Regulation of Alternative Pathway Complement Activation by Glycosaminoglycans: Specificity of the Polyanion Binding Site on Factor H

ABSTRACT Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

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Modulation of complement activation on hemodialysis membranes by immobilized heparin.

Journal of the American Society of Nephrology ◽

10.1681/asn.v281328 ◽

1992 ◽

Vol 2 (8) ◽

pp. 1328-1337

Author(s):

A K Cheung ◽

C J Parker ◽

J Janatova ◽

E Brynda

Keyword(s):

Complement Activation ◽

Alternative Pathway ◽

Membrane Surface ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Fluid Phase ◽

Factor H ◽

Binding Studies ◽

Factor B ◽

Pathway Activity

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

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Control of Complement Activation by the Long Pentraxin PTX3: Implications in Age-Related Macular Degeneration

Frontiers in Pharmacology ◽

10.3389/fphar.2020.591908 ◽

2020 ◽

Vol 11 ◽

Author(s):

Matteo Stravalaci ◽

Francesca Davi ◽

Raffaella Parente ◽

Marco Gobbi ◽

Barbara Bottazzi ◽

...

Keyword(s):

Macular Degeneration ◽

Complement Activation ◽

Hot Spot ◽

Alternative Pathway ◽

Retinal Pigment ◽

Factor H ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Rpe Cells ◽

Age Related

Dysregulation of the complement system is central to age-related macular degeneration (AMD), the leading cause of blindness in the developed world. Most of the genetic variation associated with AMD resides in complement genes, with the greatest risk associated with polymorphisms in the complement factor H (CFH) gene; factor H (FH) is the major inhibitor of the alternative pathway (AP) of complement that specifically targets C3b and the AP C3 convertase. Long pentraxin 3 (PTX3) is a soluble pattern recognition molecule that has been proposed to inhibit AP activation via recruitment of FH. Although present in the human retina, if and how PTX3 plays a role in AMD is still unclear. In this work we demonstrated the presence of PTX3 in the human vitreous and studied the PTX3-FH-C3b crosstalk and its effects on complement activation in a model of retinal pigment epithelium (RPE). RPE cells cultured in inflammatory AMD-like conditions overexpressed the PTX3 protein, and up-regulated AP activating genes. PTX3 bound RPE cells in a physiological setting, however this interaction was reduced in inflammatory conditions, whereby PTX3 had no complement-inhibiting activity on inflamed RPE. However, on non-cellular surfaces, PTX3 formed a stable ternary complex with FH and C3b that acted as a “hot spot” for complement inhibition. Our findings suggest a protective role for PTX3 in response to complement dysregulation in AMD and point to a novel mechanism of complement regulation by this pentraxin with potential implications in pathology and pharmacology of AMD.

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Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb

Journal of Biological Chemistry ◽

10.1074/jbc.m116.719443 ◽

2016 ◽

Vol 291 (33) ◽

pp. 17122-17132 ◽

Cited By ~ 6

Author(s):

Xueqin Li ◽

Peng Liu ◽

Shuzhen Gan ◽

Chunmao Zhang ◽

Yuling Zheng ◽

...

Keyword(s):

Immune Complex ◽

Complement Activation ◽

Hydrophobic Interactions ◽

Cell Envelope ◽

Alternative Pathway ◽

Streptococcus Suis ◽

Factor H ◽

Immune Recognition ◽

Bacterial Surface ◽

Pathogen Protein

Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood.

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Amnion epithelial cells are an effective source of factor H and prevent kidney complement deposition in factor H-deficient mice

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02386-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Federica Casiraghi ◽

Pamela Yossenaidy Rodriguez Ordonez ◽

Nadia Azzollini ◽

Marta Todeschini ◽

Daniela Rottoli ◽

...

Keyword(s):

Kidney Transplantation ◽

Epithelial Cells ◽

Complement Activation ◽

Therapeutic Option ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Factor H ◽

Complement Factor ◽

Complement Deposition ◽

Amnion Epithelial Cells

AbstractComplement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh−/− mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh−/− mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh−/− mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.

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Complement Factor H Family Proteins Modulate Monocyte and Neutrophil Granulocyte Functions

Frontiers in Immunology ◽

10.3389/fimmu.2021.660852 ◽

2021 ◽

Vol 12 ◽

Author(s):

Éva Kárpáti ◽

Mariann Kremlitzka ◽

Noémi Sándor ◽

Dávid Hajnal ◽

Andrea E. Schneider ◽

...

