Molecular bases for the association of FHR-1 with atypical hemolytic uremic syndrome and other diseases.

Factor H (FH)-related proteins are a group of partly characterized complement proteins thought to promote complement activation by competing with FH in binding to surface-bound C3b. Among them, FH-related protein 1 (FHR-1) is remarkable because of its association with atypical hemolytic uremic syndrome (aHUS) and other important diseases. Using a combination of biochemical, immunological, nuclear magnetic resonance, and computational approaches, we characterized a series of FHR-1 mutants (including 2 associated with aHUS) and unraveled the molecular bases of the so-called deregulation activity of FHR-1. In contrast with FH, FHR-1 lacks the capacity to bind sialic acids, which prevents C3b-binding competition between FH and FHR-1 in host-cell surfaces. aHUS-associated FHR-1 mutants are pathogenic because they have acquired the capacity to bind sialic acids, which increases FHR-1 avidity for surface-bound C3-activated fragments and results in C3b-binding competition with FH. FHR-1 binds to native C3, in addition to C3b, iC3b, and C3dg. This unexpected finding suggests that the mechanism by which surface-bound FHR-1 promotes complement activation is the attraction of native C3 to the cell surface. Although C3b-binding competition with FH is limited to aHUS-associated mutants, all surface-bound FHR-1 promotes complement activation, which is delimited by the FHR-1/FH activity ratio. Our data indicate that FHR-1 deregulation activity is important to sustain complement activation and C3 deposition at complement-activating surfaces. They also support that abnormally elevated FHR-1/FH activity ratios would perpetuate pathological complement dysregulation at complement-activating surfaces, which may explain the association of FHR-1 quantitative variations with diseases.

Inhibition of the Complement Alternative Pathway by Chemically Modified DNA Aptamers That Bind with Picomolar Affinity to Factor B.

The complement system is a conserved component of innate immunity that fulfills diverse roles in defense and homeostasis. Inappropriate activation of complement contributes to many inflammatory diseases, however, which has led to a renewed emphasis on development of therapeutic complement inhibitors. Activation of complement component C3 is required for amplification of complement and is achieved through two multisubunit proteases called C3 convertases. Of these, the alternative pathway (AP) C3 convertase is responsible for a majority of the C3 activation products in vivo, which renders it an attractive target for inhibitor discovery. In this study, we report the identification and characterization of two related slow off-rate modified DNA aptamers (SOMAmer) reagents that inhibit formation of the AP C3 convertase by binding to the proprotease, factor B (FB). These aptamers, known as SL1102 (31 bases) and SL1103 (29 bases), contain uniform substitutions of 5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine for deoxythymidine. SL1102 and SL1103 bind FB with K d values of 49 and 88 pM, respectively, and inhibit activation of C3 and lysis of rabbit erythrocytes under AP-specific conditions. Cocrystal structures of SL1102 (3.4 Å) and SL1103 (3.1 Å) bound to human FB revealed that SL1102 and SL1103 recognize a site at the juncture of the CCP1, CCP3, and vWF domains of FB. Consistent with these structures and previously published information, these aptamers inhibited FB binding to C3b and blocked formation of the AP C3 convertase. Together, these results demonstrate potent AP inhibition by modified DNA aptamers and expand the pipeline of FB-binding molecules with favorable pharmacologic properties.

Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor.

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.

Substituting the Thiol Ester of Human A2M or C3 with a Disulfide Produces Native Proteins with Altered Proteolysis-Induced Conformational Changes.

Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H2O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.

Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.

Dysregulation of the complement system is involved in the pathogenesis of several diseases, and its inhibition has been shown to be a feasible therapeutic option. Therefore, there is an interest in the development of complement modulators to treat complement activation-related inflammatory pathologies. Mannose-binding lectin (MBL)/ficolin/collectin-associated protein-1 (MAP-1) is a regulatory molecule of the lectin pathway (LP), whereas complement receptor 1 (CD35) and decay-accelerating factor (CD55) are membrane-anchored regulators with effects on the central effector molecule C3. In this study, we developed 2 novel soluble chimeric inhibitors by fusing MAP-1 to the 3 first domains of CD35 (CD351-3) or the 4 domains of CD55 (CD551-4) to modulate the complement cascade at 2 different stages. The constructs showed biologic properties similar to those of the parent molecules. In functional complement activation assays, the constructs were very efficient in inhibiting LP activation at the level of C3 and in the formation of terminal complement complex. This activity was enhanced when coincubated with recombinant LP recognition molecules MBL and ficolin-3. Recombinant MAP-1 fusion proteins, combined with recombinant LP recognition molecules to target sites of inflammation, represent a novel and effective therapeutic approach involving the initiation and the central and terminal effector functions of the complement cascade.-Pérez-Alós, L., Bayarri-Olmos, R., Skjoedt, M.-O., Garred, P. Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.

