Identification of novel and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries.

In vitro screening of large compound libraries with automated high-throughput screening is expensive and time-consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECLs) can be rapidly performed with routine equipment available in most laboratories. In this study, we identified novel inhibitors of SARS-CoV-2 main protease (Mpro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of Mpro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 (IC50 = 30 nM), proving that the rapid exploration of large chemical spaces enabled by DECL technology allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. As demonstrated further by x-ray crystallography, SLL11 was found to adopt a highly unique U-shaped binding conformation, which allows the N-terminal electrophilic group to loop back to the S1' subsite while the C-terminal amino acid sits in the S1 subsite. MP1, a close analog of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC50 = 2.3 µM) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds will be explored in the future to improve their antiviral activity.

RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo.

Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins overexpressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of α-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.

Membrane-type matrix metalloproteases as diverse effectors of cancer progression.

Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Human monoclonal Fab and human plasma antibodies to carbamyl-epitopes cross-react with malondialdehyde-adducts.

Oxidized low-density lipoprotein (OxLDL) plays a crucial role in the development of atherosclerosis. Carbamylated LDL has been suggested to promote atherogenesis in patients with chronic kidney disease. Here we observed that plasma IgG and IgM antibodies to carbamylated epitopes were associated with IgG and IgM antibodies to oxidation-specific epitopes (ρ = 0·65-0·86, P < 0·001) in healthy adults, suggesting a cross-reaction between antibodies recognizing carbamyl-epitopes and malondialdehyde (MDA)/malondialdehyde acetaldehyde (MAA) -adducts. We used a phage display technique to clone a human Fab antibody that bound to carbamylated LDL and other carbamylated proteins. Anti-carbamyl-Fab (Fab106) cross-reacted with oxidation-specific epitopes, especially with MDA-LDL and MAA-LDL. We showed that Fab106 bound to apoptotic Jurkat cells known to contain these oxidation-specific epitopes, and the binding was competed with soluble carbamylated and MDA-/MAA-modified LDL and BSA. In addition, Fab106 was able to block the uptake of carbamyl-LDL and MDA-LDL by macrophages and stained mouse atherosclerotic lesions. The observed cross-reaction between carbamylated and MDA-/MAA-modified LDL and its contribution to enhanced atherogenesis in uraemic patients require further investigation.

Natural antibodies of newborns recognize oxidative stress-related malondialdehyde acetaldehyde adducts on apoptotic cells and atherosclerotic plaques.

Malondialdehyde acetaldehyde (MAA) adducts are generated under oxidative stress and shown to be highly immunogenic. Our aim was to investigate the recognition of MAA adducts by human natural antibodies in newborns before or at the time of full-term pregnancy. Plasma samples of pre-term (n = 11) and full-term (n = 36) newborns were enriched in specific IgM binding to MAA adducts compared with the maternal plasma IgM levels. Umbilical cord blood lymphocyte phage display library was generated to clone Fabs that specifically recognized MAA adducts without cross-reactivity to malondialdehyde. Fab clones from the antibody libraries of the pre-term and full-term newborns showed high sequence homology to the germline genes encoding the variable regions of antibodies, confirming that these Fabs represented the natural antibody repertoire of human fetuses. The MAA-specific umbilical cord blood Fabs bound to apoptotic human endothelial cells and the binding was efficiently competed with MAA adducts. The MAA-specific Fabs also recognized epitopes on advanced atherosclerotic lesions, and the uptake of infrared (IR)-labeled MAA-low-density lipoprotein by mouse J774A.1 macrophages was significantly reduced in the presence of these Fabs. In conclusion, MAA adducts were identified as one of the major antigenic targets for human natural antibodies already before the time of birth. MAA-specific natural antibodies are suggested to regulate apoptotic cell clearance starting from fetal development and to participate in the immunomodulation of atherosclerosis development during adulthood.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping.

