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Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis. Pilzer David,Fishelson Zvi International immunology The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis. 10.1093/intimm/dxh300
Extracellular Vesicle-Induced Classical Complement Activation Leads to Retinal Endothelial Cell Damage via MAC Deposition. Huang Chao,Fisher Kiera P,Hammer Sandra S,Busik Julia V International journal of molecular sciences Several studies have suggested that there is a link between membrane attack complex (MAC) deposition in the retina and the progression of diabetic retinopathy (DR). Our recent investigation demonstrated that circulating IgG-laden extracellular vesicles contribute to an increase in retinal vascular permeability in DR through activation of the complement system. However, the mechanism through which extracellular vesicle-induced complement activation contributes to retinal vascular cytolytic damage in DR is not well understood. In this study, we demonstrate that IgG-laden extracellular vesicles in rat plasma activate the classical complement pathway, and in vitro Streptozotocin (STZ)-induced rat diabetic plasma results in MAC deposition and cytolytic damage in human retinal endothelial cells (HRECs). Moreover, removal of the plasma extracellular vesicles reduced the MAC deposition and abrogated cytolytic damage seen in HRECs. Together, the results of this study demonstrate that complement activation by IgG-laden extracellular vesicles in plasma could lead to MAC deposition and contribute to endothelium damage and progression of DR. 10.3390/ijms21051693
Emission of membrane vesicles: roles in complement resistance, immunity and cancer. Pilzer David,Gasser Olivier,Moskovich Oren,Schifferli Jurg A,Fishelson Zvi Springer seminars in immunopathology Complement-mediated cell death is caused by C5b-9, the membrane attack complex (MAC) composed of the five complement proteins C5b, C6, C7, C8, and C9. Assembly of the C5b-9 complex initiates oligomerization of C9 and production of a transmembrane protein channel that inflicts damage to target cells. For protection, cells eliminate the MAC from their surface either by ectocytosis (direct emission of membrane vesicles) or by endocytosis (internalization). The process of ectosome release is rapid and involves cytosolic Ca(2+) and activation of protein kinases, such as protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK). Recently, the involvement of mortalin (also known as GRP75 and mitochondrial hsp70) in MAC elimination has been suggested. Extracellular application of antibodies directed to mortalin increases cell sensitivity to MAC-mediated lysis. Release of membrane vesicles is ubiquitous and enhanced in apoptotic or tumor cells and upon cell activation. Composition of the ectosomes (also often referred to as microparticles) membrane proteins and lipids appears to be different from those of the original plasma membrane, indicating involvement of a selective sorting process during ectosome formation. Exosomes (unlike ectosomes) are membrane vesicles generated by endocytosis, endosome sorting into perinuclear multivesicular bodies (MVB) and exocytosis of MVBs. Exosomes appear to be different in size and composition from ectosomes. Exosome-associated MAC has also been described. Although research on ectosomes and exosomes is still limited, physiological roles in coagulation, vascular functions, angiogenesis, wound healing and development have been attributed to these shed membrane vesicles. On the other hand, there are indications that elevated levels of ectosomes and exosomes may predispose to morbidity. Membrane vesicles released by cells exposed to complement MAC may play roles in health and disease beyond protection from cell death. 10.1007/s00281-005-0004-1
Complement-mediated release of fibroblast growth factor 2 from human RPE cells. Yang Ping,Neal Samantha E,Buehne Kristen L,Tewkesbury Grace M,Klingeborn Mikael,Yang Yae-Young,Baciu Peter,Jaffe Glenn J Experimental eye research PURPOSE:Complement activation is associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Fibroblast growth factor 2 (FGF2) and membrane attack complex (MAC) are present in eyes of patients with CNV. Herein, we investigated the effect of complement activation on FGF2 release in human retinal pigment epithelial (RPE) cells. METHODS:Cultured human RPE cells were primed with an anti-RPE antibody and then treated with C1q-depleted human serum in the presence or absence of Tec kinases inhibitor (LFM-A13). 