C1q Subunit Research Articles

Transplacental SARS-CoV-2 protein ORF8 binds to complement C1q to trigger fetal inflammation

Prenatal SARS-CoV-2 infection is associated with higher rates of pregnancy and birth complications, despite that vertical transmission rates are thought to be low. Here, multi-omics analyses of human placental tissues, cord tissues/plasma, and amniotic fluid from 23 COVID-19 mother-infant pairs revealed robust inflammatory responses in both maternal and fetal compartments. Pronounced expression of complement proteins (C1q, C3, C3b, C4, C5) and inflammatory cytokines (TNF, IL-1α, and IL-17A/E) was detected in the fetal compartment of COVID-19-affected pregnancies. While ~26% of fetal tissues were positive for SARS-CoV-2 RNA, more than 60% of fetal tissues contained SARS-CoV-2 ORF8 proteins, suggesting transplacental transfer of this viral accessory protein. ORF8-positive fetal compartments exhibited increased inflammation and complement activation compared to ORF8-negative COVID-19 pregnancies. In human placental trophoblasts in vitro, exogenous ORF8 exposure resulted in complement activation and inflammatory responses. Co-immunoprecipitation analysis demonstrated that ORF8 binds to C1q specifically by interacting with a 15-peptide region on ORF8 (C37-A51) and the globular domain of C1q subunit A. In conclusion, an ORF8-C1q-dependent complement activation pathway was identified in COVID-19-affected pregnancies, likely contributing to fetal inflammation independently of fetal virus exposure.

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20‐30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SignificanceOesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of ‘treatment response prediction assays’ for stratification of OAC patients prior to chemotherapy.

Isolevuglandins disrupt PU.1-mediated C1q expression and promote autoimmunity and hypertension in systemic lupus erythematosus.

We describe a mechanism responsible for systemic lupus erythematosus (SLE). In humans with SLE and in 2 SLE murine models, there was marked enrichment of isolevuglandin-adducted proteins (isoLG adducts) in monocytes and dendritic cells. We found that antibodies formed against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduced transcription of all C1q subunits. Treatment of SLE-prone mice with the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA) ameliorated parameters of autoimmunity, including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowered blood pressure, attenuated renal injury, and reduced inflammatory gene expression uniquely in C1q-expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.

Association of Complement and MAPK Activation With SARS-CoV-2–Associated Myocardial Inflammation

Myocardial injury is a common feature of patients with SARS-CoV-2 infection. However, the cardiac inflammatory processes associated with SARS-CoV-2 infection are not completely understood. To investigate the inflammatory cardiac phenotype associated with SARS-CoV-2 infection compared with viral myocarditis, immune-mediated myocarditis, and noninflammatory cardiomyopathy by integrating histologic, transcriptomic, and proteomic profiling. This case series was a cooperative study between the Ludwig Maximilian University Hospital Munich and the Cardiopathology Referral Center at the University of Tübingen in Germany. A cohort of 19 patients with suspected myocarditis was examined; of those, 5 patients were hospitalized with SARS-CoV-2 infection between March and May 2020. Cardiac tissue specimens from those 5 patients were compared with specimens from 5 patients with immune-mediated myocarditis, 4 patients with non-SARS-CoV-2 viral myocarditis, and 5 patients with noninflammatory cardiomyopathy, collected from January to August 2019. Endomyocardial biopsy. The inflammatory cardiac phenotypes were measured by immunohistologic analysis, RNA exome capture sequencing, and mass spectrometry-based proteomic analysis of endomyocardial biopsy specimens. Among 19 participants, the median age was 58 years (range, 37-76 years), and 15 individuals (79%) were male. Data on race and ethnicity were not collected. The abundance of CD163+ macrophages was generally higher in the cardiac tissue of patients with myocarditis, whereas lymphocyte counts were lower in the tissue of patients with SARS-CoV-2 infection vs patients with non-SARS-CoV-2 virus-associated and immune-mediated myocarditis. Among those with SARS-CoV-2 infection, components of the complement cascade, including C1q subunits (transcriptomic analysis: 2.5-fold to 3.6-fold increase; proteomic analysis: 2.0-fold to 3.4-fold increase) and serine/cysteine proteinase inhibitor clade G member 1 (transcriptomic analysis: 1.7-fold increase; proteomic analysis: 2.6-fold increase), belonged to the most commonly upregulated transcripts and differentially abundant proteins. In cardiac macrophages, the abundance of C1q was highest in SARS-CoV-2 infection. Assessment of important signaling cascades identified an upregulation of the serine/threonine mitogen-activated protein kinase pathways. This case series found that the cardiac immune signature varied in inflammatory conditions with different etiologic characteristics. Future studies are needed to examine the role of these immune pathways in myocardial inflammation.

