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

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. 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. Cellular membranes are crucial for functional compartmentalization and the survival of cells. Membrane transporters including the solute carrier superfamily act in uptake of small substances such as cell nutrients glucose and amino acids (1Saier Jr., M.H. Transport protein evolution deduced from analysis of sequence, topology and structure.Curr. Opin. Struct. Biol. 2016; 38: 9-17Crossref PubMed Scopus (33) Google Scholar, 2Pizzagalli M.D. Bensimon A. Superti-Furga G. A guide to plasma membrane solute carrier proteins.FEBS J. 2021; 288: 2784-2835Crossref PubMed Scopus (112) Google Scholar). Additionally, endolysosomal pathways are essential for cells to import diverse insoluble and macromolecular materials including proteins and fatty acid nutrients (3Chantranupong L. Wolfson R.L. Sabatini D.M. Nutrient-sensing mechanisms across evolution.Cell. 2015; 161: 67-83Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 4Palm W. Thompson C.B. Nutrient acquisition strategies of mammalian cells.Nature. 2017; 546: 234-242Crossref PubMed Scopus (241) Google Scholar). Pore-forming proteins (PFPs) in various families with structural folds are widely distributed in all kingdoms of life (5Benton J.T. Bayly-Jones C. Challenges and approaches to studying pore-forming proteins.Biochem. Soc. Trans. 2021; 49: 2749-2765Crossref PubMed Scopus (8) Google Scholar, 6Dal Peraro M. van der Goot F.G. Pore-forming toxins: ancient, but never really out of fashion.Nat. Rev. Microbiol. 2016; 14: 77-92Crossref PubMed Scopus (475) Google Scholar, 7Daskalov A. Glass N.L. Gasdermin and gasdermin-like pore-forming proteins in invertebrates, fungi and bacteria.J. Mol. Biol. 2022; 434167273Crossref PubMed Scopus (14) Google Scholar, 8Kristan K.C. Viero G. Dalla Serra M. Macek P. Anderluh G. Molecular mechanism of pore formation by actinoporins.Toxicon. 2009; 54: 1125-1134Crossref PubMed Scopus (126) Google Scholar, 9Parker M.W. Feil S.C. Pore-forming protein toxins: from structure to function.Prog. Biophys. Mol. Biol. 2005; 88: 91-142Crossref PubMed Scopus (386) Google Scholar). PFPs have long been recognized as either pore-forming toxin for microbial infection (6Dal Peraro M. van der Goot F.G. Pore-forming toxins: ancient, but never really out of fashion.Nat. Rev. Microbiol. 2016; 14: 77-92Crossref PubMed Scopus (475) Google Scholar, 10Verma P. Gandhi S. Lata K. Chattopadhyay K. Pore-forming toxins in infection and immunity.Biochem. Soc. Trans. 2021; 49: 455-465Crossref PubMed Scopus (19) Google Scholar, 11Sannigrahi A. Chattopadhyay K. Pore formation by pore forming membrane proteins towards infections.Adv. Protein Chem. Struct. Biol. 2022; 128: 79-111Crossref PubMed Scopus (1) Google Scholar) or host immune executors (12Broz P. Pelegrín P. Shao F. The gasdermins, a protein family executing cell death and inflammation.Nat. Rev. Immunol. 2020; 20: 143-157Crossref PubMed Scopus (665) Google Scholar, 13Lassalle D. Tetreau G. Pinaud S. Galinier R. Crickmore N. Gourbal B. et al.Glabralysins, potential new β-pore-forming toxin family members from the schistosomiasis vector snail Biomphalaria glabrata.Genes (Basel). 2020; 11: 65Crossref PubMed Scopus (5) Google Scholar, 14Liu L. Deng C.J. Duan Y.L. Ye C.J. Gong D.H. Guo X.L. et al.An aerolysin-like pore-forming protein complex targets viral envelope to inactivate herpes simplex virus type 1.J. Immunol. 2021; 207: 888-901Crossref PubMed Scopus (6) Google Scholar, 15Mesa-Galloso H. Pedrera L. Ros U. Pore-forming proteins: from defense factors to endogenous executors of cell death.Chem. Phys. Lipids. 