Anti-β2-glycoprotein I paratopes and β2-glycoprotein I epitopes characterization using random peptide libraries

Abstract

Studies concerning interactions between anti-β2-glycoprotein I antibodies (anti-β2GPI) and β2-glycoprotein I (β2GPI) suggest relevance of charge interactions and hydrogen bonds. However, paratope of diagnostically and clinically relevant anti-β2GPI and epitope characteristics of β2GPI, still remain unclear. The aim of our study was to determine paratope characteristics of various anti-β2GPI antibodies and epitope characteristics of β2GPI using phage display. Monoclonal IgG anti-β2GPI, purified polyclonal high avidity and low avidity IgG anti-β2GPI derived from plasma of APS patients were used to screen phage display libraries. The affinity and competition ability of selected clones were evaluated. Various heptapeptides presenting putative paratopes of anti-β2GPI and specific heptapeptides presenting putative epitopes of β2GPI were determined. Epitope presenting peptides bind to the respective anti-β2GPI and consequently interrupt antibody–antigen interaction. The amino acid composition of selected peptides confirmed the importance of hydrogen bonds and charge interactions in the binding of anti-β2GPI to the antigen. Epitopes recognized by high avidity anti-β2GPI predominately contain hydrogen bond forming side chains, while in low avidity anti-β2GPI epitope the charged side chains prevail. The alignment of selected sequences to three-dimensional antigen structure revealed that polyclonal high avidity anti-β2GPI recognize native epitopes that are accessible regardless of β2GPI's conformation whereas the epitope recognized by low avidity anti-β2GPI is cryptic and cannot be accessed when β2GPI takes the closed plasma conformation.     

Anti-β2GPI/β2GPI induced TF and TNF-α expression in monocytes involving both TLR4/MyD88 and TLR4/TRIF signaling pathways

Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2-glycoprotein I (anti-β₂GPI/β₂GPI)-induced tissue factor (TF) expression in human acute monocytic leukemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MyD88 and TRIF, in anti-β₂GPI/β₂GPI-induced the activation of human blood monocytes and THP-1 cells and the relationship among TLR4, β₂GPI and ANX2 in this process. The results showed that treatment of monocytes or THP-1 cells with anti-β₂GPI/β₂GPI complex could increase TF, MyD88, TRIF as well as TNF-α (tumor necrosis factor alpha) expression. These effects were blocked by addition of TAK-242, a blocker of signaling transduction mediated by the intracellular domain of TLR4. Moreover, TLR4/β₂GPI/ANX2 complex could be detected in THP-1 cell lysates. Overall, our results indicate that anti-β₂GPI/β₂GPI complex induced TF and TNF-α expression involving both TLR4/MyD88 and TLR4/TRIF signaling pathways and TLR4 and its adaptors might be molecular targets for therapy of antiphospholipid syndrome (APS).     

Anti-β(2)GPI/β(2)GPI induced TF and TNF-α expression in monocytes involving both TLR4/MyD88 and TLR4/TRIF signaling pathways

Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2-glycoprotein I (anti-β(2)GPI/β(2)GPI)-induced tissue factor (TF) expression in human acute monocytic leukemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MyD88 and TRIF, in anti-β(2)GPI/β(2)GPI-induced the activation of human blood monocytes and THP-1 cells and the relationship among TLR4, β(2)GPI and ANX2 in this process. The results showed that treatment of monocytes or THP-1 cells with anti-β(2)GPI/β(2)GPI complex could increase TF, MyD88, TRIF as well as TNF-α (tumor necrosis factor alpha) expression. These effects were blocked by addition of TAK-242, a blocker of signaling transduction mediated by the intracellular domain of TLR4. Moreover, TLR4/β(2)GPI/ANX2 complex could be detected in THP-1 cell lysates. Overall, our results indicate that anti-β(2)GPI/β(2)GPI complex induced TF and TNF-α expression involving both TLR4/MyD88 and TLR4/TRIF signaling pathways and TLR4 and its adaptors might be molecular targets for therapy of antiphospholipid syndrome (APS).     

Clusterin induces matrix metalloproteinase-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways in monocytes/macrophages

Most solid tumor tissues possess a significant population of macrophages, which are known to be closely linked with tumor progression and metastasis. Clusterin has been reported to be overexpressed in various tumors and to have a tumor-promoting role. As clusterin induction and macrophage infiltration occur concurrently at the tumor site, it raises a possibility that clusterin may regulate the function of macrophages via facilitating ECM remodeling. Here, we demonstrate for the first time the expression of MMP-9 by clusterin in human primary monocytes as well as human and murine macrophage cell lines, THP-1, and Raw264.7. MMP-9 expression was accompanied by increased enzymatic activity, as revealed by gelatin zymography. The MMP-9 activity promoted by clusterin was found to be dependent on the activation of ERK1/2 and PI3K/Akt but not p38 or JNK pathways. Inhibition of PI3K activity did not affect the activation of ERK1/2 and vice versa, indicating that the two pathways were independently operated to stimulate MMP-9 activity. Moreover, clusterin facilitated nuclear translocation of NF-κB p65 along with IκB-α degradation and phosphorylation, which was critical for MMP-9 expression. As NF-κB is a central regulator of inflammation, clusterin may provide a molecular link between inflammation and cancer via up-regulating NF-κB and MMP-9. Collectively, these data highlight a novel role of clusterin as a stimulator for MMP-9 expression in macrophages, which may contribute to the tissue reorganization by serving as a modulator for ECM degradation.     

