A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions
2020; Elsevier BV; Volume: 295; Issue: 30 Linguagem: Inglês
10.1074/jbc.ra120.013556
ISSN1083-351X
AutoresQiquan Wang, Xian‐Ling Bian, Lin Zeng, Fei Pan, Ling‐Zhen Liu, Jin-Yang Liang, Lingyan Wang, Kaifeng Zhou, Wen‐Hui Lee, Yang Xiang, Sheng’an Li, Maikun Teng, Li Xu, Xiaolong Guo, Yun Zhang,
Tópico(s)Vector-borne infectious diseases
ResumoEndolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins. Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins. Cellular membranes are essential for defining the border and ensuring the function of all living cells (1Bischofberger M. Gonzalez M.R. van der Goot F.G. Membrane injury by pore-forming proteins.Curr. Opin. Cell Biol. 2009; 21 (19442503): 589-59510.1016/j.ceb.2009.04.003Crossref PubMed Scopus (103) Google Scholar, 2Bischofberger M. Iacovache I. van der Goot F.G. 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Toad skin is naked and is constantly confronted by a complex mixture of potentially injurious factors as it interacts with the environment to ensure sufficient uptake of water, electrolytes, and oxygen (16Xu X. Lai R. The chemistry and biological activities of peptides from amphibian skin secretions.Chem. Rev. 2015; 115 (25594509): 1760-184610.1021/cr4006704Crossref PubMed Scopus (144) Google Scholar, 17Jared S.R. Rao J.P. Transepithelial sodium transport across frog skin.Adv. Physiol. Educ. 2017; 41 (28679586): 444-44710.1152/advan.00115.2016Crossref PubMed Scopus (2) Google Scholar). Recently, an ALP complex named βγ-CAT was purified and isolated from the skin secretions of the firebelly toad (Bombina maxima). It consists of two subunits, of which BmALP1 (α-subunit) is a βγ-crystallin domain fused with an aerolysin domain and BmTFF3 (β-subunit) is a three-domain trefoil factor (TFF) (18Liu S.B. He Y.Y. Zhang Y. Lee W.H. Qian J.Q. Lai R. Jin Y. A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS ONE. 2008; 3 (18335045): e177010.1371/journal.pone.0001770Crossref PubMed Scopus (46) Google Scholar, 19Gao Q. Xiang Y. Zeng L. Ma X.T. Lee W.H. Zhang Y. Characterization of the βγ-crystallin domains of βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima.Biochimie. 2011; 93 (21784123): 1865-187210.1016/j.biochi.2011.07.013Crossref PubMed Scopus (12) Google Scholar). The cellular acting pathway of βγ-CAT is characterized by targeting of acidic glycosphingolipids in lipid rafts via a double-receptor binding model to initiate the endocytosis of its BmALP1 subunit and the subsequent oligomerization and pore formation of BmALP1 along the cellular endolysosomal pathways (20Guo X.L. Liu L.Z. Wang Q.Q. Liang J.Y. Lee W.H. Xiang Y. Li S.A. Zhang Y. Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.Commun. Biol. 2019; 2 (30775460): 5910.1038/s42003-019-0304-yCrossref PubMed Scopus (7) Google Scholar). This action results in changes in the biochemical properties of these intracellular vesicles, including increased acidification, which leads to diverse cellular responses and outcomes depending on various cell contexts (21Xiang Y. Yan C. Guo X. Zhou K. Li S. Gao Q. Wang X. Zhao F. Liu J. Lee W.-H. Zhang Y. 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 (24733922): 6702-670710.1073/pnas.1321317111Crossref PubMed Scopus (30) Google Scholar, 22Li S.A. Liu L. Guo X.L. Zhang Y.Y. Xiang Y. Wang Q.Q. Lee W.H. Zhang Y. Host pore-forming protein complex neutralizes the acidification of endocytic organelles to counteract intracellular pathogens.J. Infect. 