IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex βγ-CAT evolved to drive cell vesicular delivery and transport

Abstract

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. βγ-CAT, a complex of pore-forming protein BmALP1 (two βγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the βγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for βγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the βγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of βγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

Keywords: IgG Fc-binding protein; cell delivery; endolysosomal systems; pore-forming protein; trefoil factor.

Figure 1

Bombina maxima FCGBP interacts with BmALP1 and BmTFF3 (subunits of βγ-CAT).A, the IP result using rabbit IgG and anti-βγ-CAT antibodies. B, identified proteins from IP results by LC-MS. C, expression profiles of FCGBP, BmALP1, and BmTFF3 mRNAs in tissues (skin, intestine, stomach, and heart). β-Tubulin was used as the control. D, hemolysis assays of the elution fraction from the pull-down assay of the Sepharose 4B-anti-FCGBP antibody column with B. maxima secretions as the input. E, Western blots showing the results of Co-IP assays using anti-FCGBP, anti-βγ-CAT, and anti-BmTFF3 antibodies. B. maxima secretions as the input were incubated with protein A slurry beads (left) and outputted after eluting using elution buffer (right). Rabbit IgG was used as the negative control. F, association and Dissociation data of FCGBP and βγ-CAT at serial concentrations were analyzed by the BLI method of a global fit and a 1:1 binding model, with K_D at 2.12 × 10⁻⁸ M, K_D error at 1.37 × 10⁻⁹ M, full X² at 0.6163, and full R² at 0.999. Ab, antibody; BLI, bio-layer interferometry; IP, immunoprecipitation; TFF, trefoil factor.

Figure 2

Purification and identification of FCGBP from Bombina maxima secretions.A, isolation of FCGBP from B. maxima secretions by a Sephadex G-100 column. The arrow indicates FCGBP that was mainly found in peak I. B, resource Q anion-exchange column of peak I from gel filtration was found in peak III containing the target proteins (arrow). C, silver staining of the final product from antibody affinity chromatography of anti-FCGBP. SDS-PAGE was performed under reducing and nonreducing conditions. D, B. maxima skin FCGBP from transcriptome and proteosome analyses, its full-length amino acid sequence included vWD domains (blue), C8 cys-rich domains (green), TIL domains (orange), and an IgG-Fc-binding domain (yellow), which had two GDPH autocatalytic cleavage sites (arrow). E, sequence alignments of the autocatalytic cleavage sites of GDPH between FCGBP and human FCGBP. vWD, von Willebrand type D.

Figure 3

FCGBP restores hemolytic activity of the βγ–CAT complex.A, βγ-CAT (5 nM) and various concentrations of BmALP3 were incubated with human red blood cells (RBCs) at 37 °C for 30 min and then hemolysis was assessed. B, various concentrations of FCGBP were incubated with BmALP3 (6 μM) and βγ-CAT (5 nM) and then incubated with RBCs for hemolysis assays. C, changes of BmALP1 monomers band in the presence of FCGBP were analyzed by Western blotting under nonreducing conditions (left). The change in the amount of BmALP1 monomers was semiquantified by ImageJ (right). D, hemolytic activity of βγ-CAT stored at 4 °C for various times was assessed by reacting with human RBCs at 37 °C. E, 240-day-stored βγ-CAT was incubated with fresh FCGBP at various concentrations and then incubated with RBCs for hemolysis assays. Data represent the mean ± SD of triplicate samples. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Ordinary one-way ANOVA test. ALP, aerolysin-like protein.

Figure 4

FCGBP assembles the active βγ–CAT complex.A, hemolysis assays in which polymer (20 nM) and BmTFF3 (300 nM) were added to RBCs in the presence of FCGBP at various concentrations. B, changes of BmALP1 monomer bands were identified under nonreducing conditions in the presence of various concentrations of FCGBP by Western blotting. Left: Western blot results; Right: semiquantified amount of BmALP1 monomers bands were analyzed by ImageJ. C, dye release at which samples reacted for 2 min with liposomes encapsulating calcein was analyzed by the fluorescence intensity at 518 nm. All data significance represents the mean ± SD of triplicate samples. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Ordinary one-way ANOVA test. D, the number of peritoneal bacteria were counted in mice injected peritoneally with Escherichia. coli (ATCC 25922). See details in Experiment Procedures. At least five mice were included in each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by unpaired t test. ALP, aerolysin-like protein; RBC, red blood cell.

Figure 5

Prdx6 or Trx restores the function FCGBP in βγ-CAT formation.A, functional activity of FCGBP stored at 4 °C for various time was assessed by hemolysis assays. FCGBP mixed with natural BmALP1 polymers and BmTFF3 was reacted with RBCs. B, functional activity of rePrdx6 was assessed by hemolysis with the mutant as the control. rePrdx6 mixed with FCGBP, polymers, and BmTFF3 was reacted with RBCs to determine whether hemolytic activity of βγ-CAT had increased. See details in Hemolysis assays of Experimental procedures. C, functional activity of reTrx was assessed by hemolysis with the mutant as the control. reTrx mixed with FCGBP, polymers, and BmTFF3 was reacted with RBCs to determine whether hemolytic activity of βγ-CAT had increased. See details in Hemolysis assays of Experimental procedures. D, dye release of samples reacted for 2 min with liposomes encapsulating calcein was indicated by the fluorescence intensity at 518 nm. Data represents the mean ± SD of triplicate samples. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Ordinary ANOVA test. ALP, aerolysin-like protein; RBC, red blood cell; TFF, trefoil factor.

Figure 6

Proposed model of FCGBP in positively regulating assembly of the PFP complex βγ-CAT. Because of a conserved cysteine in the C terminal of af-PFPs in toad Bombina maxima, BmALP1 can be reversibly converted between active and inactive forms via redox reactions. BmALP3 senses environmental oxidative conditions (O₂ tension and ROS levels), which converts the protein to a disulfide bond-linked homodimer. BmALP3 homodimer oxidizes BmALP1 via disulfide bond exchange, leading to BmALP1 polymer formation. The action negatively regulates the assembly and biological functions of the βγ–CAT complex (32). Conversely, FCGBP possesses the capacity to reduce oxidized BmALP1 and serves as a scaffold for the interaction of BmALP1 with BmTFF3 to form the βγ–CAT complex. Additionally, Prdx6 or Trx restore and enhance the effect of FCGBP on βγ-CAT assembly. ALP, aerolysin-like protein; PFP, pore-forming protein; ROS, reactive oxygen species.