Role of molecular mimicry and polyclonal cell activation in the induction of pathogenic β2-glycoprotein I–directed immune response in Balb/c mice upon hyperimmunization with tetanus toxoid

It is known that tetanus toxoid (TTd)-hyperimmunization induces increased titer of sera β2-glycoprotein I (β2GPI)-specific antibodies (Abs) in Balb/c mice. The concentrations of such induced anti-β2GPI Abs as well as their pathogenic potential are strongly influenced by the context of TTd application. β2GPI-specific immune response is established as a part of TTd-specific immune response by molecular mimicry mechanism due to structural homology between TTd and β2GPI. This finding is supported by the following facts: (1) cross-reactive Abs that recognize both TTd and β2GPI epitopes are present in Balb/c mice sera; (2) anti-TTd Abs secretion in splenic cultures is induced after β2GPI stimulation and vice versa. However, analyses of (1) IL-10 production following in vitro stimulation of immunized Balb/c mice splenocytes by TTd, β2GPI or glutaraldehyde-treated β2GPI and (2) specific impact of ConA and agonists of TLR2, TLR4, and TLR9 on anti-TTd and autoreactive Abs secretion strongly imply that these two branches of the TTd-induced immune response do not use identical cell populations and are regulated in a different way. Results presented in this paper describe that structural homology between foreign and self-antigens could focus mounted autoreactive immune response toward specific self-structure, but the context of antigen application, including a history of previous immune stimulations and adjuvants applied together with the antigen, are the main factors which determine the outcome of the induced immune response.     

Transforming growth factor-β1 induces glutathione peroxidase-1 and protects from H2O2-induced cell death in colon cancer cells via the Smad2/ERK1/2/HIF-1α pathway

Recent studies have shown that transforming growth factor-β1 (TGF-β1) signaling plays important roles in the redox system in benign and malignant cells. Whether TGF-β mediates an antioxidative damage response in colorectal cancer cells is largely unknown. Herein, using the human colorectal cancer cell lines we found that TGF-β1 induced glutathione peroxidase-1 (GPx-1) expression and enzyme activity, and that the upregulation of GPx-1 by TGF-β1 could protect colorectal cell lines from H2O2-induced oxidation damage. Further, we used loss- and gain-function approaches to elucidate the underlying mechanism and found that TGF-β1 induced GPx-1 through activation of the TGF-β receptor type?I (TGF-βRI)/Smad2/extracellular-signal-regulated kinase 1/2 (ERK1/2)/hypoxia-inducible factor-1α (HIF-1α) signaling pathway. This cascade could be blocked by the TGF-βRI inhibitor or ERK1/2 inhibitor. Taken together, our data demonstrated that TGF-β1 induced GPx-1 expression and enzymatic activity via the TGF-βRI/Smad2/ERK1/2/HIF-1α signaling pathway, suggesting a novel antioxidative protective function of TGF-β1 in colorectal cancer cells.     

12-deoxyphorbol-13-O-phenylacetate 20 acetate [an agonist of protein kinase Cβ1 (PKCβ1)] induces DNA synthesis,interleukin-2 (IL-2) production,IL-2 receptor α-chain (CD25) and β-chain (CD122) expression,and translocation of PKCβ isozyme in human peripheral blood lymphocytes: Evidence for a role of PKCβ1 in human T cell activation

To determine a role of protein kinase C (PKC) isozymes in lymphocyte activation, human peripheral blood mononuclear cells were activated with 12-deoxyphorbol-13-O-phenylacetate (dPP; an agonist of both calcium-dependent and calcium-independent PKC isozymes), thymeleatoxin (TX; an activator of calcium-dependent PKC, , and ), and 12-deoxyphorbol-13-O-phenylacetate 20 acetate (dPPA; an activator of PKC1 isozyme) and examined for DNA synthesis, lymphocyte proliferation, interleukin-2 (IL-2) production, expression of IL-2 receptor and chains on CD3+, CD4+, and CD8+ T lymphocytes and CD20+ B lymphocytes, and translocation of PKC isozyme from cytosol to membrane fraction. The results show that dPPA activates lymphocytes by inducing the above changes in a manner analogous to that of dPP, TX, and phorbol myristate acetate. These data suggest that PKC1 is involved in the activation of human peripheral blood T and B lymphocytes.