Complement C1q subunit molecules from Xenopus laevis possess conserved function in C1q-immunoglobulin interaction

Complement component 1q (C1q), together with C1r and C1s to form C1, recognize and bind immune complex to initiate the classical complement pathway. In this study, C1q subunit molecules (XlC1qA, XlC1qB, XlC1qC) were cloned and analyzed from Xenopus laevis (X. laevis). The open reading frame (ORF) of XlC1qA is 819 bp of nucleotide sequence encoding 272 amino acids, the ORF of XlC1qB is 711 bp encoding 236 aa, and the XlC1qC is consists of 732 bp encoding 243 aa. The deduced amino acid sequences contain a collagen-like region (CLR), Gly-X-Y repeats in the N-terminus and a C1q family domain at the C-terminus. Phylogenetic analysis revealed that the XlC1qs are clustered with the amphibian clade. Expression analysis indicated that the XlC1qs exhibited constitutive expression in all examined tissues, with the highest expression in liver. Additionally, XlC1q could interact with heat-aggregated mouse IgG and IgM, Xenopus IgM and Nile tilapia IgM, respectively, indicating the functional conservation of XlC1q binding to immunoglobulins. Further, XlC1qs can inhibit C1q-dependent hemolysis of sensitized sheep red blood cells with concentration-dependent manner. These data collectively suggest that the function of C1qs in X. laevis may be conserved in interaction with immunoglobulins, as that of mammals and teleosts.

Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

SummaryBackgroundThe incidence of epidermal keratinocyte‐derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide.ObjectivesTo study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC.MethodsThe mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real‐time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s.ResultsSignificantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa‐associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal‐related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo.ConclusionsThese results provide evidence for the role of tumour‐cell‐derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally.Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

Modification of the plasma complement protein profile by exogenous estrogens is indicative of a compromised immune competence in marine medaka (Oryzias melastigma)

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming “feminised”. Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens.

Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Abstract 2842: A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adjustable cytokine activity.

Abstract The clinical use of interleukin 2 (IL2) for cancer immune therapy has been limited by a lack of tumor targeting that requires the administration of dose levels sufficient to activate anti-tumor effector cells in the tumor microenvironment. These doses, in turn, are limited by several side effects including vascular toxicities of hypotension and vascular leak syndrome. One way to increase the accumulation of IL2 at the tumor site is to genetically fuse it to the heavy chains of a tumor specific antibody, and several such molecules (immunocytokines or ICs) have been shown to have improved anti-tumor activity compared to free IL2, the targeting antibody, or the combination. This is especially true of ICs with longer circulating half lives that increase the amount of drug delivered to the tumor. Unfortunately, this increases the vascular exposure and toxicity of the IL2 component that, again, limits the amount that can be dosed and the rate of infusion. One way to reduce the toxicity of long-acting ICs is to dose them subcutaneously (greatly reducing Cmax levels in the blood) rather than intravenously. Another way is to make the IC more selective for the high affinity IL2 receptor (IL2Rhi) than the intermediate form (IL2Rint) by mutagenesis. Both approaches are based on the hypothesis that activating cells expressing IL2Rint should be limited to reduce toxicity, while activating cells expressing IL2Rhi should be maintained for anti-tumor activity. I have recently developed a novel method for constructing ICs that utilizes fusion of cytokines to the C terminus of the Ig light (L) chain, rather than the traditional method of fusing to the heavy (H) chain. The fusion junction of IL2, in this case, is located adjacent to the antibody hinge region, where the L chain forms a disulfide bond through its C-terminal cysteine residue to the H chain. This constrains both the N-terminal region of IL2, as well as the hinge-CH2 region of the H chain where both Fc receptors and the C1q subunit of complement bind when they mediate antibody-dependent cytotoxic activities (ADCC and CDC). Surprisingly, such ICs are expressed well in transfected cells, are very stable in normal buffers and have biological properties that are superior to ICs made by fusion to the H chain. These properties include longer circulating half life, increased uptake following subcutaneous dosing, and similar or improved antibody effector activities of ADCC and CDC, respectively. Furthermore, the sequestering effect of this fusion junction allows one to adjust IL2Rint binding and activation by shortening the N-terminus of IL2 at the fusion point. This appears to limit access of the critical contact residue Asp20 of IL2 to the β chain of IL2Rint, while maintaining binding and activation of IL2Rhi expressing cells as well as antibody effector functions. Citation Format: Stephen D. Gillies. A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adjustable cytokine activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2842. doi:10.1158/1538-7445.AM2013-2842