Estimating Constraints for Protection Factors from HDX-MS Data.

Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level).

A potent complement factor C3-specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement.

The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance, and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with subnanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable with that of C3, and hC3Nb1 is shown to prevent proconvertase assembly, as well as binding of the C3 substrate to C3 convertases. Our crystal structure of the C3b-hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b, rationalizing its inhibition of factor I activity. Our results identify hC3Nb1 as a versatile, inexpensive, and powerful inhibitor of the alternative pathway in both human and murine in vitro model systems of complement activation.

Mutations in CPAMD8 Cause a Unique Form of Autosomal-Recessive Anterior Segment Dysgenesis.

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs∗23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD.

Molecular cloning of complement component C3 gene from pearl mussel, Hyriopsis cumingii and analysis of the gene expression in response to tissue transplantation.

Complement component C3 is well recognized as the central mediator of complement system, whose activation is responsible for the immune surveillance and elimination of non-self-antigens. In this study, C3 gene (HcC3) from a pearl making mussel, Hyriopsis cumingii, was successfully identified. The putative HcC3 possessed the canonical domains and highly conserved functional residues of C3 family members. In phylogenetic analysis, HcC3 was also clustered into C3 subfamily and separated from α2 macroglobulin clade. HcC3 gene was constitutively expressed in a wide range of tissues of pearl mussels, among which the immune-related tissues like hemocytes got highest expression. After allograft surgery of mantle tissues for aquaculture pearl production, the gene expression of HcC3 exhibited a rapid upregulation on day 1, dropped back on day 3, peaked the value on day 7, and restored to the level similar to control samples on day 14 after mantle allograft. The biphasic expression within the two weeks post the surgery suggests the important roles for HcC3 in alloimmune responses and an intricate complement activation mechanism in mollusks during tissue allograft.

A novel complement C3 like gene (Lv-C3L) from Litopenaeus vannamei with bacteriolytic and hemolytic activities and its role in antiviral immune response.

As a core component of the complement system, complement component 3 (C3) plays a central role in the opsonization of pathogens, immune defense and immune regulation in the mammalian for its activation is required to trigger classical as well as alternative complement pathways. However, the molecular mechanism underlying C3 activation in invertebrates remains unknown. Several C3 genes have been characterized in invertebrates but very few in crustacean. To understand the molecular characterization and immunological functions of shrimp C3, we characterized a novel complement C3 like gene (designated Lv-C3L) with full-length cDNA sequence identified from pacific white shrimp Litopenaeus vannamei in the present study. The full length cDNA of Lv-C3L sequence was 4769 bp (GenBank accession number: MH638255) containing a 4077 bp open reading frame (ORF), which encodes 1358 amino acids contained a putative signal peptide of 17 amino acids. Six model motifs of C3 were found in Lv-C3L including typical A2M domain, a highly conserved thioester region (GCGEQ) and proteolytic cleavage site of ANATO. In addition to typical conservative domains, Lv-C3L also contains a particular GLN-rich region which might be involved in the protein interaction and transcriptional activation. The transcripts of Lv-C3L were mainly detected in hemocytes and gill which might be involved in defense response. At 36 h post V.parahaemolyticus and B.thuringensis infection, the expression level of Lv-C3L gene in hemocytes were significantly upregulated. At 48 h and 72 h post WSSV infection, the expression level of Lv-C3L gene in hemocytes and gill were significantly upregulated. These results indicated that Lv-C3L gene play a pivotal role in innate immune responses to the WSSV and G+/G- bacterial infection. The obvious immune function of Lv-C3L was described as an effective membrane rupture in bacteriolytic and hemolytic activities on V.parahaemolyticus, V.anguillarum and rabbit erythrocytes. Combining with WSSV copy number, WSSV-VP28 gene expression profile and shrimp cumulative mortality analysis, RNAi knockdown of Lv-C3L gene could obviously promote the in vivo propagation of WSSV in shrimp. This is the first report in crustaceans that Lv-C3L, as a key complement like components, is involved in shrimp antiviral immune response. It is speculated that complicated complement response cascade may exist in shrimp. These results collectively indicated that the complement pathway in shrimp might play an important protective role against pathogenic infection and activation of complement pathway including C3 could restrict the propagation of WSSV.