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

An Approach for Systems-Level Understanding of Prostate Cancer from High-Throughput Data Integration to Pathway Modeling and Simulation.

To understand complex diseases, high-throughput data are generated at large and multiple levels. However, extracting meaningful information from large datasets for comprehensive understanding of cell phenotypes and disease pathophysiology remains a major challenge. Despite tremendous advances in understanding molecular mechanisms of cancer and its progression, current knowledge appears discrete and fragmented. In order to render this wealth of data more integrated and thus informative, we have developed a GECIP toolbox to investigate the crosstalk and the responsible genes'/proteins' connectivity of enriched pathways from gene expression data. To implement this toolbox, we used mainly gene expression datasets of prostate cancer, and the three datasets were GSE17951, GSE8218, and GSE1431. The raw samples were processed for normalization, prediction of differentially expressed genes, and the prediction of enriched pathways for the differentially expressed genes. The enriched pathways have been processed for crosstalk degree calculations for which number connections per gene, the frequency of genes in the pathways, sharing frequency, and the connectivity have been used. For network prediction, protein-protein interaction network database FunCoup2.0 was used, and cytoscape software was used for the network visualization. In our results, we found that there were enriched pathways 27, 45, and 22 for GSE17951, GSE8218, and GSE1431, respectively, and 11 pathways in common between all of them. From the crosstalk results, we observe that focal adhesion and PI3K pathways, both experimentally proven central for cellular output upon perturbation of numerous individual/distinct signaling pathways, displayed highest crosstalk degree. Moreover, we also observe that there were more critical pathways which appear to be highly significant, and these pathways are HIF1a, hippo, AMPK, and Ras. In terms of the pathways' components, GSK3B, YWHAE, HIF1A, ATP1A3, and PRKCA are shared between the aforementioned pathways and have higher connectivity with the pathways and the other pathway components. Finally, we conclude that the focal adhesion and PI3K pathways are the most critical pathways, and since for many other pathways, high-rank enrichment did not translate to high crosstalk degree, the global impact of one pathway on others appears distinct from enrichment.

Co-evolution of matrisome and adaptive adhesion dynamics drives ovarian cancer chemoresistance.

Due to its dynamic nature, the evolution of cancer cell-extracellular matrix (ECM) crosstalk, critically affecting metastasis and treatment resistance, remains elusive. Our results show that platinum-chemotherapy itself enhances resistance by progressively changing the cancer cell-intrinsic adhesion signaling and cell-surrounding ECM. Examining ovarian high-grade serous carcinoma (HGSC) transcriptome and histology, we describe the fibrotic ECM heterogeneity at primary tumors and distinct metastatic sites, prior and after chemotherapy. Using cell models from systematic ECM screen to collagen-based 2D and 3D cultures, we demonstrate that both specific ECM substrates and stiffness increase resistance to platinum-mediated, apoptosis-inducing DNA damage via FAK and ß1 integrin-pMLC-YAP signaling. Among such substrates around metastatic HGSCs, COL6 was upregulated by chemotherapy and enhanced the resistance of relapse, but not treatment-naïve, HGSC organoids. These results identify matrix adhesion as an adaptive response, driving HGSC aggressiveness via co-evolving ECM composition and sensing, suggesting stromal and tumor strategies for ECM pathway targeting.

Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer.