2021; 234105026Crossref PubMed Scopus (11) Google Scholar). In particular, aerolysin is a bacterial β-barrel pore-forming toxin (16Dang L. Rougé P. Van Damme E.J.M. Amaranthin-like proteins with aerolysin domains in plants.Front. Plant Sci. 2017; 8: 1368Crossref PubMed Scopus (17) Google Scholar, 17Szczesny P. Iacovache I. Muszewska A. Ginalski K. van der Goot F.G. Grynberg M. Extending the aerolysin family: from bacteria to vertebrates.PLoS One. 2011; 6e20349Crossref PubMed Scopus (93) Google Scholar). However, a diverse array of aerolysin family PFPs (af-PFPs; previously referred to as aerolysin-like proteins, ALPs) have been identified in various animals and plant species (16Dang L. Rougé P. Van Damme E.J.M. Amaranthin-like proteins with aerolysin domains in plants.Front. Plant Sci. 2017; 8: 1368Crossref PubMed Scopus (17) Google Scholar, 17Szczesny P. Iacovache I. Muszewska A. Ginalski K. van der Goot F.G. Grynberg M. Extending the aerolysin family: from bacteria to vertebrates.PLoS One. 2011; 6e20349Crossref PubMed Scopus (93) Google Scholar, 18Podobnik M. Kisovec M. Anderluh G. Molecular mechanism of pore formation by aerolysin-like proteins.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 37220160209Crossref PubMed Scopus (37) Google Scholar, 19Parker M.W. Van Der Goot F.G. Buckley J.T. Aerolysin—the ins and outs of a model channel-forming toxin.Mol. Microbiol. 1996; 19: 205-212Crossref PubMed Scopus (96) Google Scholar, 20Lacomel C.J. Dunstone M.A. Spicer B.A. Branching out the aerolysin, ETX/MTX-2 and Toxin_10 family of pore forming proteins.J. Invertebr. Pathol. 2021; 186107570Crossref PubMed Scopus (6) Google Scholar). BmALP1, an af-PFP in toad Bombina maxima, interacts with trefoil factor (TFF) BmTFF3 to form a membrane-active PFP complex named βγ–CAT (21Liu S.B. He Y.Y. Zhang Y. Lee W.H. Qian J.Q. Lai R. et al.A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS One. 2008; 3e1770Crossref Scopus (52) Google Scholar, 22Gao Q. Xiang Y. Zeng L. Ma X.T. Lee W.H. Zhang Y. Characterization of the betagamma-crystallin domains of betagamma-CAT, a non-lens betagamma-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima.Biochimie. 2011; 93: 1865-1872Crossref PubMed Scopus (16) Google Scholar, 23Guo X.L. Liu L.Z. Wang Q.Q. Liang J.Y. Lee W.H. Xiang Y. et al.Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.Commun. Biol. 2019; 2: 59Crossref PubMed Scopus (14) Google Scholar). This protein complex targets acidic glycosphingolipids in lipid rafts of cell plasma membranes via a double-receptor binding model (23Guo X.L. Liu L.Z. Wang Q.Q. Liang J.Y. Lee W.H. Xiang Y. et al.Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.Commun. Biol. 2019; 2: 59Crossref PubMed Scopus (14) Google Scholar). It also drives import and export of extracellular materials and/or plasma membrane components through endo-lysosomal systems (24Shi Z.H. Zhao Z. Liu L.Z. Bian X.L. Zhang Y. Pore-forming protein βγ-CAT promptly responses to fasting with capacity to deliver macromolecular nutrients.FASEB J. 2022; 36e22533Crossref Scopus (3) Google Scholar, 25Zhang Y. Wang Q.Q. Zhao Z. Deng C.J. Animal secretory endolysosome channel discovery.Zool. Res. 2021; 42: 141-152Crossref PubMed Google Scholar, 26Zhao Z. Shi Z.H. Ye C.J. Zhang Y. A pore-forming protein drives macropinocytosis to facilitate toad water maintaining.Commun. Biol. 2022; 5: 730Crossref PubMed Scopus (4) Google Scholar). This PFP oligomerizes to form channels with a functional diameter approximately 1.5 to 2.0 nm on endolysosomes, facilitating material exchange between these intracellular organelles and the cytosol (14Liu L. Deng C.J. Duan Y.L. Ye C.J. Gong D.H. Guo X.L. et al.An aerolysin-like pore-forming protein complex targets viral envelope to inactivate herpes simplex virus type 1.J. Immunol. 