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Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.FASEB J. 2019; 33 (30063438): 782-79510.1096/fj.201800087RCrossref PubMed Scopus (14) Google Scholar). Furthermore, this protein complex has been found to stimulate and to participate in the formation and release of extracellular vesicles to eliminate infecting intracellular pathogens (22Li S.A. Liu L. Guo X.L. Zhang Y.Y. Xiang Y. Wang Q.Q. Lee W.H. Zhang Y. Host pore-forming protein complex neutralizes the acidification of endocytic organelles to counteract intracellular pathogens.J. Infect. Dis. 2017; 215 (28419297): 1753-176310.1093/infdis/jix183Crossref PubMed Scopus (8) Google Scholar). These biological functions suggested that βγ-CAT could play important roles in maintaining mucosal barrier homeostasis, facilitating material exchange via vesicle formation and trafficking, and mediating immune defenses in the toad (18Liu S.B. He Y.Y. Zhang Y. Lee W.H. Qian J.Q. Lai R. Jin Y. A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS ONE. 2008; 3 (18335045): e177010.1371/journal.pone.0001770Crossref PubMed Scopus (46) Google Scholar, 21Xiang Y. Yan C. Guo X. Zhou K. Li S. Gao Q. Wang X. Zhao F. Liu J. Lee W.-H. Zhang Y. 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 (24733922): 6702-670710.1073/pnas.1321317111Crossref PubMed Scopus (30) Google Scholar, 22Li S.A. Liu L. Guo X.L. Zhang Y.Y. Xiang Y. Wang Q.Q. Lee W.H. Zhang Y. Host pore-forming protein complex neutralizes the acidification of endocytic organelles to counteract intracellular pathogens.J. Infect. Dis. 2017; 215 (28419297): 1753-176310.1093/infdis/jix183Crossref PubMed Scopus (8) Google Scholar, 23Gao Z.H. Deng C.J. Xie Y.Y. Guo X.L. Wang Q.Q. Liu L.Z. Lee W.H. Li S.A. Zhang Y. Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.FASEB J. 2019; 33 (30063438): 782-79510.1096/fj.201800087RCrossref PubMed Scopus (14) Google Scholar). Distinct from classic membrane-integrated ion channels and transporters, βγ-CAT provides the first example of an endogenous secreted β-barrel PFP that extracellularly targets cellular endocytotic pathways to modulate the biochemical contents and properties of endolysosomes. This secreted PFP-mediated action represents a hitherto unknown regulatory mechanism of cell endocytosis and exocytosis via endolysosome modulation. This cellular acting pathway should be tightly regulated, and regulatory proteins of βγ-CAT may exist in the toad. In this study, a paralog of the βγ-CAT BmALP1 subunit (named BmALP3) was identified in B. maxima. BmALP3 homodimer linked by a disulfide bond specifically oxidized BmALP1 into its own homodimer via disulfide bond formation, as well as a water-soluble higher molecular weight polymer, which negatively regulated the assembly and biological functions of the βγ-CAT complex. To identify βγ-CAT-associated proteins, skin secretions of B. maxima were prepared and subjected to a pulldown assay using anti-βγ-CAT-Sepharose 4B affinity chromatography. A specific band, in addition to the two subunits of βγ-CAT, was found in the eluted proteins of anti-βγ-CAT columns by SDS-PAGE (Fig. 1A). The unknown protein band was further digested by trypsin and analyzed by MS; three MS/MS spectra representing the main components of digest products were selected for de novo sequencing, and the sequences of these peptide fragments matched well with the theoretical sequence deduced from the skin transcriptome of B. maxima (Fig. S1A). Subsequent molecular cloning of the unknown protein showed that its full-length cDNA was 601 bp, and the open reading frame was 471 bp, which encoded 156 amino acids (GenBank accession number MN787048). Sequence alignment revealed that the protein shared 27% sequence identity with the βγ-CAT BmALP1 subunit, suggesting that it was homologous to BmALP1, and we termed it BmALP3. Further sequence analysis revealed that BmALP3 contained only the aerolysin domain; this is different from BmALP1, which contained the βγ-crystallin domain and aerolysin domain (Fig. 1B and Fig. S1B). In addition, a specific conserved cysteine in the C-terminal region was found in both BmALP1 and BmALP3 (Fig. 1B and Fig. S1B). Our previous study verified that βγ-CAT was constitutively expressed in different tissues of B. maxima (21Xiang Y. Yan C. Guo X. Zhou K. Li S. Gao Q. Wang X. Zhao F. Liu J. Lee W.-H. Zhang Y. 