Biophysical analysis of sialic acid recognition by the complement regulator Factor H.

Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.

Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.

Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein-protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which 'matchout labelled' proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system.

A method of purifying intact complement factor H from human plasma.

The aim of this study was to establish a method of purifying intact complement factor H (CFH) from human plasma. CFH was isolated from human plasma by polyethylene glycol (PEG) precipitation, following three sequential chromatographic columns, which consisted of l-lysine Sepharose column, Resource Q column and Sephacryl S-300 High Resolution HiPrep 16/60 column. All the above steps were performed at 4°C by Fast Protein Liquid Chromatography (FPLC) AKTA Purifier 10 with Frac-900. Identification of the purified CFH was confirmed by SDS-PAGE and Western blot. The following functions of the purified CFH were further analyzed compared with the commercial CFH in vitro: (1) binding ability with C3b; (2) binding ability with mCRP; (3) the protecting function of the hemolysis of sheep red blood cells; (4) the cofactor role for complement factor I-mediated proteolytic inactivation of C3b. Homogeneous CFH was purified from the plasma fraction through the above four steps. The purity and the functions of the purified CFH were comparable to the commercial CFH. The yield of CFH was 26±3% in our study. Compared with previous methods, our method was high yield with high purity. We established a stable and feasible system for purifying intact CFH, which could be used in the lab and clinical investigations.

Purification and biochemical characterization of functional complement factor H from human plasma fractions.

BACKGROUND AND OBJECTIVES: Complement factor H (CFH) acts as major regulator of the alternative pathway of complement and its mutations and polymorphisms predispose to various human diseases. We aimed to develop a scalable purification process of CFH from human plasma fractions to supply a pathogen-safe and functional CFH concentrate. MATERIALS AND METHODS: Starting with intermediates of cold ethanol fractionation of plasma, CFH purification was performed with three chromatographic steps including solvent detergent treatment and nanofiltration. CFH functionality was tested by a haemolysis assay using sheep erythrocytes, by determining decay acceleration activity on C3 convertases and cofactor activity of C3b cleavage. CFH identity was confirmed by Western blot and mass spectrometry. RESULTS: Three scalable chromatographic steps highly purified full-length and native CFH from human plasma fractions. The purification process enabled the removal of truncated and dysfunctional CFH species, yielding a native CFH concentrate as demonstrated in sensitive functional in vitro assays. CONCLUSION: This novel process provides a pathogen-safe and functional CFH concentrate that can be produced on an industrial scale and is suitable for pre-/clinical studies.

Factor H in porcine seminal plasma protects sperm against complement attack in genital tracts.

We found that factor H (FH) exists in porcine seminal plasma. Purified FH strongly inhibited serum alternative pathway complement activation against lipopolysaccharide. The molecular weight, pI, and heparin-binding activity of the purified protein were different from those of purified FH from porcine serum. The complement regulatory activity of seminal plasma FH was approximately 2-fold stronger than that of serum FH. Treatment of purified serum FH with sialidase and N-glycosidase F gave almost the same results as those of seminal plasma FH. The deletion of sialic acid from the carbohydrate chains of both FHs contributed to heparin-binding and complement regulatory activities. Results of reverse transcriptase-PCR, Western blot analysis, and immunohistochemistry showed that seminal plasma FH is mainly secreted from epithelial cells of the seminal vesicle in male genital tracts. FH was also detected in the outer acrosomal region of ejaculated sperm by immunofluorescence staining, and found that the purified FH from the sperm membrane has the same complement regulatory activity as that of seminal plasma FH. The ejaculated sperm possessing FH in the outer acrosomal region considerably evaded complement attack. We also found that there is strong complement activity in fluids from female genital tract ducts. These findings indicate that FH bound to the outer acrosomal region and soluble FH play important roles in protecting sperm against complement attack in male and female genital tracts.

Plasma kallikrein is activated on dermatan sulfate and cleaves factor H.

When human plasma is applied to a dermatan sulfate column, amidase activity is detected in the bound fraction and complement factor H is cleaved [A. Saito, H. Munakata, Factor H is a dermatan sulfate-binding protein: identification of a dermatan sulfate-mediated protease that cleaves factor H, J. Biochem. 137 (2005) 225-233]. Here, the amidase-active fraction was purified by sequential gel filtration and hydroxyapatite chromatography, and the amidase-active protein was identified to be plasma kallikrein by mass spectrometry. The activation of plasma kallikrein was further investigated by Western blotting using plasma deficient in prekallikrein or coagulation factor Xll. The dermatan sulfate column-bound fraction of the prekallikrein- and factor Xll-deficient plasmas did not show any amidase activity and factor H remained intact. Addition of kallikrein, but not activated factor Xll, to factor H purified from plasma resulted in cleavage of factor H. Thus, dermatan sulfate induces contact activation and activates kallikrein-mediated cleavage of FH.