2021; 207: 888-901Crossref PubMed Scopus (6) Google Scholar, 21Liu S.B. He Y.Y. Zhang Y. Lee W.H. Qian J.Q. Lai R. et al.A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS One. 2008; 3e1770Crossref Scopus (52) Google Scholar, 26Zhao Z. Shi Z.H. Ye C.J. Zhang Y. A pore-forming protein drives macropinocytosis to facilitate toad water maintaining.Commun. Biol. 2022; 5: 730Crossref PubMed Scopus (4) Google Scholar, 27Deng C.J. Liu L. Liu L.Z. Wang Q.Q. Guo X.L. Lee W.H. et al.A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.FASEB J. 2020; 34: 13609-13625Crossref PubMed Scopus (7) Google Scholar). Thus, the βγ–CAT complex is a multifunctional PFP that plays diverse physiological roles in B. maxima depending on distinct cell contexts and environmental cues. Accordingly, the roles of βγ–CAT in immune defense were first documented (14Liu L. Deng C.J. Duan Y.L. Ye C.J. Gong D.H. Guo X.L. et al.An aerolysin-like pore-forming protein complex targets viral envelope to inactivate herpes simplex virus type 1.J. Immunol. 2021; 207: 888-901Crossref PubMed Scopus (6) Google Scholar, 27Deng C.J. Liu L. Liu L.Z. Wang Q.Q. Guo X.L. Lee W.H. et al.A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.FASEB J. 2020; 34: 13609-13625Crossref PubMed Scopus (7) Google Scholar, 28Gao Z.H. Deng C.J. Xie Y.Y. Guo X.L. Wang Q.Q. Liu L.Z. et al.Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.FASEB J. 2019; 33: 782-795Crossref PubMed Scopus (18) Google Scholar, 29Li S.A. Liu L. Guo X.L. Zhang Y.Y. Xiang Y. Wang Q.Q. et al.Host pore-forming protein complex neutralizes the acidification of endocytic organelles to counteract intracellular pathogens.J. Infect. Dis. 2017; 215: 1753-1763Crossref PubMed Scopus (12) Google Scholar, 30Xiang Y. Yan C. Guo X.L. Zhou K.F. Li S.A. Gao Q. et al.Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6702-6707Crossref PubMed Scopus (37) Google Scholar). In starved toad cells, βγ–CAT drives the production of protein nutrient-containing vesicles, called nutrisomes. These specific vesicles fuse with lysosomes to hydrolyze imported proteins and the resulting amino acid products are released to cytosol to support the nutrient supply and energy fuel in starved cells (31Liu L.Z. Liu L. Shi Z.H. Bian X.L. Wang Q.Q. Xiang Y. et al.Pore-forming protein βγ-CAT drives extracellular nutrient scavenging under cell starvation.bioRxiv. 2022; ([preprint])https://doi.org/10.1101/2022.1107.1120.500773Crossref Google Scholar). Alternatively, specific intracellular vesicles induced by βγ–CAT, which contain imported extracellular substances, such as water with ions (26Zhao Z. Shi Z.H. Ye C.J. Zhang Y. A pore-forming protein drives macropinocytosis to facilitate toad water maintaining.Commun. Biol. 2022; 5: 730Crossref PubMed Scopus (4) Google Scholar) and albumin-bound fatty acids (24Shi Z.H. Zhao Z. Liu L.Z. Bian X.L. Zhang Y. Pore-forming protein βγ-CAT promptly responses to fasting with capacity to deliver macromolecular nutrients.FASEB J. 2022; 36e22533Crossref Scopus (3) Google Scholar), are exported out of cells by exosome release, resulting in intercellular delivery of the cargo molecules for water acquisition and tissue parenchymal cell nutrient supply, respectively. Thus, βγ–CAT defines a secretory (soluble) endolysosomal channel pathway, representing a novel PFP system evolved to mediate cell vesicular delivery and transport (24Shi Z.H. Zhao Z. Liu L.Z. Bian X.L. Zhang Y. Pore-forming protein βγ-CAT promptly responses to fasting with capacity to deliver macromolecular nutrients.FASEB J. 2022; 36e22533Crossref Scopus (3) Google Scholar, 25Zhang Y. Wang Q.Q. Zhao Z. Deng C.J. Animal secretory endolysosome channel discovery.Zool. Res. 2021; 42: 141-152Crossref PubMed Google Scholar, 