An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.

Retinal pigment epithelial (RPE) cell death is a hallmark of age-related macular degeneration. The alternative pathway of complement activation is strongly implicated in RPE cell dysfunction and loss in age-related macular degeneration; therefore, it is critical that RPE cells use molecular strategies to mitigate the potentially harmful effects of complement attack. We show that the terminal complement complex C5b-9 assembles rapidly on the basal surface of cultured primary porcine RPE cells but disappears over 48 h without any discernable adverse effects on the cells. However, in the presence of the dynamin inhibitor dynasore, C5b-9 was almost completely retained at the cell surface, suggesting that, under normal circumstances, it is eliminated via the endocytic pathway. In support of this idea, we observed that C5b-9 colocalizes with the early endosome marker EEA1 and that, in the presence of protease inhibitors, it can be detected in lysosomes. Preventing the endocytosis of C5b-9 by RPE cells led to structural defects in mitochondrial morphology consistent with cell stress. We conclude that RPE cells use the endocytic pathway to prevent the accumulation of C5b-9 on the cell surface and that processing and destruction of C5b-9 by this route are essential for RPE cell survival.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

The complement system is an important aspect of immune defense against microbial invasion. Eukaryotic cells express various complement regulatory proteins to protect them from uncontrolled complement activation. However, some eukaryotic cells possess constitutive complement system activation that does not require specific triggering factors, which is known to have unexpected effects on cell proliferation and survival. This area of research is still preliminary and a standard method to measure complement system activation in eukaryotic cells has yet to be identified. Here, we present a quantitative in vitro method to measure complement system activation in eukaryotic cells by detecting C5b-9, the membrane attack complex, on cell surfaces. The results obtained using this assay correlated with C3b deposition measured using flow cytometry and C5b-9 deposition detected using an immunofluorescence assay. Furthermore, we showed that various cancer cell lines displayed different levels of complement system activation by using this assay.

The co-inhibitory molecule B7-H1 has been broadly detectable on human inflammatory renal tubular epithelial cells (TECs) and is proposed to limit tubular damage through down-regulation of tubulointerstitial infiltration T cell activation. Nevertheless, factors that initiate B7-H1 expression on TECs remain unclarified. The terminal complement complex C5b-9, which deposits diffusely on tubules and glomerules of diseased kidneys, is now recognized as a mediator that triggers cellular activation rather than inducing cell death. Whether the up-regulation of B7-H1 on tubules is also induced by C5b-9 is uncertain. Here, after assembling functional sublytic C5b-9 on the membranes of TECs based on purified complement components, we found that B7-H1 gene transcription and protein synthesis was enhanced by C5b-9. Promoter constructs in a luciferase assay, site-directed mutagenesis and laser scanning confocal microscopy assay (LSCM) revealed that the transcription factor NF-kappaB is primarily responsible for C5b-9-mediated B7-H1 expression. To further detect the physiologic function of B7-H1, triggering B7-H1 with its agonist mAb (clone 5H1) profoundly enhanced Fas expression on C5b-9-treated TECs and thus induced TEC apoptosis. Interestingly, pretreatment of TECs with Fas blocking antibodies prevented this effect. Our results propose that C5b-9 regulates tubular pathogenesis in glomerulonephritis or other renal autoimmune diseases, possibly through enhances cell apoptosis mediated by B7-H1 signals, in addition to it directly promotes tubular damage.

Trichloroethylene (TCE), a commonly used industrial solvent and degreasing agent, is known to cause trichloroethylene hypersensitivity syndrome (THS) with multi-system damage, including skin, liver and kidney. Clinical evidence have shown that the kidney injury occurs in THS and our previous studies suggested that the terminal complement complex C5b-9 deposited in impaired renal tubules induced by TCE with unclear mechanisms. In the present study, we questioned whether activation of the complement system with renal deposition of C5b-9 contributes to TCE-induced kidney injury in THS. We established a BALB/c mouse model of TCE sensitization with or without pretreatment of exogenous CD59, a C5b-9 inhibitory protein. H&E staining, PAS staining, and biochemical detection of urinary proteins were performed to assess renal function. Deposition of C5b-9 and expression of CD59 were evaluated by immunohistochemistry. Sub-lytic effects of C5b-9 in tubular epithelial cells were assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Expression of endocytosis receptors megalin and cubilin on proximal tubules were assessed by immunofluorescence and qRT-PCR. We found that TCE sensitization induced structural and functional changes of renal tubules in mice, associated with the deposition of sub-lytic C5b-9 on proximal tubular epithelial cells. TCE sensitization decreased proximal tubule uptake of filtered proteins and renal expression of megalin and cubilin, phenotypes that were attenuated by pretreatment with exogenous CD59. Overall, our findings reveal a novel mechanism underlying sub-lytic C5b-9 acting on megalin and cubilin, contributes to the renal tubules damage by TCE exposure.

