Complement C4A Regulates Autoreactive B Cells in Murine Lupus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus.

The mechanism and modulation of complement activation on polymer grafted cells.

Cell surface engineering using polymers is a promising approach to address unmet needs and adverse immune reactions in the fields of transfusion, transplantation, and cell-based therapies. Furthermore, cell surface modification may minimize or prevent adverse immune reactions to homologous incompatible cells as the interface between the host immune system and the cell surface is modified. In this report, we investigate the immune system reaction, precisely the complement binding and activation on cell surfaces modified with a functional polymer, hyperbranched polyglycerol (HPG). We used red blood cells (RBCs) as a model system to investigate the mechanism of complement activation on cell surfaces modified with various forms of HPG. Using a battery of in vitro assays including: traditional diagnostic hemolytic assays involving sheep and rabbit erythrocytes, ELISAs and flow cytometry, we show that HPG modified RBCs at certain concentrations and molecular weights activate complement via the alternative pathway. We show that by varying the grafting concentration, molecular weight and the number of cell surface reactive groups of HPG, the complement activity on the cell surface can be modulated. HPGs with molecular weights greater than 28kDa and grafting concentrations greater than 1.0mM, as well as a high degree of HPG functionalization with cell surface reactive groups result in the activation of the complement system via the alternative pathway. No complement activation observed when these threshold levels are not exceeded. These insights may have an impact on devising key strategies in developing novel next generation cell-surface engineered therapeutic products for applications in the fields of cell therapy, transfusion and drug delivery. STATEMENT OF SIGNIFICANCE: Cell-surface engineering using functional polymers is a fast emerging area of research. Importantly modified cells are used in many experimental therapeutics, transplantation and in transfusion. The success of such therapies depend on the ability of modified products to avoid immune detection and subsequent rejection or removal. Polymer grafting has been shown to modulate immune response, however, there is limited knowledge available. Thus in this manuscript, we investigated the interaction of human complement, part of our innate immune system, by polymer modified cells. Our results provide important evidences on the mechanism of complement activation by the modified cells and also found ways to modulate the innate immune response. These results will have implications in development of next generation cell-based therapies.

Selective enrichment and sensitive detection of peptide and protein biomarkers in human serum using polymeric reverse micelles and MALDI-MS.

Reverse-micelle forming amphiphilic homopolymers with carboxylic acid and quaternary amine substituents are used to selectively enrich biomarker peptides and protein fragments from human serum prior to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. After depletion of human serum albumin (HSA) and immunoglobulin G (IgG), low abundance peptide biomarkers can be selectively enriched and detected by MALDI-MS at clinically relevant concentrations by using the appropriate homopolymer(s) and extraction pH value(s). Three breast cancer peptide biomarkers, bradykinin, C4a, and ITIH(4), were chosen to test this new approach, and detection limits of 0.5 ng mL(-1), 0.08 ng mL(-1), and 0.2 ng mL(-1), respectively, were obtained. In addition, the amphiphilic homopolymers were used to detect prostate specific antigen (PSA) at concentrations as low as 0.5 ng mL(-1) by targeting a surrogate peptide fragment of this protein biomarker. Selective enrichment and sensitive MS detection of low abundance peptide/protein biomarkers by these polymeric reverse micelles should be a sensitive and straightforward approach for biomarker screening in human serum.

A review of the Chido/Rodgers blood group.

The C4 protein plays an important role in maintaining health and, in some situations complicated by poor expression of the C4 protein, may lead to or exacerbate certain diseases. The blood groups Chido and Rodgers are epitopes on the C4 protein, and polymorphisms associated with these epitopes may lead to the formation of antibodies to the Chido or Rodgers antigens in transfused patients. Identification of anti-Ch or anti-Rg is still based on the antibody neutralization with plasma from Ch-positive or Rg-positive individuals and lack of reactivity with qualified Ch-negative or Rg-negative RBCs. These antibodies may be useful in genetic studies of C4 polymorphisms or, in the case of C4-deficient patients, a signal of the potential for serious illnesses. The recognition of the extreme polymorphism of the C4 gene and the gene complex RCCX should lead to more insights in the understanding of disease risk and potential treatment.

Proteomic analysis of peritoneal dialysate fluid in patients with different types of peritoneal membranes.