Metastatic cancers commonly activate adaptive chemotherapy resistance, attributed to both microenvironment-dependent phenotypic plasticity and genetic characteristics of cancer cells. However, the contribution of chemotherapy itself to the non-genetic resistance mechanisms was long neglected. Using high-grade serous ovarian cancer (HGSC) patient material and cell lines, we describe here an unexpectedly robust cisplatin and carboplatin chemotherapy-induced ERK1/2-RSK1/2-EphA2-GPRC5A signaling switch associated with cancer cell intrinsic and acquired chemoresistance. Mechanistically, pharmacological inhibition or knockdown of RSK1/2 prevented oncogenic EphA2-S897 phosphorylation and EphA2-GPRC5A co-regulation, thereby facilitating a signaling shift to the canonical tumor-suppressive tyrosine phosphorylation and consequent downregulation of EphA2. In combination with platinum, RSK inhibitors effectively sensitized even the most platinum-resistant EphA2high , GPRC5Ahigh cells to the therapy-induced apoptosis. In HGSC patient tumors, this orphan receptor GPRC5A was expressed exclusively in cancer cells and associated with chemotherapy resistance and poor survival. Our results reveal a kinase signaling pathway uniquely activated by platinum to elicit adaptive resistance. They further identify GPRC5A as a marker for abysmal HGSC outcome and putative vulnerability of the chemo-resistant cells to RSK1/2-EphA2-pS897 pathway inhibition.

Cerebrospinal fluid antibodies to oxidized LDL are increased in Alzheimer's disease.

Lipoprotein oxidation may play an important role in the pathogenesis of Alzheimer's Disease (AD), and therefore, we investigated cerebrospinal fluid (CSF) antibodies to oxidized low-density lipoprotein (OxLDL) in patients with AD and other neurodegenerative dementias. IgM and IgG antibody titers to OxLDL were measured in 50 CSF samples and 11 plasma samples using chemiluminescent ELISA. All CSF samples contained IgG antibodies, and also most IgM, binding to OxLDL. CSF antibodies to OxLDL were not related to CSF protein or albumin concentrations or plasma antibodies to OxLDL. Competition immunoassay for specificity demonstrated that about 50% of the CSF IgG binding to OxLDL was inhibited by soluble OxLDL. CSF IgG antibodies to OxLDL were significantly increased in AD patients compared to controls and to patients with frontotemporal lobar degeneration. The role of these antibodies in CSF is unknown and further investigations are needed.

FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis.

Cancer cells balance with the equilibrium of cell death and growth to expand and metastasize. The activity of mammalian sterile20-like kinases (MST1/2) has been linked to apoptosis and tumor suppression via YAP/Hippo pathway-independent and -dependent mechanisms. Using a kinase substrate screen, we identified here MST1 and MST2 among the top substrates for fibroblast growth factor receptor 4 (FGFR4). In COS-1 cells, MST1 was phosphorylated at Y433 residue in an FGFR4 kinase activity-dependent manner, as assessed by mass spectrometry. Blockade of this phosphorylation by Y433F mutation induced MST1 activation, as indicated by increased threonine phosphorylation of MST1/2, and the downstream substrate MOB1, in FGFR4-overexpressing T47D and MDA-MB-231 breast cancer cells. Importantly, the specific knockdown or short-term inhibition of FGFR4 in endogenous models of human HER2+ breast cancer cells likewise led to increased MST1/2 activation, in conjunction with enhanced MST1 nuclear localization and generation of N-terminal cleaved and autophosphorylated MST1. Unexpectedly, MST2 was also essential for this MST1/N activation and coincident apoptosis induction, although these two kinases, as well as YAP, were differentially regulated in the breast cancer models analyzed. Moreover, pharmacological FGFR4 inhibition specifically sensitized the HER2+ MDA-MB-453 breast cancer cells, not only to HER2/EGFR and AKT/mTOR inhibitors, but also to clinically relevant apoptosis modulators. In TCGA cohort, FGFR4 overexpression correlated with abysmal HER2+ breast carcinoma patient outcome. Therefore, our results uncover a clinically relevant, targetable mechanism of FGFR4 oncogenic activity via suppression of the stress-associated MST1/2-induced apoptosis machinery in tumor cells with prominent HER/ERBB and FGFR4 signaling-driven proliferation.

Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilization.