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.

The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

Antibodies against Rho GDP-dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti-RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement-fixing properties of anti-RhoGDI2 antibodies were studied using imaging flow cytometry. Anti-RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co-localized with C3 on ECs. Binding of human IgG1 monoclonal anti-RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti-RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury.

Rare mutations in the human RGR gene lead to autosomal recessive retinitis pigmentosa or dominantly inherited peripapillary choroidal atrophy. Here, we analyze a common exon-skipping isoform of the human retinal G protein-coupled receptor opsin (RGR-d) to determine differences in subcellular targeting between RGR-d and normal RGR and possible association with abnormal traits in the human eye.

We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-1 (SDF-1). As SDF-1 is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) increase in the BM after conditioning for transplantation and that both S1P and, as we show here for the first time, C1P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD11b/CD18)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-1 by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

Terminal complement complex deposition in endomysial capillaries detected by a C5b-9 immunostain is considered a diagnostic feature for dermatomyositis. However, we found widespread microvascular C5b-9 reactivity in a substantial subset of muscle biopsies with denervation changes, and in nerve biopsies of peripheral neuropathies, particularly in patients with diabetes. It is unclear whether the presence of C5b-9 deposition signifies active immune-mediated vascular injury that requires immune suppression therapy. We retrospectively identified 63 nerve biopsies in patients with a documented history of diabetes, 26 of which had concomitant muscle biopsies, as well as 54 control nerve biopsies in patients without a documented diabetes history, 18 of which had concomitant muscle biopsies. C5b-9 immunostain was performed on all cases. 87% of the nerve biopsies and 92% of the muscle biopsies from diabetic patients showed microvascular C5b-9 reactivity, compared to 34% and 50% in non-diabetic patients. The differences were statistically significant (p < 0.0001 for nerve and p = 0.002 for muscle). The C5b-9 reactivity was generally proportional to the extent of microvascular sclerosis in diabetic patients, but unrelated to inflammation or vasculitis. C5b-9 deposition in micro-vasculature in both muscle and nerve is therefore a common feature in patients with diabetic neuropathies and may have diagnostic utility. Precaution needs to be taken before using muscle capillary C5b-9 reactivity as evidence of myositis.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Trichloroethylene (TCE), a commonly used organic solvent, is known to cause trichloroethylene hypersensitivity syndrome (THS), also called occupational medicamentosa-like dermatitis due to TCE (OMDT) in China. OMDT patients presented with severe inflammatory kidney damage, and we have previously shown that the renal damage is related to the terminal complement complex C5b-9. Here, we sought to determine whether C5b-9 participated in TCE-induced immune kidney injury by promoting pyroptosis, a new form of programed cell death linked to inflammatory response, with underlying molecular mechanisms involving the NLRP3 inflammasome. A BALB/c mouse-based model of OMDT was established by dermal TCE sensitization in the presence or absence of C5b-9 inhibitor (sCD59-Cys, 25μg/mouse) and NLRP3 antagonist (MCC950, 10 mg/kg). Kidney histopathology, renal function, expression of inflammatory mediators and the pyroptosis executive protein gasdermin D (GSDMD), and the activation of pyroptosis canonical NLRP3/caspase-1 pathway were examined in the mouse model. Renal tubular damage was observed in TCE-sensitized mice. GSDMD was mainly expressed on renal tubular epithelial cells (RTECs). The caspase-1-dependent canonical pathway of pyroptosis was activated in TCE-induced renal damage. Pharmacological inhibition of C5b-9 could restrain the caspase-1-dependent canonical pathway and rescued the renal tubular damage. Taken together, our results demonstrated that complement C5b-9 plays a central role in TCE-induced immune kidney damage, and the underlying mechanisms involve NLRP3-mediated pyroptosis.