38 cytokines/chemokines levels were measured by Luminex technology. Secretion of FGF2 and interleukin (IL)-6 was assessed by ELISA. Tec protein was measured by Western blot. mRNA expression of FGF2, chemokine (C-X-C motif) ligand 1 (CXCL-1), and family members of Tec kinases was evaluated by qPCR. Cell viability and MAC deposition were determined by WST-1 assay and flow cytometry, respectively. RESULTS:Complement activation caused increased FGF2 and IL-6 release. FGF2 was released when C6-depleted human serum was reconstituted with C6. Anti-C5 antibody significantly attenuated complement-mediated FGF2 release, but not IL-6. FGF2 mRNA levels were not affected, while CXCL-1 mRNA levels were increased by complement activation. FGF2-containing extracellular vesicles were detected in response to complement challenge. Tec mRNA and protein were expressed in RPE cells. In the presence of LFM-A13, secretion of FGF2, but not IL-6, and MAC deposition were significantly decreased and cell viability was significantly increased in complement-treated cells when compared to controls. CONCLUSIONS:Complement plays an important role to release FGF2 from RPE cells. Tec kinase is involved in MAC formation and complement-mediated FGF2 release. This information suggests a role for complement activation to mediate neovascularization in conditions such as AMD, and may elucidate potential therapeutic targets. 10.1016/j.exer.2021.108471
Quantitative Analysis of Complement Membrane Attack Complex Proteins Associated with Extracellular Vesicles. Proteomes Extracellular vesicles (EVs) represent a universal mechanism of intercellular communication in normal and pathological conditions. There are reports showing the presence of complement proteins in EV preparations, specifically those that can form a membrane attack complex (MAC). In the present work, we have used a quantitative mass spectrometry method that allows for the measurement of multiple targeted proteins in one experimental run. The quantification of MAC-forming proteins, namely C5b, C6, C7, C8, and C9, in highly purified EVs from normal human plasma revealed the presence of MAC proteins at approximately equal stoichiometry that does not fit the expected stoichiometry of preformed MAC. We concluded that while MAC proteins can be associated with EVs from normal plasma and presumably can be delivered to the recipient cells, there is no evidence that the EVs carry preformed MAC. 10.3390/proteomes12030021
Therapy-induced senescent cancer cells exhibit complement activation and increased complement regulatory protein expression. Immunology and cell biology Therapy-induced senescence (TIS) is a primary response to chemotherapy, contributing to untoward treatment outcomes such as evasion of immunosurveillance. Despite the established role of the complement system in the immune response to cancer, the role of complement in mediating the immune response against senescent tumor cells remains poorly understood. To explore this relationship, we exposed lung adenocarcinoma (A549), breast adenocarcinoma (MCF7) and pancreatic carcinoma (Panc-1) cell lines to sublethal doses of either etoposide or doxorubicin to trigger TIS. Identification of TIS was based on morphological changes, upregulation of the senescence-associated β-galactosidase, p21 induction and lamin B1 downregulation. Using immunofluorescence microscopy, quantitative PCR, ELISA of conditioned media and in silico analysis, we investigated complement activation, complement protein expression, C3 levels in the conditioned media of senescent cells and secreted complement proteins as part of the senescence-associated secretory phenotype (SASP), respectively. In cell lines undergoing TIS, complement-related changes included (i) activation of the terminal pathway, evidenced by the deposition of C5b-9 on senescent cells; (ii) an increase in the expression of CD59 and complement factor H and (iii) in A549 cells, an elevation in the expression of C3 with its secretion into the medium. In addition, increased C3 expression was observed in breast cancer samples expressing TIS hallmarks following exposure to neoadjuvant chemotherapy. In conclusion, TIS led to the activation of complement, upregulation of complement regulatory proteins and increased C3 expression. Complement appears to play a role in shaping the cancer microenvironment upon senescence induction. 10.1111/imcb.12727