Efficacy of peritoneal dialysis is determined by solute transport through peritoneal membranes. With the use of the peritoneal equilibration test (PET), peritoneal membranes can be classified as high (H), high average (HA), low average (LA), and low (L) transporters, based on the removal or transport rate of solutes, which are small molecules. Whether there is any difference in macromolecules (i.e., proteins) removed by different types of peritoneal membranes remains unclear. We performed a gel-based differential proteomics study of peritoneal dialysate effluents (PDE) obtained from chronic peritoneal dialysis (CPD) patients with H, HA, LA, and L transport rates (n=5 for each group; total n=20). Quantitative analysis and ANOVA with Tukey's posthoc multiple comparisons revealed five proteins whose abundance in PDE significantly differed among groups. These proteins were successfully identified by matrix-assisted laser desorption ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses, including serum albumin in a complex with myristic acid and triiodobenzoic acid, alpha1-antitrypsin, complement component C4A, immunoglobulin kappa light chain, and apolipoprotein A-I. The differences among groups in PDE levels of C4A and immunoglobulin kappa were clearly confirmed in a validation set of the other 24 patients (n=6 for each group) using ELISA. These data may lead to better understanding of the physiology of peritoneal membrane transport in CPD patients. Extending the study to a larger number of patients with subgroup analyses may yield additional information of the peritoneal dialysate proteins in association with dialysis adequacy, residual renal function, nutritional status, and risk of peritoneal infection.

[Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms].

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.

Preservation of antimicrobial properties of complement peptide C3a, from invertebrates to humans.

The human anaphylatoxin peptide C3a, generated during complement activation, exerts antimicrobial effects. Phylogenetic analysis, sequence analyses, and structural modeling studies paired with antimicrobial assays of peptides from known C3a sequences showed that, in particular in vertebrate C3a, crucial structural determinants governing antimicrobial activity have been conserved during the evolution of C3a. Thus, regions of the ancient C3a from Carcinoscorpius rotundicauda as well as corresponding parts of human C3a exhibited helical structures upon binding to bacterial lipopolysaccharide permeabilized liposomes and were antimicrobial against gram-negative and gram-positive bacteria. Human C3a and C4a (but not C5a) were antimicrobial, in concert with the separate evolutionary development of the chemotactic C5a. Thus, the results demonstrate that, notwithstanding a significant sequence variation, functional and structural constraints imposed on C3a during evolution have preserved critical properties governing antimicrobial activity.

Identification of plasma protein biomarkers associated with idiopathic pulmonary arterial hypertension.

We have employed SELDI-TOF MS to screen for differentially expressed proteins in plasma samples from 27 patients with idiopathic pulmonary arterial hypertension (IPAH) and 26 healthy controls. One ion (m/z approximately 8600) that was found to be elevated in IPAH was validated by SELDI-TOF MS analysis of a second and separate set of plasma samples comprising 30 IPAH patients and 19 controls. The m/z 8600 was purified from plasma by sequential ion exchange and reverse-phase chromatographies and SDS-PAGE. It was identified, following trypsin digestion, by MS peptide analysis as the complement component, complement 4a (C4a) des Arg. Plasma levels of C4a des Arg measured by ELISA confirmed that the levels were significantly higher (p < 0.0001) in IPAH patients (2.12 +/- 0.27 microg/mL) compared with normal controls (0.53 +/- 0.05 microg/mL). A cut-off level of 0.6 microg/mL correctly classified 92% of IPAH patients and 80% of controls. Further studies will be needed to determine its performance as a diagnostic biomarker, whether used alone or in combination with other biomarkers. Nevertheless, this study demonstrates that putative biomarkers characteristic of IPAH can be identified using a conjoint SELDI-TOF MS - proteomics approach.

Structural comparison of human C4A3 and C4B1 after proteolytic activation by C1s.

The fourth component of human complement is an essential part of the classical and lectin pathways performing multifunctional roles in both host defense and immune regulation. C4 is the most polymorphic member of the complement proteins, and complete deficiency is strongly associated with autoimmune disease, especially, systemic lupus erythematosus (SLE). Of the two C4 genes C4A, but not C4B, null alleles have been implicated as important independent disease susceptibility genes occurring in more than half of SLE patients. Whether and how this deficiency contributes to the development or pathology remains unclear. We do know that activation of C4 by C1s cleaves the thioester bond, thus inducing a conformational change that exposes numerous ligand-binding sites involved in functional activity. Structural comparison, among many other tools, plays an important role in predicting function. In this report, the tertiary structures of C4A and C4B were compared using near and far-UV circular dichroism, ANS fluorescence, site-specific monoclonal antibodies and isoelectric focusing. Negligible differences in the native proteins were found. However, the activated proteins were dissimilar in secondary and tertiary structure that was accompanied by significant differences in charge distribution and surface hydrophobicity. These conformational differences, together with known acceptor preferences, have functional implications for the association between C4A null alleles and SLE.