26Zhao Z. Shi Z.H. Ye C.J. Zhang Y. A pore-forming protein drives macropinocytosis to facilitate toad water maintaining.Commun. Biol. 2022; 5: 730Crossref PubMed Scopus (4) Google Scholar). Reasonably, the βγ–CAT pathway should be regulated tightly. Interestingly, af-PFPs in toad B. maxima contain a conserved C-terminal cysteine that is a key regulatory site via redox reactions. Consequently, BmALP1 is oxidized into a homodimer and water-soluble high molecular weight polymer by BmALP3, a paralog of BmALP1, via disulfide bond exchange, which negatively regulates the assembly and biological functions of the βγ–CAT complex (32Wang Q.Q. Bian X.L. Zeng L. Pan F. Liu L.Z. Liang J.Y. et al.A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions.J. Biol. Chem. 2020; 295: 10293-10306Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Considering the important physiological roles of βγ–CAT, positive regulatory elements should exist in the toad to promote activation and assembly of βγ–CAT. Here, by screening potential βγ-CAT–associated proteins, we found that IgG Fc-binding protein (FCGBP), a mucin-like protein highly conserved in vertebrates with unknown functions (33Wang K. Guan C. Shang X. Ying X. Mei S. Zhu H. et al.A bioinformatic analysis: the overexpression and clinical significance of FCGBP in ovarian cancer.Aging (Albany NY). 2021; 13: 7416-7429Crossref PubMed Scopus (18) Google Scholar, 34Ehrencrona E. van der Post S. Gallego P. Recktenwald C.V. Rodriguez-Pineiro A.M. Garcia-Bonete M.J. et al.The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.J. Biol. Chem. 2021; 297100871Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), interacted physically with βγ-CAT subunits in toad B. maxima skin secretions. Further experiments demonstrated that FCGBP possessed the capacity to reduce oxidized BmALP1 and served as a scaffold for the interaction of BmALP1 with BmTFF3 to form the βγ–CAT complex. Additionally, B. maxima peroxiredoxin 6 (Prdx6) and thioredoxin (Trx) restored and enhanced the effect of FCGBP on βγ-CAT assembly. To identify proteins that interact with βγ-CAT, B. maxima skin secretions were subjected to protein A columns, respectively coupled with antibodies for immunoprecipitation (IP) assays. Some protein bands were found by comparing the products of the protein A column coupled with polyclonal anti-βγ-CAT antibodies with those of the protein A column coupled with rabbit IgG (Fig. 1A). These unknown proteins that indirectly or directly interacted with βγ-CAT were identified and listed as potential candidates with high abundance and oxidoreductases through an IP/LC-MS method (Fig. 1B). In particular, except for BmALP3 (a negative regulator of βγ-CAT) and the two BmALP1 and BmTFF3 subunits of βγ-CAT, FCGBP with high abundance and some oxidoreductases were identified such as Prdx6 and Trx (Fig. 1B). Transcriptomic analysis of the toad B. maxima skin (35Zhao F. Yan C. Wang X. Yang Y. Wang G. Lee W. et al.Comprehensive transcriptome profiling and functional analysis of the frog (Bombina maxima) immune system.DNA Res. 2014; 21: 1-13Crossref PubMed Scopus (37) Google Scholar) revealed a cDNA sequence encoding FCGBP. Semiquantitative PCR of FCGBP showed that the mRNA displayed a tissue distribution pattern and was abundant in skin and intestines. Reported FCGBP was widely expressed in mucosal surfaces and external secretions in humans and mice (36Kobayashi K. Ogata H. Morikawa M. Iijima S. Harada N. Yoshida T. et al.Distribution and partial characterisation of IgG Fc binding protein in various mucin producing cells and body fluids.Gut. 2002; 51: 169-176Crossref PubMed Scopus (80) Google Scholar). It was also distributed in the secretory system. FCGBP was co-expressed with the BmALP1 and BmTFF3 