Molecular characterization and expression analysis of complement component C3 in southern catfish (Silurus meridionalis) and a whole mount in situ hybridization study on its ontogeny.

The complement system plays an important role in protecting fish against attack by pathogens early in life. Complement component C3 is a central component in the complement system. The present work aimed to clone the full length C3 cDNA sequence of southern catfish (Silurus meridionalis), detect the tissue expression patterns of C3, investigate the ontogeny of C3 in embryo and larva, and assess the expression of C3 in response to pathogen infection. The full length C3 cDNA sequence of 5157 bp with an open reading frame (ORF) of 4938 bp was cloned from southern catfish. The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C3 was detected in liver, spleen, stomach, intestine, and head kidney with RT-PCR and in situ hybridization. Whole mount in situ hybridization results revealed that C3 was first expressed in the yolk syncytial layer at 34 h post fertilization (hpf), followed by the liver at 36 h post hatching (hph). When challenged with Aeromonas hydrophila, the transcripts of C3 showed a significant up-regulation in liver and spleen at 24 h. The results suggested that complement C3 played a key role in defense against invading pathogens in the early development stages of southern catfish. Therefore, these results provide important information to understand the functions of C3 during fish early development in Siluriformes.

Aliskiren inhibits renin-mediated complement activation.

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.

Molecular characterization and expression analysis of complement component 3 in dojo loach (Misgurnus anguillicaudatus).

The complement component 3 (C3) is a central component of complement system. All three pathways converge at formation of C3 convertases and share the terminal pathways of membrane attack complex (MAC) formation. In this study, three isoforms of C3 were discovered in Misgurnus anguillicaudatus, named "C3-1", "C3-2" and "C3-3", respectively. The full-length of C3-1 cDNA sequence was firstly identified and analyzed from dojo loach (Misgurnus anguillicaudatus). The Ma-C3-1 cDNA sequence comprised of 4509 bp encoding 1454 amino acids with a putative signal peptide of 20 amino acid residues. The deduced amino acid sequence showed that Ma-C3-1 has conserved residues and domain, which are known to be crucial for C3 function. Interestingly, an amino acid substitution of the highly conserved GCGEQ was discovered in Ma-C3-1. Phylogenetic analysis showed that Ma-C3-1 was closely related to Cyprinidae. The mRNA expression levels of three isoforms of C3 were detected in kidney, eye, spleen, gonad, heart, fin ray, gut, muscle, brain, gill, skin, blood and liver. The expression of Ma-C3-1 and Ma-C3-3 were mainly detected in liver, followed by spleen, gonad. However, the high expression of Ma-C3-2 was found in kidney, followed by blood and gonad. The morphological changes of gill and skin, and the expression pattern of these three isoforms C3 molecular following the infection with Aeromonas hydrophila were investigated. The mRNA expression levels of three C3 isoforms were up-regulated in the gill, skin, liver and spleen after infection with A.hydrophila. Similarly, challenge experiments resulted in significant up-regulated expression of other complement-relevant genes in gill, liver and skin, such as C4, C5, C8b, especially at 24 h and 36 h. These results suggest that complement system might play an important role not only in liver, but also in the mucosal tissues as gill and skin of teleost fish.

Molecular characterization of complement component 3 (C3) in Mytilus coruscus improves our understanding of bivalve complement system.