Functional and structural implications of the complement factor H Y402H polymorphism associated with age-related macular degeneration.

PURPOSE: A Tyr-to-His (Y402H) sequence variant in the factor H (FH) and factor H-like protein (FHL-1) gene is strongly associated with an increased susceptibility for age-related macular degeneration (AMD). The purpose of this study was to understand how the Y402H variant in FH/FHL-1 contributes to the pathogenesis of AMD and, in particular, whether interactions mediated by FH/FHL-1, including binding to C-reactive protein (CRP), group A streptococcal M protein (GAS M6), heparin, and retinal pigment epithelial cells (RPE), are affected. METHODS: FH was purified from sera of patients homozygous for FH(Y402) or (H402), and recombinant FH fragments representing FHL-1 were generated. Proteins were analyzed for binding to CRP, GAS M6, heparin, and RPE cells. RESULTS: Binding of the FH and FH1 to seven polymorphic variants to CRP and M protein was reduced. The variant did not influence the interaction of FH with heparin but did reduce binding of FHL-1. Binding of the FH and FHL-1 polymorphic variant to RPE cells was not affected. CONCLUSIONS: The FH Y402H polymorphism associated with AMD causes a reduction in binding of FH and FHL-1 to CRP and M protein. Both variants show comparable binding to RPE cells, indicating that AMD is unlikely to manifest as a result of impaired host cell-surface recognition. The decreased interaction between FH and CRP, which is essential for the anti-inflammatory function of CRP, provides a possible pathophysiological explanation for the association of the Y402H variant with AMD.

Hereditary porcine membranoproliferative glomerulonephritis type II is caused by factor H deficiency.

We have recently described hereditary membranoproliferative glomerulonephritis type II in the pig. All affected animals had excessive complement activation, revealed as low plasma C3, elevated plasma terminal complement complex, and massive deposits of complement in the renal glomeruli, and eventually died of renal failure within 11 wk of birth. The aim of the present study was to investigate the cause of complement activation in this disease. Transfusion of normal porcine plasma to affected piglets inhibited complement activation and increased survival. Plasma was successively fractionated and the complement inhibitory effect of each fraction tested in vivo. A single chain 150-kD protein which showed the same complement inhibitory effect as whole plasma was finally isolated. Immunologic cross-reactivity, functional properties, and NH2-terminal sequence identified the protein as factor H. By Western blotting and enzyme immunoassay, membranoproliferative glomerulonephritis-affected piglets were demonstrated to be subtotally deficient in factor H. At 1 wk of age, median (range) factor H concentration was 1.6 mg/liter (1.1-2.3) in deficient animals (n = 13) and 51 mg/liter (26-98) in healthy littermates (n = 52). Our data show that hereditary porcine membrano-proliferative glomerulonephritis type II is caused by factor H deficiency.

Expression, purification, cocrystallization and preliminary crystallographic analysis of sucrose octasulfate/human complement regulator factor H SCRs 6-8.

Human plasma protein complement factor H (FH) is an inhibitor of the spontaneously activated alternative complement pathway. An allotypic variant of FH, 402His, has been associated with age-related macular degeneration, the leading cause of blindness in the elderly. Crystals of FH domains 6-8 (FH678) containing 402His have been grown in the presence of a polyanionic sucrose octasulfate ligand (an analogue of the natural glycosaminoglycan ligands of FH) using both native and selenomethionine-derivatized protein. Native data sets diffracting to 2.3 A and SeMet data sets of up to 2.8 A resolution have been collected. An anomalous difference Patterson map reveals self- and cross-peaks from two incorporated Se atoms. The corresponding selenium substructure has been solved.

Surface-attached PEO in the form of activated Pluronic with immobilized factor H reduces both coagulation and complement activation in a whole-blood model.