CD59 double knockout mice express a CD59ba hybrid fusion protein that mediates insulin secretion. FASEB journal : official publication of the Federation of American Societies for Experimental Biology CD59 is a cell-surface inhibitor of the terminal step in the complement cascade. However, in addition to its complement inhibitory function, a non-canonical role of CD59 in pancreatic beta cells has been identified. Two recently discovered intracellular alternative splice forms of CD59, IRIS-1 and IRIS-2, are involved in insulin exocytosis through interactions with SNARE-complex components. In mice, the CD59 gene has undergone duplication and to further explore the role of CD59 in insulin secretion, blood glucose homeostasis was studied in a CD59 double knockout (CD59abKO) mouse model. However, no phenotypic deviation related to insulin secretion or blood glucose homeostasis was observed for the CD59abKO mice. Instead, a CD59ba hybrid transcript formed as a consequence of the mutation induced to generate the model was identified. This hybrid transcript is expressed in pancreatic islets of the CD59abKO mice and is comprised of the remaining exons of the two CD59 genes spliced together. Similar to canonical CD59, the CD59ba hybrid was found to be glycosylated and present on the cell surface when exogenously expressed in INS-1 832/13 cells. Furthermore, INS-1 832/13 cells over-expressing the mouse CD59ba hybrid retained normal insulin secretion following siRNA-mediated knockdown of canonical CD59. Hence, although the CD59ba hybrid has lost the complement inhibitory function, the intracellular insulin secretory function remains. These results provide further information concerning the structural requirements of CD59 in its intracellular role relative to its role as a complement inhibitor. It also highlights the importance of carefully assessing plausible consequences of induced mutations in research models. 10.1096/fj.202401808R
The role of complement inhibitors beyond controlling inflammation. Blom A M Journal of internal medicine The complement system is an arm of innate immunity that aids in the removal of pathogens and dying cells. Due to its harmful, pro-inflammatory potential, complement is controlled by several soluble and membrane-bound inhibitors. This family of complement regulators has been recently extended by the discovery of several new members, and it is becoming apparent that these proteins harbour additional functions. In this review, the current state of knowledge of the physiological functions of four complement regulators will be described: cartilage oligomeric matrix protein (COMP), CUB and sushi multiple domains 1 (CSMD1), sushi domain-containing protein 4 (SUSD4) and CD59. Complement activation is involved in both the development of and defence against cancer. COMP expression is pro-oncogenic, whereas CSMD1 and SUSD4 act as tumour suppressors. These effects may be related in part to the complex influence of complement on cancer but also depend on unrelated functions such as the protection of cells from endoplasmic reticulum stress conveyed by intracellular COMP. CD59 is the main inhibitor of the membrane attack complex, and its deficiency leads to complement attack on erythrocytes and severe haemolytic anaemia, which is now amenable to treatment with an inhibitor of C5 cleavage. Unexpectedly, the intracellular pool of CD59 is crucial for insulin secretion from pancreatic β-cells. This finding is one of several relating to the intracellular functions of complement proteins, which until recently were only considered to be present in the extracellular space. Understanding the alternative functions of complement inhibitors may unravel unexpected links between complement and other physiological systems, but is also important for better design of therapeutic complement inhibition. 10.1111/joim.12606
Targeting complement in therapy. Kirschfink M Immunological reviews With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane-bound complement regulators (decay-accelerating factor (CD55), membrane co-factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody-based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement-mediated tumor cell destruction.
Defining the CD59-C9 binding interaction. Huang Yuxiang,Qiao Fei,Abagyan Ruben,Hazard Starr,Tomlinson Stephen The Journal of biological chemistry CD59 is a membrane glycoprotein that regulates formation of the cytolytic membrane attack complex (MAC or C5b-9) on host cell membranes. It functions by binding to C8 (alpha chain) and C9 after their structural rearrangement during MAC assembly. Previous studies indicated that the CD59 binding site in C9 was located within a 25-residue disulfide-bonded loop, and in C8alpha was located within a 51-residue sequence that overlaps the CD59 binding region of C9. By peptide screens and the use of peptides in binding assays, functional assays, and computer modeling and docking studies, we have identified a 6-residue sequence of human C9, spanning residues 365-371, as the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. The data also indicate that both C8alpha and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59-peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational and modeling studies. 10.1074/jbc.M603690200