Complement component 3 (C3) plays a central role in the complement system whose activation is essential for all the important functions performed by this system. Here, a novel C3 gene, termed Mc-C3, was identified from thick shell mussel (Mytilus coruscus). The deduced Mc-C3 protein possessed the characteristic structure features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Mc-C3 gene constitutively expressed in all examined tissues and predominantly expressed in immune-related tissues such as gills, hemocytes and hepatopancreas. After stimulation with lipopolysaccharide or Cu2+, the expression of Mc-C3 was significantly induced in gills. Further luciferase reporter assays showed the ability for activation of NF-κB signaling transduction of Mc-C3a. Taken together, these results show that C3 may play an essential role in the immune defense of M. coruscus. The present data therefore provide a more detailed insight into the functional activities of the bivalve complement system.

Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling.

The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation.

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

The solvent at antigen-binding site regulated C3d-CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation.

BACKGROUND: The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 play important links between the innate and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines and inhibitors, a number of studies have been performed to investigate C3d-CR2 interaction. Many studies have indicated C3d-CR2 interactions are ionic strength-dependent. METHODS: To investigate the molecular mechanism of C3d-CR2 interaction and the origin of effects of ionic strength, molecular dynamics simulations for C3d-CR2 complex together with the energetic and structural analysis were performed. RESULTS: Our results revealed the increased interactions between charged protein and ions weaken C3d-CR2 association, as ionic strengths increase. Moreover, ion strengths have similar effects on antigen-binding site and CR2 binding site. Meanwhile, Ala17 and Gln20 will transform between the activated and non-activated states mediated by His133 and Glu135 at different ion strengths. CONCLUSIONS: Our results reveal the origins of the effects of ionic strengths on C3d-CR2 interactions are due to the changes of water, ion occupancies and distributions. GENERAL SIGNIFICANCE: This study uncovers the origin of the effect of ionic strength on C3d-CR2 interaction and deepens the understanding of the molecular mechanism of their interaction, which is valuable for the design of vaccines and small molecule inhibitors.

Rare loss-of-function mutation in complement component C3 provides insight into molecular and pathophysiological determinants of complement activity.

The plasma protein C3 is a central element in the activation and effector functions of the complement system. A hereditary dysfunction of C3 that prevents complement activation via the alternative pathway (AP) was described previously in a Swedish family, but its genetic cause and molecular consequences have remained elusive. In this study, we provide these missing links by pinpointing the dysfunction to a point mutation in the ß-chain of C3 (c.1180T > C; p.Met(373)Thr). In the patient's plasma, AP activity was completely abolished and could only be reconstituted with the addition of normal C3. The M373T mutation was localized to the macroglobulin domain 4 of C3, which contains a binding site for the complement inhibitor compstatin and is considered critical for the interaction of C3 with the AP C3 convertase. Structural analyses suggested that the mutation disturbs the integrity of macroglobulin domain 4 and induces conformational changes that propagate into adjacent regions. Indeed, C3 M373T showed an altered binding pattern for compstatin and surface-bound C3b, and the presence of Thr(373) in either the C3 substrate or convertase-affiliated C3b impaired C3 activation and opsonization. In contrast to known gain-of-function mutations in C3, patients affected by this loss-of-function mutation did not develop familial disease, but rather showed diverse and mostly episodic symptoms. Our study therefore reveals the molecular mechanism of a relevant loss-of-function mutation in C3 and provides insight into the function of the C3 convertase, the differential involvement of C3 activity in clinical conditions, and some potential implications of therapeutic complement inhibition.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Neisseria meningitidis NalP cleaves human complement C3, facilitating degradation of C3b and survival in human serum.

The complement system is a crucial component of the innate immune response against invading bacterial pathogens. The human pathogen Neisseria meningitidis (Nm) is known to possess several mechanisms to evade the complement system, including binding to complement inhibitors. In this study, we describe an additional mechanism used by Nm to evade the complement system and survive in human blood. Using an isogenic NalP deletion mutant and NalP complementing strains, we show that the autotransporter protease NalP cleaves C3, the central component of the complement cascade. The cleavage occurs 4 aa upstream from the natural C3 cleavage site and produces shorter C3a-like and longer C3b-like fragments. The C3b-like fragment is degraded in the presence of the complement regulators (factors H and I), and this degradation results in lower deposition of C3b on the bacterial surface. We conclude that NalP is an important factor to increase the survival of Nm in human serum.