or control RGE peptide.

Figure 3—figure supplement 2.

The bars and errors (1 SD) are for 2–5 biological replicates from a single integrin preparations. **p<0.01; assessed by unpaired, two-tailed Student’s t-test.