subunits of βγ-CAT in various tissues, which was highly expressed and consistent with βγ-CAT in toad skin (Fig. 1C). Additionally, after pull-down assays using anti-FCGBP antibodies, the eluted solution induced hemolysis of human red blood cells (RBCs), which was also blocked by anti-βγ-CAT antibodies (Fig. 1D). This strongly suggested that FCGBP had a relationship with βγ-CAT. Furthermore, Co-IP assays using various antibodies, including anti-FCGBP, anti-CAT, and anti-BmTFF3, revealed significant interactions between FCGBP and βγ-CAT subunits (BmALP1 and BmTFF3) (Fig. 1E). The peptide fragment of BmALP1 was also determined (Fig. S1A). The bio-layer interferometry (BLI) method showed that FCGBP and βγ-CAT had a strong molecular interaction with a KD value up to 2.12 × 10−8 M (Fig. 1F). These findings indicated an interaction between FCGBP and βγ-CAT subunits, suggesting that the newly identified FCGBP in toad skin secretions may be involved in the assembly and biological functions of βγ-CAT. FCGBP was highly present in the skin of B. maxima. Thus, to better understand the molecular characteristics and functions of FCGBP, B. maxima skin secretions were separated on a Sephadex G-100 column. FCGBP was distributed in peaks I and II (Figs. 2A and S2A). The peak containing FCGBP was collected for isolation by an anion exchange column which produced three protein peaks (Fig. 2B). Peak III of anion exchanges containing FCGBP was injected into a Sepharose-4B-anti-FCGBP affinity column. The purified FCGBP had an apparent weight of 200 kDa in nonreduced SDS-PAGE. It could break different fragments in reduced SDS-PAGE and identified by MS (Figs. 2C and S2B). Data analysis of the B. maxima skin transcriptome and proteome indicated that FCGBP was 1774 amino acids with a theoretic molecular weight of 195.25 kDa. The domain architecture of FCGBP was similarly conserved to human FCGBP (Fig. S2D), which included an IgG Fc-binding domain at the N terminus and four repeated units that were composed of von Willebrand type D (vWD) and C8 cysteine-rich and trypsin inhibitor-like domains (Fig. 2D). Furthermore, sequence alignments revealed that the protein shared 35.4% sequence identity with human FCGBP, indicating conservation of FCGBPs in vertebrates (Fig. S2D). The FCGBP sequence was shared two auto-cleaved sites of GDPH at amino acids 445–448 and 845–848. (Fig. 2E), which was autocatalytically cleaved to generate different fragments and was connected by disulfide bonds as reported previously (34Ehrencrona E. van der Post S. Gallego P. Recktenwald C.V. Rodriguez-Pineiro A.M. Garcia-Bonete M.J. et al.The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.J. Biol. Chem. 2021; 297100871Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). A paralog of the BmALP1 subunit (named BmALP3) of βγ-CAT has been identified in B. maxima. These two β-PFPs (i.e., BmALP1 and BmALP3) contain a conserved cysteine in their C-terminal regions, which is also highly conserved in vertebrate β-PFPs from fish to reptiles (32Wang Q.Q. Bian X.L. Zeng L. Pan F. Liu L.Z. Liang J.Y. et al.A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions.J. Biol. Chem. 2020; 295: 10293-10306Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The BmALP3 homodimer linked by disulfide bonds specifically oxidizes BmALP1 into its own homodimer via disulfide bond formation as well as water-soluble higher molecular weight polymers, which leads to disassociation of the βγ–CAT complex and loss of its biological activity. Additionally, the hemolytic activity is an indication of the presence of biologically active βγ–CAT complex, which is a best and fast method. In a preliminary experiment, BmALP3 did inhibit βγ-CAT activity in a dose-dependent manner (Fig. 