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 (24733922): 6702-670710.1073/pnas.1321317111Crossref PubMed Scopus (30) Google Scholar). Here, tissue localization analysis of BmALP3 at the mRNA and protein levels revealed that its tissue distribution pattern was consistent with that of βγ-CAT (Fig. 1C and Fig. S1C). Immunofluorescence staining also displayed strong colocalization of BmALP3 with βγ-CAT in different tissues of toad (Fig. 1D). In addition, a coimmunoprecipitation assay was performed and showed a significant interaction between BmALP3 and βγ-CAT in toad skin secretions (Fig. 1E). All of these findings showed that BmALP3 and βγ-CAT existed together, suggesting that the newly identified ALP paralog was involved in the functions of βγ-CAT. As mentioned above, BmALP3 existed mainly in the skin secretions of B. maxima. Thus, in the subsequent study, natural BmALP3 was purified to better understand its molecular characteristics and functions. B. maxima skin secretions were separated by a DEAE Sephadex A-50 column, and seven protein peaks were obtained; βγ-CAT was distributed in peaks I–VII and BmALP3 was distributed in peaks IV–VII (Fig. 2A). The hemolytic activity of skin secretions is mainly induced by βγ-CAT, as confirmed by an anti-βγ-CAT antibody blocking assay (Fig. S2A). Interestingly, the hemolytic activity of βγ-CAT in each fraction was decreased suddenly in association with the appearance of BmALP3 (Fig. 2A), suggesting that the function of βγ-CAT might be negatively regulated by BmALP3. Since free cysteine was easier to oxidize and BmALP3 contained its only cysteine in its C-terminal region, the reducing agent DTT was added in the following purification. DEAE Sephadex A-50 column peak VII was used in subsequent gel filtration and resulted in the separation of two protein peaks. Peak II was highly purified BmALP3, with an apparent weight of 18 kDa in SDS-PAGE (Fig. 2B). In the following study, Western blotting revealed that a new 35-kDa band was found in nonreducing SDS-PAGE when purified BmALP3 was exposed to air for different times (Fig. 2C) or treated with H2O2 at different concentrations (Fig. 2D), whereas the 35-kDa band disappeared under reducing conditions (Fig. 2, C and D). A reasonable explanation was that the 35-kDa band was the dimeric form of BmALP3 under oxidative conditions. A point mutation assay also verified that the conserved C-terminal cysteine (Cys141) was responsible for BmALP3 homodimer formation under oxidative conditions (Fig. 2E). To further study whether BmALP3 dimerization was mediated by a disulfide bond, a MS assay was performed and showed that a peptide at m/z 6108.4 in the homodimer was observed after enzyme digestion, whereas this ion disappeared along with the appearance of a new ion with m/z 3055.1, which is close to the theoretical m/z value of the peptide that contains Cys141, when DTT was added (Fig. S2B). This implied that a disulfide bond may exist in the homodimer. Unfortunately, we failed to obtain the MALDI-postsource decay spectra of both ions because the ion intensity was too low for MS/MS analysis. To increase the signal intensity of the enzyme-digested peptide that contained Cys141, Glu150 was mutated to Arg. Similar to BmALP3, rBmALP3E150R could also be oxidized to a homodimer. A disulfide bond-linked peptide was found in the rBmALP3E150R homodimer by MS/MS (Fig. S2C). These results demonstrated that BmALP3 could be oxidized to a disulfide bond-linked homodimer via the conserved C-terminal cysteine under oxidative conditions. Since BmALP3 belongs to the ALP family, its oligomerization and pore-forming capacity were detected in a subsequent study. Given that BmALP3 existed in both the monomeric and dimeric states, we first detected the proportion of prepared BmALP3 monomer and homodimer before the following assay (Fig. S2D). A hemolytic assay on human red blood cells (RBCs) was performed to reflect pore-forming ability and revealed that there was no significant hemolysis when the concentration of BmALP3 was up to 1.28 mg/ml, while high hemolytic activity was observed after treatment with βγ-CAT (Fig. S2E). Oligomerization was the key step for ALP pore formation (14Szczesny 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; 6 (21687664): e2034910.1371/journal.pone.0020349Crossref PubMed Scopus (74) Google Scholar, 24Cirauqui N. Abriata L.A. van der Goot F.G. Dal Peraro M. Structural, physicochemical and dynamic features conserved within