Atomic resolution model of the antibody Fc interaction with the complement C1q component

The globular C1q heterotrimer is a subunit of the C1 complement factor. Binding of the C1q subunit to the constant (Fc) part of antibody molecules is a first step and key event of complement activation. Although three-dimensional structures of C1q and antibody Fc subunits have been determined experimentally no atomic resolution structure of the C1q–Fc complex is known so far. Based on systematic protein–protein docking searches and Molecular Dynamics simulations a structural model of the C1q–IgG1–Fc-binding geometry has been obtained. The structural model is compatible with available experimental data on the interaction between the two partner proteins. It predicts a binding geometry that involves mainly the B-subunit of the C1q-trimer and both subunits of the IgG1–Fc-dimer with small conformational adjustments with respect to the unbound partners to achieve high surface complementarity. In addition to several charge–charge and polar contacts in the rim region of the interface it also involves nonpolar contacts between the two proteins and is compatible with the carbohydrate moiety of the Fc subunit. The model for the complex structure provides a working model for rationalizing available biochemical data on this important interaction and can form the basis for the design of Fc variants with a greater capacity to activate the complement system for example on binding to cancer cells or other target structures.

Enzymatically Modified Low-Density Lipoprotein Is Recognized by C1q and Activates the Classical Complement Pathway

Several studies suggest that the complement system is involved in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of LDL to bind and activate C1, the complex protease that triggers the classical pathway of complement. Unlike native LDL, oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were both recognized by the C1q subunit of C1, but only E-LDL particles, obtained by sequential treatment with a protease and then with cholesterol esterase, had the ability to trigger C1 activation. Further investigations revealed that C1q recognizes a lipid component of E-LDL. Several approaches, including reconstitution of model lipid vesicles, cosedimentation, and electron microscopy analyses, provided evidence that C1 binding to E-LDL particles is mediated by the C1q globular domain, which senses unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed.

Low-molecular-weight heparin inhibition in classical complement activaton pathway during pregnancy

Introduction Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications. Materials and Methods The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH 50 assay). Results The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03 ± 1.20 μg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 µg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG. Conclusions We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.

Inflammation and tissue repair markers distinguish the nodular sclerosis and mixed cellularity subtypes of classical Hodgkin's lymphoma

Background:Classical Hodgkin's lymphoma (cHL), although a malignant disease, has many features in common with an inflammatory condition. The aim of this study was to establish the molecular characteristics of the two most common cHL subtypes, nodular sclerosis (NS) and mixed cellularity (MC), based on molecular profiling and immunohistochemistry, with special reference to the inflammatory microenvironment.Methods:We analysed 44 gene expression profiles of cHL whole tumour tissues, 25 cases of NS and 19 cases of MC, using Affymetrix chip technology and immunohistochemistry.Results:In the NS subtype, 152 genes showed a significantly higher expression, including genes involved in extracellular matrix (ECM) remodelling and ECM deposition similar to wound healing. Among these were SPARC, CTSK and COLI. Immunohistochemistry revealed that the NS-related genes were mainly expressed by macrophages and fibroblasts. Fifty-three genes had a higher expression in the MC subtype, including several inflammation-related genes, such as C1Qα, C1Qβ and CXCL9. In MC tissues, the C1Q subunits were mainly expressed by infiltrating macrophages.Conclusions and interpretations:We suggest that the identified subtype-specific genes could reflect different phases of wound healing. Our study underlines the potential function of infiltrating macrophages in shaping the cHL tumour microenvironment.

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS–PAGE experiments.

Immune Function of C1q and Its Modulators CD91 and CD93

C1q is a subcomponent of the first component of complement C1, which is a multimolecular complex comprising one molecule of C1q and two molecules each of the autoreactive proteases, C1r and C1s. This multimolecular complex triggers the classical pathway of complement. Advances in the past several years have provided a partial crystal structure of the C1q subunit. This, together with gene deletion of C1q, has allowed further insight into the multifunctional immune aspects of this molecule. Two C1q-mediated functions that have received intense scrutiny recently are C1q-mediated apoptotic clearance of cell debris and phagocytosis. This has led to a heightened search for specific receptors for the collagen-like region (CLR) as well as the globular heads. Two transmembrane proteins, CD91 and CD93, have been proposed to interact indirectly with the CLR of C1q, promoting apoptotic clearance and phagocytosis, respectively. The aim of this article is to provide an overview of the structural and functional information that implicates CD91 and CD93 in C1q-mediated functional effects.

The mechanism of inhibition of the activation of the complement first component by polyanions and polycations

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.

Mapping of the interaction between the immunodominant loop of the ectodomain of HIV-1 gp41 and human complement protein C1q.