3A). Interestingly, the negative regulatory action of BmALP3 (6 μM) on hemolytic activity of βγ-CAT was dose-dependently restored by FCGBPs, whereas a single FCGBP had no hemolytic activity (Figs. 3B and S3A). In the presence of 10 and 30 nM FCGBP, the hemolytic activity was recovered by 27% and 36%, respectively (Fig. 3B). Conversely, rePrdx6 or reTrx alone instead of FCGBP did not restore βγ-CAT hemolytic activity, even at a dose up to approximately 3.1 μM (Fig. S3B). Western blotting was used to detect the generation of BmALP1 monomers. In the presence of FCGBP, BmALP1 monomer bands were significantly observed (Fig. 3C). These data suggested that FCGBP had the ability to reverse the inhibition of βγ-CAT by BmALP3 and restore hemolytic activity of the complex. Purified βγ–CAT complex underwent denaturation when stored alone at 4 °C, resulting in a dramatic loss of its biological activity. When purified βγ-CAT was stored alone for 240 days at 4 °C, hemolytic activity was strongly decreased to approximately 13% (Fig. 3D). Intriguingly, when various concentrations of fresh FCGBP were incubated with 240-day-stored βγ-CAT, the hemolytic activity of βγ-CAT was augmented in a dose-dependent manner (Fig. 3E). However, rePrdx6 or reTrx alone instead of FCGBP had no effect on βγ-CAT hemolytic activity (Fig. S3C). These findings revealed that FCGBP reversed the negative effect of BmALP3 on active βγ-CAT and restored the hemolytic activity of oxidized βγ-CAT. During collection and isolation processes, because of the presence of BmALP3, a fraction of BmALP1 had been oxidized to BmALP1 polymers, which appeared in collected samples of toad B. maxima skin secretions. Thus, natural BmALP1 polymers were isolated by a series of purification procedures including gel filtration, anion exchanges, and affinity chromatography (Fig. S4, A–C). In hemolytic assays, isolated natural polymers at approximately 21 μg/ml did not display hemolytic activity and BmTFF3 itself and the mixture of natural polymers with BmTFF3 also did not possess any hemolytic activity (Fig. S4, D and E). However, interestingly, in the presence of BmTFF3, significantly increased hemolytic activity appeared in a dose-dependent manner after addition of various concentrations of FCGBP (Fig. 4A), which was also blocked by anti-βγ-CAT antibodies (Fig. S4E). This result indicated the appearance of the βγ–CAT complex. Moreover, BmALP1 monomer bands were significantly observed in Western blots (Fig. 4B). Because βγ-CAT has traditional pore-forming activity on cell membranes, various concentrations of FCGBP, BmTFF3, and BmALP1 polymers were premixed and added to 0.1 μm liposomes encapsulating 120 mM calcein which selfquenched at high concentrations and fluoresced at low concentrations, leading to dye release. The mean fluorescence intensity of maximum was measured up to 76.8% at 518 nm emission when FCGBP was added at 150 nM (Fig. 4C), suggesting generation of active βγ-CAT. Active βγ-CAT protects the host against microbial infection (30Xiang Y. Yan C. Guo X.L. Zhou K.F. Li S.A. Gao Q. et al.Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6702-6707Crossref PubMed Scopus (37) Google Scholar). Thus, we assessed in vivo FCGBP activity in mice. Bacterial counting in mice peritoneal fluids showed that mice injected FCGBP, polymers, and BmTFF3 had significant clearance on Escherichia coli and active βγ-CAT–injected mice as the positive control (Fig. 4D), suggesting that FCGBP assembled active βγ-CAT to stimulate mice anti-bacterial immune reactions in vivo. These findings implied that FCGBP reduced polymers to generate active βγ-CAT. We also assessed the possible functions of Prdx6 and Trx in BmALP1 polymers reduction and complex formation under the same conditions as the