The blocking effect of zinc on complement factor H in vitro: further proof by the hemolytic assay of Pilar Sánchez-Corral.

Dear Editor, The only method that is available to study the control of alternative C3/C5convertase by the complement factor H (CFH) was developed by the team of Pilar Sánchez-Corral in 2003 using sheep erythrocytes. Nonetheless, we recall that the control of alternative C3/C5-convertase activity by CFH depends on the trimolecular complex formation of C3b-CFH-Factor I (FI) that itself depend on the binding capacity of CFH to cell membranes. Meanwhile, defective interactions at CFH–heparin sites reduce the CFH activity on surface-bound C3b, while the fluid phase activity continues to prevent C3/C5-convertase formation. On the other hand, Zn2+ that ranges between 2 and 15 μM in physiology, has the capacity to inhibit cell lysis induced by activated complement when its concentrations exceed 50 μM as initially described by Gotze et al. on red cells. The most relevant comprehensive mechanism of this hemolysis inhibition was deciphered by the team of Stephen J. Perkins who are shaping an emerging view based on the capacity of zinc to induce nonphysiologically complex between C3 molecules and complement factor H (CFH). Nevertheless, all these previous reports regarding Zn/CFH interaction were limited to purified systems using biophysical methods and fluid-phase degradation assays, and no study has yet addressed this question using Pilar Sánchez-Corral assay that uses sheep erythrocytes as a non-activator surface. To study CFH interaction with Zn2+, we have used two hemolytic assays (AP50 using rabbit red cells as activator surface and Pilar Sánchez-Corral’s CFH Functional assays), which are based on the formation of hydrophilic pores through which hemoglobin is able to pass and assessed spectrophotometrically as described previously. Indeed, we have succeeded to evidence the opposite and reversible effects of Zn2+ on ACP by demonstrating that physiological and micromolar concentrations of Zn2+ exert an inhibitory action on ACP simultaneously to a slight enhancement of CFH effectiveness in a dose-dependent manner (Figure 1). By using analytical ultracentrifugation and x-ray scattering, Nan et al. (2013) explain that in the presence of excess zinc above 100 μM zinc, very large complexes of CFH and C3b with zinc precipitate out of solution, thus reducing the availability of C3b to mediate its normal ACP response as evidenced by the functional test based on the lysis of chicken erythrocytes in an agarose gel. JOURNAL OF IMMUNOASSAY AND IMMUNOCHEMISTRY 2023, VOL. 44, NO. 3, 309–312 https://doi.org/10.1080/15321819.2023.2173529