Purification and functional assessment of C3a, C4a and C5a of the common carp (Cyprinus carpio) complement.

Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5-1 nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs.

Genetic, structural and functional diversities of human complement components C4A and C4B and their mouse homologues, Slp and C4.

The complement protein C4 is a non-enzymatic component of the C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. The covalent binding of C4 to immunoglobulins and immune complexes (IC) also enhances the solubilization of immune aggregates, and the clearance of IC through complement receptor one (CR1) on erythrocytes. Human C4 is the most polymorphic protein of the complement system. In this review, we summarize the current concepts on the 1-2-3 loci model of C4A and C4B genes in the population, factors affecting the expression levels of C4 transcripts and proteins, and the structural, functional and serological diversities of the C4A and C4B proteins. The diversities and polymorphisms of the mouse homologues Slp and C4 proteins are described and contrasted with their human homologues. The human C4 genes are located in the MHC class III region on chromosome 6. Each human C4 gene consists of 41 exons coding for a 5.4-kb transcript. The long gene is 20.6 kb and the short gene is 14.2 kb. In the Caucasian population 55% of the MHC haplotypes have the 2-locus, C4A-C4B configurations and 45% have an unequal number of C4A and C4B genes. Moreover, three-quarters of C4 genes harbor the 6.4 kb endogenous retrovirus HERV-K(C4) in the intron 9 of the long genes. Duplication of a C4 gene always concurs with its adjacent genes RP, CYP21 and TNX, which together form a genetic unit termed an RCCX module. Monomodular, bimodular and trimodular RCCX structures with 1, 2 and 3 complement C4 genes have frequencies of 17%, 69% and 14%, respectively. Partial deficiencies of C4A and C4B, primarily due to the presence of monomodular haplotypes and homo-expression of C4A proteins from bimodular structures, have a combined frequency of 31.6%. Multiple structural isoforms of each C4A and C4B allotype exist in the circulation because of the imperfect and incomplete proteolytic processing of the precursor protein to form the beta-alpha-gamma structures. Immunofixation experiments of C4A and C4B demonstrate > 41 allotypes in the two classes of proteins. A compilation of polymorphic sites from limited C4 sequences revealed the presence of 24 polymophic residues, mostly clustered C-terminal to the thioester bond within the C4d region of the alpha-chain. The covalent binding affinities of the thioester carbonyl group of C4A and C4B appear to be modulated by four isotypic residues at positions 1101, 1102, 1105 and 1106. Site directed mutagenesis experiments revealed that D1106 is responsible for the effective binding of C4A to form amide bonds with immune aggregates or protein antigens, and H1106 of C4B catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens. The expression of C4 is inducible or enhanced by gamma-interferon. The liver is the main organ that synthesizes and secretes C4A and C4B to the circulation but there are many extra-hepatic sites producing moderate quantities of C4 for local defense. The plasma protein levels of C4A and C4B are mainly determined by the corresponding gene dosage. However, C4B proteins encoded by monomodular short genes may have relatively higher concentrations than those from long C4A genes. The 5' regulatory sequence of a C4 gene contains a Spl site, three E-boxes but no TATA box. The sequences beyond--1524 nt may be completely different as the C4 genes at RCCX module I have RPI-specific sequences, while those at Modules II, III and IV have TNXA-specific sequences. The remarkable genetic diversity of human C4A and C4B probably promotes the exchange of genetic information to create and maintain the quantitative and qualitative variations of C4A and C4B proteins in the population, as driven by the selection pressure against a great variety of microbes. An undesirable accompanying byproduct of this phenomenon is the inherent deleterious recombinations among the RCCX constituents leading to autoimmune and genetic disorders.

Complement factors and their receptors.