In the present work we have bound Pluronic, a class of triblock copolymers consisting of a block of polypropylene oxide (PPO) surrounded on each side by polyethylene oxide (PEO) blocks, to polystyrene surfaces and investigated the thrombogenicity and complement activation of this construct upon exposure to whole blood. The surface was highly inert towards coagulation, unfortunately at the expense of increased complement activation. We, therefore, as an alternative approach, used End-Group Activated Pluronic to conjugate factor H, a regulator of complement activation (RCA), to the surface. The bound factor H did not detach from the surface upon incubation with human serum. Furthermore, factor H bound in a physiological conformation could to a significant degree attenuate complement activation at the Pluronic surface. Thus, we have created a hybrid surface in which the coagulation-inert properties of the original Pluronic are supplemented with a specific complement-inhibitory effect. Medical device technology includes numerous potential applications for crosslinkers that are capable of specifically binding biomolecules to surfaces with retained activity. These applications include coupling of functional biomolecules to biomedical devices such as stents and grafts. The biomolecule may be an RCA, antibody, or other beneficial ligand.

Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms.

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test.

Factor H co-purifies with thrombospondin isolated from platelet secretate.

Thrombospondin is a trimeric glycoprotein that has several known functions, including roles in platelet aggregation, phagocytosis and an inhibitor of angiogenesis. Typically the molecule is isolated from platelet secretate by heparin affinity followed by sizing chromatography. In this study, purity is analysed by 7.5% SDS-PAGE under reducing conditions when thrombospondin monomers run as a band at around 180 kDa. Under nonreducing conditions of 7.5% SDS-PAGE, thrombospondin does not penetrate beyond the stacking gel; however, under these conditions a major contaminating band can be seen which, upon reduction, merges into the thrombospondin band. Further purification of this contaminating protein was achieved by DEAE chromatography and it was identified as Factor H by peptide sequencing and immunoblotting. Factor H function was demonstrated by the ability of the protein to function as a cofactor in the Factor-I-mediated cleavage of C3b. Since Factor H has several known functions, such contamination could confound functional studies of thrombospondin thus purified and a pre-elution step of the heparin affinity column is recommended.

Complement factor H or a related protein is a marker for transitional cell cancer of the bladder.

The BTAstat and BTA TRAK tests are new immunoassays that detect and measure an antigen in the urine of individuals diagnosed with bladder cancer. As described in this report, the monoclonal antibodies used in these kits were developed by immunizing mice with partially purified protein preparations derived from the urine of patients with bladder cancer. The antigen that is recognized by the monoclonal antibodies was purified from the urine of bladder cancer patients by immunoaffinity chromatography and identified as being either complement factor H (FH) or a closely related protein (CFHrp) by partial amino acid sequence analysis. Like serum FH, the urine antigen was demonstrated to have a complement factor C3b binding site and to accelerate the degradation of C3b in the presence of complement factor I. The culture supernatants from several human bladder, cervical, and renal cancer cell lines contained antigen as determined by immunoassay, and antigen affinity-purified from HeLaS3 culture media was shown to have FH activity. Moreover, the cell lines were shown to make products of the expected sizes by reverse transcription-PCR using FH-specific primers. In contrast, normal human epithelial keratinocytes, a myeloid leukemia cell line, and the colon cancer line LS174T were negative for production of a FH-like protein (CFHrp). We propose that the expression of proteins with FH-like activities may confer a selective growth advantage to cancer cells in vivo by decreasing complement activity, thus aiding their escape from lysis by immune surveillance. Identification of these proteins as cancer products also suggests avenues of chemotherapy or immunotherapy of some cancers.

Functional properties of the allotypes of mouse complement regulatory protein, factor H: difference of compatibility of each allotype with human factor I.

Three allotypes of mouse factor H, H.1, H.2, and H.3 were purified from the sera of mice with different factor H allotypes, and their functional properties were investigated. The three allotypes all bound to heparin, DNA, Con A, and methylamine-treated mouse C3 (C3(MA)mo) with similar affinities for each protein immobilized, showed identical mobilities on SDS-PAGE, and were reacted well with rabbit polyclonal antibody against H.1 and H.2. Factor I-cofactor activity of these factor H allotypes was measured using highly purified material of mouse, guinea-pig, and human origin. In a homologous system, these allotypes expressed indistinguishable mouse factor I (Imo)-cofactor activity for the cleavage of C3(MA)mo. Imo-cofactor activity was again indistinguishable in these allotypes when methylamine-treated human C3 (C3(MA)hu) or methylamine-treated guinea-pig C3 (C3(MA)gp) was substituted for the C3(MA)mo substrate. The cofactor activity of these factor H allotypes, however, was augmented 4-5 times if C3(MA)hu) was used instead of C3(MA)mo, and was barely detected if C3(MA)gp was employed. In contrast, differences in the potency of the cofactor activity for the three allotypes were revealed if human factor 1 (Ihu) was substituted for Imo: the order of the efficiency for the cleavage of C3(MA)hu was H.2 > H.1 = H.3. These results, taken together with the finding that the homologous combinations of mouse and human factors H and I expressed greater activity for the cleavage of C3(MA)hu than did the heterologous combinations of factor H and factor I, suggest that mouse factor H allotypes discriminate species of protease factor I but not those of substrate (C3(MA), and H.2 possesses the best compatibility for Ihu in C3(MA)hu inactivation.