The current clinical care of glioblastomas leaves behind invasive, radio- and chemo-resistant cells. We recently identified mammary-derived growth inhibitor (MDGI/FABP3) as a biomarker for invasive gliomas. Here, we demonstrate a novel function for MDGI in the maintenance of lysosomal membrane integrity, thus rendering invasive glioma cells unexpectedly vulnerable to lysosomal membrane destabilization. MDGI silencing impaired trafficking of polyunsaturated fatty acids into cells resulting in significant alterations in the lipid composition of lysosomal membranes, and subsequent death of the patient-derived glioma cells via lysosomal membrane permeabilization (LMP). In a preclinical model, treatment of glioma-bearing mice with an antihistaminergic LMP-inducing drug efficiently eradicated invasive glioma cells and secondary tumours within the brain. This unexpected fragility of the aggressive infiltrating cells to LMP provides new opportunities for clinical interventions, such as re-positioning of an established antihistamine drug, to eradicate the inoperable, invasive, and chemo-resistant glioma cells from sustaining disease progression and recurrence.

Immunization with gingipain A hemagglutinin domain of Porphyromonas gingivalis induces IgM antibodies binding to malondialdehyde-acetaldehyde modified low-density lipoprotein.

Treatment of periodontitis has beneficial effects on systemic inflammation markers that relate to progression of atherosclerosis. We aimed to investigate whether immunization with A hemagglutinin domain (Rgp44) of Porphyromonas gingivalis (Pg), a major etiologic agent of periodontitis, would lead to an antibody response cross-reacting with oxidized low-density lipoprotein (OxLDL) and how it would affect the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLR-/-) mice. The data revealed a prominent IgM but not IgG response to malondialdehyde-acetaldehyde modified LDL (MAA-LDL) after Rgp44 and Pg immunizations, implying that Rgp44/Pg and MAA adducts may share cross-reactive epitopes that prompt IgM antibody production and consequently confer atheroprotection. A significant negative association was observed between atherosclerotic lesion and plasma IgA to Rgp44 in Rgp44 immunized mice, supporting further the anti-atherogenic effect of Rgp44 immunization. Plasma IgA levels to Rgp44 and to Pg in both Rgp44- and Pg-immunized mice were significantly higher than those in saline control, suggesting that IgA to Rgp44 could be a surrogate marker of immunization in Pg-immunized mice. Distinct antibody responses in plasma IgA levels to MAA-LDL, to Pg lipopolysaccharides (Pg-LPS), and to phosphocholine (PCho) were observed after Rgp44 and Pg immunizations, indicating that different immunogenic components between Rpg44 and Pg may behave differently in regard of their roles in the development of atherosclerosis. Immunization with Rgp44 also displayed atheroprotective features in modulation of plaque size through association with plasma levels of IL-1α whereas whole Pg bacteria achieved through regulation of anti-inflammatory cytokine levels of IL-5 and IL-10. The present study may contribute to refining therapeutic approaches aiming to modulate immune responses and inflammatory/anti-inflammatory processes in atherosclerosis.

Characterization of a natural mouse monoclonal antibody recognizing epitopes shared by oxidized low-density lipoprotein and chaperonin 60 of Aggregatibacter actinomycetemcomitans.

Natural antibodies are predominantly antibodies of the IgM isotype present in the circulation of all vertebrates that have not been previously exposed to exogenous antigens. They are often directed against highly conserved epitopes and bind to ligands of varying chemical composition with low affinity. In this study we cloned and characterized a natural mouse monoclonal IgM antibody selected by binding to malondialdehyde acetaldehyde epitopes on low-density lipoprotein (LDL). Interestingly, the IgM antibody cross-reacted with Aggregatibacter actinomycetemcomitans (Aa) bacteria, a key pathogenic microbe in periodontitis reported to be associated with risk factor for atherosclerosis, thus being named as Aa_Mab. It is more intriguing that the binding molecule of Aa to Aa_Mab IgM was found to be Aa chaperonin 60 or HSP60, a member of heat-shock protein family, behaving not only as a chaperone for correct protein folding but also as a powerful virulence factor of the bacteria for inducing bone resorption and as a putative pathogenic factor in atherosclerosis. The findings will highlight the question of whether molecular mimicry between pathogen components and oxidized LDL could lead to atheroprotective immune activity, and also would be of great importance in potential application of immune response-based preventive and therapeutic strategies against atherosclerosis and periodontal disease.