the aerolysin pore-forming toxin family.Sci. Rep. 2017; 7 (29066778): 1393210.1038/s41598-017-13714-4Crossref PubMed Scopus (18) Google Scholar). Western blotting showed that there was no oligomer observed after cells (RBCs and toad peritoneal cells) or liposomes were treated with BmALP3, whereas the SDS-stable oligomer was observed in βγ-CAT-treated samples (Fig. S2, F–H). These findings indicated that BmALP3 lacks the typical pore-forming ability, which is different from previously reported ALPs. As a novel ALP that lacks the pore-forming ability, the three-dimensional structure of the BmALP3 monomer at 1.73 Å was next determined by the single-wavelength anomalous diffraction method (Table S1). The whole structure of BmALP3 could be divided into two distinct β-sandwiches; the N-terminal β-sandwich was formed by a twisted antiparallel five-stranded β-sheet together with a long hairpin, whereas the C-terminal β-sandwich comprised a three‐stranded antiparallel β‐sheet packing against a two-stranded β-sheet (Fig. 2F).The structural comparison revealed that it harbored the conserved aerolysin fold, which was similar to typical ALP family members, such as Dln1 (25Jia N. Liu N. Cheng W. Jiang Y.L. Sun H. Chen L.L. Peng J. Zhang Y. Ding Y.H. Zhang Z.H. Wang X. Cai G. Wang J. Dong M.Q. Zhang Z. et al.Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.EMBO Rep. 2016; 17 (26711430): 235-24810.15252/embr.201540851Crossref PubMed Scopus (32) Google Scholar) and aerolysin (26Parker M.W. Buckley J.T. Postma J.P.M. Tucker A.D. Leonard K. Pattus F. Tsernoglou D. Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.Nature. 1994; 367 (7510043): 292-29510.1038/367292a0Crossref PubMed Scopus (351) Google Scholar) (Fig. S2I). However, the long hairpin of BmALP3 between strand β2 and strand β3 was enriched in hydrophilic amino acids (Fig. 2G), which was significantly different from the amphipathic pre-stem loop of previously reported ALPs (14Szczesny 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; 6 (21687664): e2034910.1371/journal.pone.0020349Crossref PubMed Scopus (74) Google Scholar, 24Cirauqui N. Abriata L.A. van der Goot F.G. Dal Peraro M. Structural, physicochemical and dynamic features conserved within the aerolysin pore-forming toxin family.Sci. Rep. 2017; 7 (29066778): 1393210.1038/s41598-017-13714-4Crossref PubMed Scopus (18) Google Scholar). Notably, the conserved amphipathic pre-stem loop is pivotal for the pore-forming capacity of ALPs (14Szczesny 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; 6 (21687664): e2034910.1371/journal.pone.0020349Crossref PubMed Scopus (74) Google Scholar). The hydrophilic hairpin may explain the poor pore-forming ability of BmALP3. All of the above findings revealed that, unlike typical ALP members, BmALP3 could be oxidized to a disulfide-linked homodimer but lacked the pore-forming ability, suggesting that it performs some novel functions that are different from those of classic ALPs, such as negative regulation. To probe the direct effects of BmALP3 on βγ-CAT, natural purified BmALP3, recombinant rBmALP3, and mutant rBmALP3C141A were used, and the hemolytic activity and oligomerization of βγ-CAT on RBCs and toad peritoneal cells were detected in vitro. Interestingly, both the hemolytic activity and oligomerization of βγ-CAT were largely attenuated by BmALP3 and rBmALP3 in a dose-dependent manner (Fig. 3, A and B, and Fig. S3, A–C). However, these behaviors were not affected by rBmALP3C141A (Fig. 3A and Fig. S3, D and E). In contrast to natural BmALP3 or recombinant rBmALP3, rBmALP3C141A could not be oxidized to form a homodimer. The above findings suggested that the functions of βγ-CAT could be negatively regulated by oxidized BmALP3 homodimer rather than reduced monomer. To validate this hypothesis, fully reduced BmALP3 was prepared through pretreatment with DTT and applied in the in vitro activity inhibition assay. As expected, similar to rBmALP3C141A, the hemolytic activity and oligomerization of βγ-CAT were not affected by reduced BmALP3 under DTT treatment (Fig. 3, C and D, and Fig. S3F). The above findings indicated that BmALP3 negatively regulated the functions of βγ-CAT under oxidative conditions rather than under reducing conditions. A previous study demonstrated that βγ-CAT could reduce the infectious bacterial load in the toad peritoneum and protect toads against bacterial infection (21Xiang Y. Yan C. Guo X. Zhou K. Li S. Gao Q. Wang X. Zhao F. Liu J. Lee W.