Some patients with antiphospholipid syndrome express hitherto undescribed antibodies to cardiolipin-binding proteins.

Contrary to infective anticardiolipin (aCL) antibodies, autoimmune aCL antibodies react with phospholipids (PL) mainly via binding to the plasma glycoprotein cofactor beta2-Glycoprotein I (beta2GPI). While there is a well-documented link between the risk of thrombosis and the presence of beta2GPI-dependent anticardiolipin antibodies, the pathological impact of other antiphospholipid antibodies is less clear. By means of cardiolipin affinity-chromatography, we isolated and identified 3 CL-binding proteins, complement component C4, complement factor H and a kallikrein-sensitive glycoprotein, and tested for the presence of autoantibodies against these proteins in patients with antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and other autoimmune diseases. High titers of autoantibodies to C4 as compared to age- and sex-matched healthy controls were present in 3 of 26 patients with APS, and weak titers were found in 2 of 26 patients with SLE and in none of 26 patients with other autoimmune diseases. Autoantibodies to complement factor H were found in 4 APS, 3 SLE and none of the other autoimmune patients. Autoantibodies to kallikrein-sensitive glycoprotein were detected in 6 APS patients, 1 SLE patient, and 1 patient with another autoimmune disease. A close relationship between these antibodies was found, suggesting their origin from a common macromolecular complex. However, no relationship with anti-beta2GPI antibodies was found, with the three patients with higher levels of autoantibodies having a low titer of anti-beta2GPI antibodies. In conclusion, some patients with APS harbor circulating antibodies to other CL-binding proteins which might be useful to further characterize these patients.

Direct interaction of complement factor H with the C1 domain of HIV type 1 glycoprotein 120.

A protein that binds specifically to Env 105-119 (HEDIISLWDQSLKPC) was found in pools of normal human plasma when this peptide was used in affinity chromatography procedures. These samples represented the negative control in experiments aimed at the purification of putative human antibodies to the Env 105-119 region from AIDS sera. In this article we describe the biochemical characterization of this protein, which turned out to be complement factor H (CFH). We propose a functional role for this protein in the complex, early steps of CD4-dependent HIV infection.

Enhancement of complement-mediated lysis by a peptide derived from SCR 13 of complement factor H.

Complement factor H (fH) is an important regulator of complement activation. It contributes to protection of cells against homologous complement attack. In this study we tested the effect of fH-depletion of normal human serum (NHS) on lysis of antibody-coated sheep and human erythrocytes (EshA and EhuA). In the absence of fH, lysis of sensitised Esh and Ehu was clearly increased. Addition of fH to fH-depleted serum re-established protection of cells against complement similar to that seen with NHS. A fH-derived peptide (pepAred), covering the N-terminal half of SCR 13 in fH, was able to enhance complement-mediated lysis of EhuA significantly. However, the oxidised form of this peptide (pepAox) had no effect. Biotinylated pepAred, but not pepAox, was able to directly bind to cells. Additionally, pepAred competed with direct fH-cell interaction which was observable only after treatment of purified fH with mercaptoethanol. Only pepAred increased the amount of C3 fragments and reduced levels of fH detectable on cells as shown by FACS analysis and radio-immuno assay. Furthermore, fH and factor I (fI)-mediated cleavage of agarose bound C3b into iC3b was decreased in the presence of pepAred. These data indicate that a fH-derived peptide can enhance complement-mediated lysis. We will continue to investigate whether the use of a fH peptide can be exploited for therapeutical purposes.

Purification, quantification, and functional analysis of Complement Factor H.

Complement Factor H (FH) is an abundant, non-enzymic plasma/serum glycoprotein, which has a major role in regulating activation of the complement system. It can be purified from human plasma/serum by affinity chromatography, using a monoclonal anti-FH antibody as ligand. Other affinity chromatography ligands, including cardiolipin and trinitrophenyl-bovine serum albumin (TNP-BSA), can be used to purify human FH and also FH from a wide range of vertebrates, including mammals, birds, bony fish. Human FH protein concentration can be quantified by sandwich ELISA. The activity of FH is generally measured by assays which detect the cleavage, by complement factor I, of the complement protein C3b to form iC3b. Cleavage occurs only in the presence of a cofactor, and FH is one of a small number of cofactors for this reaction.

Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.