Immunization with malondialdehyde-modified low-density lipoprotein (LDL) reduces atherosclerosis in LDL receptor-deficient mice challenged with Porphyromonas gingivalis.

Periodontal infections increase the risk of atherosclerotic vascular disease via partly unresolved mechanisms. Of the natural IgM Abs that recognize molecular mimicry on bacterial epitopes and modified lipid and protein structures, IgM directed against oxidized low-density lipoprotein (LDL) is associated with atheroprotective properties. Here, the effect of natural immune responses to malondialdehyde-modified LDL (MDA-LDL) in conferring protection against atherosclerosis, which was accelerated by the major periodontopathogen Porphyromonas gingivalis, was investigated. LDL receptor-deficient (LDLR(-/-)) mice were immunized with mouse MDA-LDL without adjuvant before topical application challenge with live P. gingivalis. Atherosclerosis was analyzed after a high-fat diet, and plasma IgG and IgM Ab levels were measured throughout the study, and the secretion of IL-5, IL-10 and IFN-γ in splenocytes stimulated with MDA-LDL was determined. LDLR(-/-) mice immunized with MDA-LDL had elevated IgM and IgG levels to MDA-LDL compared with saline-treated controls. MDA-LDL immunization diminished aortic lipid depositions after challenge with P. gingivalis compared with mice receiving only P. gingivalis challenge. Immunization of LDLR(-/-) mice with homologous MDA-LDL stimulated the production of IL-5, implicating general activation of B-1 cells. Immune responses to MDA-LDL protected from the P. gingivalis-accelerated atherosclerosis. Thus, the linkage between bacterial infectious burden and atherogenesis is suggested to be modulated via natural IgM directed against cross-reactive epitopes on bacteria and modified LDL.

Carbamyl adducts on low-density lipoprotein induce IgG response in LDLR-/- mice and bind plasma autoantibodies in humans under enhanced carbamylation.

AIMS: Post-translational modification of proteins via carbamylation predicts increased risk for coronary artery disease. Uremia and smoke exposure are known to increase carbamylation. The aim was to investigate the role of carbamylated low-density lipoprotein (LDL) immunization on antibody formation and atherogenesis in LDL receptor-deficient (LDLR-/-) mice, and to study autoantibodies to carbamylated proteins in humans with carbamylative load. RESULTS: LDLR-/- mice immunized with carbamylated mouse LDL (msLDL; n=10) without adjuvant showed specific immunoglobulin G (IgG) antibody levels to carbamyl-LDL and malondialdehyde-modified LDL (MDA-LDL) but not to oxidized LDL or native LDL. Immunization did not influence the atherosclerotic plaque area compared with control LDLR-/- mice immunized with native msLDL (n=10) or phosphate-buffered saline (n=11). Humans with high plasma urea levels, as well as smokers, had increased IgG autoantibody levels to carbamyl-modified proteins compared to the subjects with normal plasma urea levels, or to nonsmokers. INNOVATION: Carbamyl-LDL induced specific IgG antibody response cross-reactive with MDA-LDL in mice. IgG antibodies to carbamyl-LDL were also found in human plasma and related to conditions known to have increased carbamylation, such as uremia and smoking. Plasma antibodies to carbamylated proteins may serve as new indicator of in vivo carbamylation. CONCLUSION: These data give insight into mechanisms of in vivo humoral recognition of post-translationally modified structures. Humoral IgG immune response to carbamylated proteins is suggested to play a role in conditions leading to enhanced carbamylation, such as uremia and smoking.

Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein.

OBJECTIVE: Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. METHODS AND RESULTS: Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR(-/-) mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. CONCLUSION: Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a.

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.