The human immunodeficiency virus type 1 transmembrane envelope glycoprotein gp41 has been previously shown to activate the C1 complex of human complement through direct interaction with its C1q subunit. The major interaction site has been located within the gp41 immunodominant region (residues 590-620), and a synthetic peptide overlapping residues 601-613 of gp41 (sequence GIWGCSGKLICTT) was shown to inhibit binding of gp41 to C1q in vitro (Thielens, N.M., Bally, I.M., Ebenbichler, C.F., Dierich, M.P. & Arlaud, G.J. (1993) J. Immunol. 151, 6583-6592). The ectodomain of gp41 (s-gp41) was secreted from the methylotrophic yeast Pichia pastoris and purified by immunoaffinity chromatography. Enzymatic deglycosylation of the recombinant s-gp41 was necessary to allow its in vitro interaction with C1q. A solid-phase competition assay was used to monitor the effect of mutant peptides derived from segment 601-613 of gp41 on the binding of deglycosylated s-gp41 to C1q. Whereas mutation of Ser606 had no effect, replacement of Ile602, Trp603, Lys608, Leu609 and Ile610 by Ala abolished the ability of the resulting peptides to inhibit binding of s-gp41 to C1q, suggesting that these residues participate in the interaction between gp41 and C1q. These findings are discussed in the light of a structural model of the immunodominant loop of gp41. It is proposed that the recognition of gp41 by C1q is driven by hydrophobic interactions, and that the sites of gp41 responsible for interaction with gp120 and C1q partly overlap.

Type XVII collagen and collagen-like molecules: related by more than a common motif

Abstract Historically, collagens have been defined as extracellular matrix structural proteins whose sequence is characterized by many repeats of a three amino acid unit, GLY-X-Y. However, the latter motif has been identified in certain domains of a number of other proteins including the C1q subunit of complement, the collectins, the asymmetric form of acetylcholinesterase, ficolin and the macrophage scavenger receptors. We describe these molecules and discuss both their structure and their putative functions. Intriguingly, these proteins show similarity to a 180 kD protein component of an epithelial cell/matrix anchorage device called the hemidesmosome. The latter protein is now considered a bona fide member of the collagen superfamily and has been termed type XVII collagen, despite the fact that it is a non-secreted transmembrane protein. We review recent studies on type XVII collagen which provide clues to its potential protein–protein interactions as well as its role in the pathogenesis of two human skin diseases.

Angioedema With Acquired Deficiency of the C Inhibitor: A Constellation of Syndromes

Patients with angioedema are often referred to an allergist to rule out an allergic cause. In most of these cases, no allergic cause is identified, and the cases are labeled "idiopathic." Occasionally, a deficiency of the inhibitor of the first component of complement (C1INH) is discovered, which may be either hereditary or acquired. In comparison with the hereditary variant, the acquired deficiency of C1INH is extremely rare, approximately 40 cases having been reported to date. Measurement of the C1q subunit is the key to the differential diagnosis between hereditary and acquired deficiencies of C1INH--it is normal in the former and decreased in the latter. The acquired deficiency of C1INH is usually found in association with benign or malignant B-cell lymphoproliferative disorders, and the angioedema responds to therapy with androgens. A subset of six patients with acquired C1INH deficiency has been reported recently; they had anti-C1INH autoantibodies, no evidence of an underlying disease, a benign course, and variable responses to therapy. Two new cases of angioedema in patients with acquired C1INH deficiency are described in this report. One patient had no evidence of an underlying disease 11 years after the onset of angioedema. The other case was associated with a B-cell lymphoproliferative disorder that became evident 9 months after C1INH deficiency was diagnosed, and androgen therapy stopped the attacks of angioedema. In this second case, the functional activity of C1INH mirrored the clinical response to therapy.

Subunit interactions in the first component of complement, C1

Interactions between C1q and other subunits of C1 were analyzed by sucrose gradient ultracentrifugation. A zone of dilute, radioiodine labelled C1q was sedimented through uniform concentrations of either C1r 2C1s 2, C1r 2, C1r 2 or C1s (2) . The dissociation constants were found to be 3×10 −9M and 6×10 −9M for C1r 2C1s 2 and C1r 2 binding respectively. Hill coefficients of 1 indicated no cooperativity in these bindings. Positive cooperativity was found in binding of C1s to C1q. Dissociation constants of 2×10 −6M and 5×10 −8M were obtained from computer modelling of a two step binding mechanism. No inter-action was detected between C1q and activated C1r 2 . The data indicate that most of the interactions between C1q and C1r 2C1s 2 originates from a strong binding to the C1r 2 moiety of the zymogen complex. This interaction is lost upon activation of C1r 2.