The apoptosis of glomerular mesangial cells (GMCs) in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis (MsPGN), is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 induces GMC apoptosis is unclear. In the present studies, the effect of X-linked inhibitor of apoptosis-associated factor 1 (XAF1) expression on GMC apoptosis and the role of p300 and interferon regulatory factor-1 (IRF-1) in mediating XAF1 gene activation were determined, both in the GMCs induced by sublytic C5b-9 (in vitro) and in the renal tissues of rats with Thy-1N (in vivo). The in vitro studies demonstrated that IRF-1-enhanced XAF1 gene activation and its regulation by p300-mediated IRF-1 acetylation were involved in GMC apoptosis induced by sublytic C5b-9. The element of IRF-1 binding to XAF1 promoter and two acetylated sites of IRF-1 protein were also revealed. In vivo, silence of p300, IRF-1 or XAF1 genes in the renal tissues diminished GMC apoptosis and secondary GMC proliferation as well as urinary protein secretion in Thy-1N rats. Together, these data implicate that sublytic C5b-9 induces the expression of both p300 and IRF-1, as well as p300-dependent IRF-1 acetylation that may contribute to XAF1 gene activation and subsequent GMC apoptosis in Thy-1N rats.

We searched for indicators to predict the prognosis of infectious peritonitis by measuring levels of complement proteins and activation products in peritoneal dialysis (PD) fluid (PDF) of patients at early stages of peritonitis. We retrospectively analyzed the relationship between the levels of sC5b-9, C3 and C4 in PDF and the subsequent clinical prognosis.

Podocytes are an important part of the glomerular filtration barrier and the key player in the development of proteinuria, which is an early feature of complement mediated renal diseases. Complement factors are mainly liver-born and present in circulation. Nevertheless, there is a growing body of evidence for additional sites of complement protein synthesis, including various cell types in the kidney. We hypothesized that podocytes are able to produce complement components and contribute to the local balance of complement activation and regulation. To investigate the relevant balance between inhibiting and activating sides, our studies focused on complement factor H (CFH), an important complement regulator, and on C3, the early key component for complement activation. We characterized human cultured podocytes for the expression and secretion of activating and regulating complement factors, and analyzed the secretion pathway and functional activity. We studied glomerular CFH and C3 expression in puromycin aminonucleoside (PAN) -treated rats, a model for proteinuria, and the physiological mRNA-expression of both factors in murine kidneys. We found, that C3 and CFH were expressed in cultured podocytes and expression levels differed from those in cultivated glomerular endothelial cells. The process of secretion in podocytes was stimulated with interferon gamma and located in the Golgi apparatus. Cultured podocytes could initiate the complement cascade by the splitting of C3, which can be shown by the generation of C3a, a functional C3 split product. C3 contributed to external complement activation. Podocyte-secreted CFH, in conjunction with factor I, was able to split C3b. Podocytes derived from a patient with a CFH mutation displayed impaired cell surface complement regulation. CFH and C3 were synthesized in podocytes of healthy C57Bl/6-mice and were upregulated in podocytes of PAN treated rats. These data show that podocytes produce functionally active complement components, and could therefore influence the local glomerular complement activation and regulation. This modulating effect should therefore be considered in all diseases where glomerular complement activation occurs. Furthermore, our data indicate a potential novel role of podocytes in the innate immune system.

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b-9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy-1 nephritis (Thy-1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC-32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal-regulated kinase 5 (phosphorylated ERK5, p-ERK5), F-box only protein 28 (FBXO28) and TNF receptor-associated factor 6 (TRAF6) were all markedly up-regulated both in the renal tissues of rats with Thy-1N (in vivo) and in the GMCs upon sublytic C5b-9 stimulation (in vitro). Further in vitro experiments revealed that up-regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63-ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1-dependent RGC-32 up-regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1-binding element within RGC-32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC-32 genes respectively markedly reduced GMC proliferation and ECM production in Thy-1N rats. Together, these findings indicate that sublytic C5b-9 induces GMC proliferative changes in rat Thy-1N through ERK5/MZF1/RGC-32 axis activated by the FBXO28-TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.

The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future.