-H. Zhang Y. 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 (24733922): 6702-670710.1073/pnas.1321317111Crossref PubMed Scopus (30) Google Scholar). To further examine whether BmALP3 could inhibit βγ-CAT in vivo and affect the microbial clearance of toads, a toad peritoneal bacterial infection model was used. The results showed that the ability of βγ-CAT to induce rapid bacterial clearance was inhibited by intraperitoneal injection of BmALP3 and βγ-CAT together (Fig. 3E), and BmALP3 alone also attenuated the bacterial clearance ability of toads (Fig. 3F). All of these findings suggested that oxidized BmALP3 negatively regulates the function of βγ-CAT in vivo and in vitro, while reduced BmALP3 has no impact on the function of βγ-CAT. To further study the mechanism by which oxidized BmALP3 negatively regulates βγ-CAT, we first detected the states of two subunits of βγ-CAT under nonreducing conditions after treatment with BmALP3. Western blotting of BmALP1 showed that two new bands appeared in nonreducing SDS-PAGE, one band at ∼70 kDa and another at ∼170 kDa, whereas the two new bands were not found in reducing SDS-PAGE (Fig. 4A). A reasonable explanation was that the two new bands were the dimeric and polymeric forms of BmALP1 that were oxidized by BmALP3. It is worth noting that the BmALP1 high-molecular-weight polymer could be reduced to a monomeric form (Fig. 4A), indicating that the reversible polymer was obviously different from the SDS-resistant oligomer that formed in cells or liposomes, as described above; thus, we termed this form the polymer to distinguish it from the SDS-resistant oligomer. In addition to BmALP1 being oxidized to a homodimer and a polymer, we next observed that the BmALP3 homodimer was reduced to its monomeric form in nonreducing SDS-PAGE after incubation with βγ-CAT (Fig. 4B). Previous studies demonstrated that the BmTFF3 subunit has no free cysteine (18Liu S.B. He Y.Y. Zhang Y. Lee W.H. Qian J.Q. Lai R. Jin Y. A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS ONE. 2008; 3 (18335045): e177010.1371/journal.pone.0001770Crossref PubMed Scopus (46) Google Scholar, 20Guo X.L. Liu L.Z. Wang Q.Q. Liang J.Y. Lee W.H. Xiang Y. Li S.A. Zhang Y. Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.Commun. Biol. 2019; 2 (30775460): 5910.1038/s42003-019-0304-yCrossref PubMed Scopus (7) Google Scholar). Accordingly, Western blotting of BmTFF3 revealed that there was no obvious band position change in BmTFF3 in reducing or nonreducing SDS-PAGE after treatment with BmALP3 (Fig. S4A). A point mutation assay was used to probe which cysteine was responsible for BmALP1 dimerization and polymerization, and it showed that the formation of the BmALP1 homodimer and polymer were both dependent on the conserved C-terminal cysteine Cys321 (Fig. 4C). Further MS detection also verified that both the BmALP1 dimeric band and the polymeric band were linked by a disulfide bond via Cys321 (Fig. S4B). These results suggested that BmALP1 could be oxidized by the BmALP3 homodimer and form its own homodimer, as well as a polymer, via disulfide bond exchange. A previous study revealed that both the BmALP1 and BmTFF3 subunits were required for the functions of βγ-CAT and the dissociation of the BmALP1 and BmTFF3 subunits would lead to functional loss of βγ-CAT (18Liu S.B. He Y.Y. Zhang Y. Lee W.H. Qian J.Q. Lai R. Jin Y. A novel non-lens βγ-crystallin and trefoil factor complex from amphibian skin and its functional implications.PLoS ONE. 2008; 3 (18335045): e177010.1371/journal.pone.0001770Crossref PubMed Scopus (46) Google Scholar, 20Guo X.L. Liu L.Z. Wang Q.Q. Liang J.Y. Lee W.H. Xiang Y. Li S.A. Zhang Y. Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome reg
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