In summary, recent advances in molecular cloning of anaphylatoxins and the anaphylatoxin receptors add new dimensions to our investigations and understanding of the molecular mechanisms involved in anaphylatoxin action. Combining knowledge accumulated from peptide modeling of the ligands with mutagenesis studies of these ligands and their receptors makes it possible to more accurately model interactive sites and understand the sequence of molecular interactions required for cellular activation. In addition, these new developments provide valuable tools for investigating, yet unknown, activities and cellular targets of the anaphylatoxin molecules.

Evidence showing that the 1105 and 1106 isotypic residues of the fourth component of human complement, C4A, are not involved in amide bond formation.

Human C4A and C4B have different functions that may stem from their ability to bind hydroxyl or free amino groups on complement activating surfaces. Previous studies suggest that C4B binds to hydroxyl or amino groups whereas C4A binds to free amino groups on acceptor molecules. Comparison of the derived amino acid sequences of C4A and C4B has shown that differences exist between them at positions 1101, 1102, 1105 and 1106. These residues appear to be involved in the binding specificity of C4B. Less is known about the corresponding residues of C4A. It has been suggested that the aspartic acid of C4A at position 1106 is involved in amide bond formation by serving as a catalytic residue for the reaction or by promoting an increased interaction with amino nucleophilic groups. To examine the functional role of residues 1101-1106, we studied the effects of the C4A site-specific antipeptide mAb, AII-1 in assays dependent on the covalent binding properties of C4A; the C4 mediated inhibition of hemolysis and the C4 mediated inhibition of immune precipitation. This study shows that mAb AII-1 has no effect on C4-mediated hemolysis or its ability to inhibit the rate of immune precipitate formation. The lack of interference by AII-1 in these assays could not be explained by low affinity interaction between antibody and C4A showing that mAb AII-1 does not affect the covalent binding activity of C4A. Furthermore, results from epitope mapping studies show that AII-1 binds to Leu1105 and Asp1106 suggesting that these residues are not critical for amide bond formation by C4A.

Decreased plasma concentrations of the C4B complement protein in autism.

OBJECTIVE: To determine complement C4 protein concentrations in the plasmas of autistic subjects and their family members. DESIGN: Cross-sectional study. SETTING: Center for Persons with Disabilities and the Department of Biology, Utah State University, Logan. PARTICIPANTS: Forty-two autistic subjects (34 males [81%] and eight females [19%]), 50 of their biologic parents, 21 siblings, and 105 normal subjects (56 females [53%] and 49 males [47%]; all white) living in northern Utah. INTERVENTIONS: None. METHODS: The enzyme-linked immunosorbent assay was used to determine C4 protein concentrations in autistic subjects. MAIN RESULTS: Plasma concentration (median, 14.7 g/L of the C4B protein) in autistic patients was significantly (P = .01) decreased compared with that of normal subjects (median, 22.4 g/L). The C4B concentrations in parents and siblings of autistic children were decreased, but not significantly. The C4A protein concentrations in the plasma of autistic subjects and their family members were normal. CONCLUSION: Decreased protein concentrations of C4B may be associated with autism.

Proteins separated from human IgG molecules.

Immunoglobulin G binding proteins were separated from human IgG molecules using 1 N acetic acid followed by 5 M guanidinium chloride in 0.1 M acetic acid. The proteins thus obtained were heterogeneous as demonstrated by SDS-PAGE and reverse-phase HPLC. The isolated proteins consisted of two types: the C3a and C4a complement fragments (anaphylatoxins) and immunoglobulin peptide chain fragments V kappa I and C gamma 3. Both anaphylatoxins immobilized on cellulose nitrate membranes could reassociate with intact IgG molecules. The ubiquitous presence of C3a in IgG preparations was demonstrated using monoclonal antibodies specific for C3a. Nearly all of the bound anaphylatoxin molecules were found in the Fab fragment. These findings suggest that IgG molecules can eliminate anaphylatoxins from the circulation, and thus prevent harmful effects due to these active complement components.

Process-scale purification of immunoglobulin M concentrate.

An IgM concentrate was purified from Cohn fraction III. Efficiency of euglobin precipitation was shown to be controlled by pH and ionic strength. Prekallikrein activator activity in the product was insignificant. Overall yield from the octanoic acid supernate and purity of the concentrate were 66 +/- 8 (n = 16) and 50 +/- 5% (n = 16), respectively. Solvent-detergent treatment to inactivate lipid-enveloped viruses was demonstrated and implemented into the process. Process studies to control residual virucidal agents and C4a generating activity are presented.