Keyword(s):

Immune Cells ◽

Complement Activation ◽

Vaccine Development ◽

Alternative Pathway ◽

Factor H ◽

Neutrophil Granulocyte ◽

Complement Factor ◽

Heterogeneous Effects ◽

Enhanced Secretion ◽

Main Regulator

Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1β and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.

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Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.10.3982 ◽

1990 ◽

Vol 87 (10) ◽

pp. 3982-3986 ◽

Cited By ~ 258

Author(s):

S. Meri ◽

M. K. Pangburn

Keyword(s):

Sialic Acid ◽

Binding Site ◽

Alternative Pathway ◽

Factor H ◽

Complement Regulation ◽

Alternative Pathway Of Complement

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Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

Blood ◽

10.1182/blood.2020008248 ◽

2020 ◽

Vol 136 (18) ◽

pp. 2080-2089 ◽

Cited By ~ 13

Author(s):

Jia Yu ◽

Xuan Yuan ◽

Hang Chen ◽

Shruti Chaturvedi ◽

Evan M. Braunstein ◽

...

Keyword(s):

Complement Activation ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Factor H ◽

Spike Protein ◽

Target Cells ◽

Antiphospholipid Antibody ◽

Antiphospholipid Antibody Syndrome ◽

Complement Factor ◽

Spike Proteins

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

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Functional Analysis of the Human Antibody Response to Meningococcal Factor H Binding Protein

mBio ◽

10.1128/mbio.00842-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 13

Author(s):

Peter T. Beernink ◽

Serena Giuntini ◽

Isabella Costa ◽

Alexander H. Lucas ◽

Dan M. Granoff

Keyword(s):

Antibody Response ◽

Binding Site ◽

Bactericidal Activity ◽

Binding Protein ◽

Alternative Pathway ◽

Antibody Fragments ◽

Factor H ◽

Antibody Repertoire ◽

Human Antibody ◽

Protective Activity

ABSTRACTTwo licensed serogroup B meningococcal vaccines contain factor H binding protein (FHbp). The antigen specifically binds human FH, which downregulates complement. In wild-type mice whose mouse FH does not bind to FHbp vaccines, the serum anti-FHbp antibody response inhibited binding of human FH to FHbp. The inhibition was important for eliciting broad anti-FHbp serum bactericidal activity. In human FH transgenic mice and some nonhuman primates, FHbp was able to form a complex with FH and FHbp vaccination elicited anti-FHbp antibodies that did not inhibit FH binding. To investigate the human anti-FHbp repertoire, we cloned immunoglobulin heavy- and light-chain-variable-region genes of individual B cells from three adults immunized with FHbp vaccines and generated 10 sequence-distinct native anti-FHbp antibody fragments (Fabs). All 10 Fabs bound to live meningococci; only 1 slightly inhibited binding of human FH, while 4 enhanced FH binding. Affinity-purified anti-FHbp antibody from serum of a fourth immunized adult also enhanced binding of human FH to live meningococcal bacteria. Despite the bound FH, the affinity-purified serum anti-FHbp antibodies elicited human complement-mediated bactericidal activity that was amplified by the alternative pathway. The lack of FH inhibition by the human anti-FHbp Fabs and serum antibodies suggests that binding of human FH to the vaccine antigen skews the anti-FHbp antibody repertoire to epitopes outside the FH-binding site. Mutant FHbp vaccines with decreased FH binding may represent a means to redirect the human antibody repertoire to epitopes within the FH binding site, which can inhibit FH binding and, potentially, increase safety and protective activity.IMPORTANCETwo meningococcal vaccines contain factor H binding protein (FHbp). Immunized mice whose mouse factor H (FH) does not bind to FHbp develop serum anti-FHbp antibodies that block binding of human FH to the bacteria. With less bound FH, the bacteria become more susceptible to complement killing. To investigate human responses, we isolated 10 recombinant anti-FHbp antibody fragments (Fabs) from immune cells of three immunized adults. One slightly inhibited binding of FH to the bacteria, and four enhanced FH binding. Purified serum anti-FHbp antibodies from a fourth immunized adult also enhanced FH binding. Although bound FH would be expected to block the alternative pathway, the human anti-FHbp antibodies retained bactericidal activity and the ability to activate the alternative pathway. Mutant FHbp vaccines with decreased binding to human FH may redirect the human antibody repertoire to epitopes within the FH binding site that inhibit FH binding, which are expected to increase protective activity.

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