above assays for FCGBP. Different from the positive effects of FCGBP, in the presence of recombinant Prdx6 or Trx at concentrations used up to 3.1 μM, neither reduction of BmALP1 polymer nor βγ-CAT hemolytic activity was detected (Fig. S4F), indicating that these oxidoreductases did not directly reduce oxidized BmALP1. To further determine whether FCGBP facilitated generation of the βγ–CAT complex, reBmALP1 was expressed through the secreted expression system of Pichia pastoris. ReBmALP1 had polymeric forms of approximately 240 kDa (Fig. S5A). Hemolysis assays indicated that reBmALP1 polymers and BmTFF3 had no hemolytic activity, whereas addition of 30 nM FCGBP increased hemolysis significantly which was blocked by anti-βγ-CAT antibodies with rabbit IgG as the control. In the presence of FCGBP at serial concentrations, hemolytic activity of the mixture of FCGBP and reBmALP1 polymers with BmTFF3 on RBCs was significantly increased in a dose-dependent manner (Fig. S5B). And BmALP1 monomer bands were also significantly observed in Western blots (Fig. S5C). These results implied that FCGBP assembled reBmALP1 polymers and BmTFF3 to generate the βγ–CAT complex. Collectively, these data showed that FCGBP, but not Prdx6 or Trx, was a positive regulator of βγ–CAT complex formation and its biological functions in toad B. maxima. Purified FCGBP stored alone at 4 °C underwent denaturation with dramatic loss of its biological activity in 7 days (Fig. 5A). When FCGBP was stored for 28 days, its effect on BmALP1 polymers was decreased strongly to approximately 1% (Fig. 5A). This suggested that FCGBP was composed of easily oxidized-free cystines and the status of oxidized and reduced free cystines might be changed by certain oxidoreductases. Interestingly, the protein sequence identities of reducing enzymes Prdx6 and Trx were highly conserved between toad B. maxima (35Zhao F. Yan C. Wang X. Yang Y. Wang G. Lee W. et al.Comprehensive transcriptome profiling and functional analysis of the frog (Bombina maxima) immune system.DNA Res. 2014; 21: 1-13Crossref PubMed Scopus (37) Google Scholar) and humans (86% and 70% sequence identities, respectively). Thus, experiments were carried out to determine whether Prdx6 and Trx restored the biological activity of naturally occurring FCGBP. Interestingly, Various concentrations of reprdx6 were reacted with a mixture of inactive FCGBP, natural purified polymers, and BmTFF3 to assess hemolysis of RBCs with reprdx6(C46A, C90A) mutant as the control. rePrdx6 (3.1 μM) significantly restored the biological activity of FCGBP with being stored for 8 days (Fig. 5B). Similarly, reTrx (3.1 μM) also restored the biological activities of FCGBP (Fig. 5C). In dye release assays of pore formation in liposomes, FCGBP had no activity on liposomes. However, the fluorescence intensity was significantly increased after addition of 150 nM FCGBP. When FCGBP was reduced by Trx, significantly more dye was released than after addition of FCGBP stored for 8 days, resulting in a difference of up to 12.84% (Fig. 5D). These results suggested that the biological activity of FCGBP was restored by oxidoreductases Prdx6 and Trx via a redox reaction. These findings indicated FCGBP assembled active βγ-CAT and FCGBP with deceased activity reduced prdx6 and Trx. Cell membranes with their selective permeability form barriers for molecule exchange between the cytosol and the extracellular environment (37Watson H. Biological membranes.Essays Biochem. 2015; 59: 43-69Crossref PubMed Google Scholar, 38Gilbert R.J. Dalla Serra M. Froelich C.J. Wallace M.I. Anderluh G. Membrane pore formation at protein-lipid interfaces.Trends Biochem. Sci. 2014; 39: 510-516Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Toad B. maxima