Evaluating the conformation of recombinant domain I of β(2)-glycoprotein I and its interaction with human monoclonal antibodies

Pathogenic antiphospholipid antibodies (aPL) cause the antiphospholipid syndrome (APS) by interacting with domain I (DI) of beta-2-glycoprotein I (β(2)GPI). The aPL/β(2)GPI complex then exerts pathogenic effects on target cells. We previously described periplasmic bacterial expression of native and mutated variants of DI, and reported the presence of immunodominant epitopes at positions 8-9 (D8/D9) and position 39 (R39). Mutations at these positions strongly influenced the ability of recombinant DI to bind patient-derived IgG aPL and to inhibit pathogenic effects of these aPL in a mouse model of APS. We now describe an improved cytoplasmic bacterial expression system allowing higher yield of DI. We demonstrate that the nuclear magnetic resonance (NMR) spectra of a (15)N,(13)C-isotope-labelled sample of the recombinant DI protein exhibit properties consistent with the structure of DI in crystal structure of intact β(2)GPI. Mutations at D8/D9 and R39 had limited impact on the NMR spectrum of DI indicating maintenance of the overall fold of the DI domain. We investigated interactions between five variants of DI and ten monoclonal human IgG antibodies, all derived from the IgG aPL antibody IS4 by sequence manipulation and in vitro expression. Arginine residues at positions 100 and 100g in IS4V(H) CDR3 play a particularly important role in binding to DI, but this is unlikely to be due to electrostatic interactions with negatively charged amino acids on DI. Both the strength of binding to DI and the ability to discriminate different DI variants varies between the different IgG antibodies tested. There was no simple relationship between these binding properties and antibody pathogenicity.     

The clinical value of assays detecting antibodies against domain I of β2-glycoprotein I in the antiphospholipid syndrome

As the clinical symptoms of the antiphospholipid syndrome (APS) frequently occur irrespective of the syndrome, diagnosis predominantly depends on the laboratory assays measuring the level or function of antiphospholipid antibodies (aPLs). β2-glycoprotein I (β2GPI) is increasingly accepted as the most important target of aPLs. Anti-β2GPI antibodies constitute a heterogeneous population, but current in vivo and in vitro evidence show that especially the first domain (DI) of β2GPI contains an important pathogenic epitope. This epitope containing Glycine40-Arginine43 (G40-R43) has proven to be cryptic and only exposed when β2GPI is in its open conformation. A previous study demonstrated a highly variable exposure of the cryptic epitope in commercial anti-β2GPI assays, with implications on correct patient classification. Unexpectedly, recent unpublished data revealed impaired exposure of the pathogenic epitope in the commercially available anti-DI chemiluminescence immunoassay (CIA) assay detecting specific antibodies directed to DI.In this review we summarize the laboratory and clinical performance characteristics of the different anti-DI assays in published data and conclude with inconsistent results for both the correlation of anti-DI antibodies with clinical symptoms and the added value of anti-DI antibodies in the classification criteria of APS. Additionally, we hypothesize on possible explanations for the observed discrepancies. Finally, we highly advise manufacturers to use normal pooled plasma spiked with the monoclonal anti-DI antibodies to verify correct exposure of the cryptic epitope.     

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.     

Immunogenic Oxidized Low-density Lipoprotein/β2-glycoprotein I Complexes in the Diagnostic Management of Atherosclerosis

Oxidized low-density lipoprotein (oxLDL) promotes atherosclerosis through a complex interaction of inflammatory and immunologic factors that lead to macrophage lipid uptake and foam cell formation. OxLDL interacts with β2-glycoprotein I (β2GPI) forming oxLDL/β2GPI complexes. These complexes may be formed in the arterial intima during atherogenesis and released into the circulation. Autoantibodies against oxLDL/β2GPI complexes have been demonstrated in patients with systemic lupus erythematosus and/or antiphospholipid syndrome, and shown to be significantly associated with arterial thrombosis. The observation that monoclonal autoantibodies against oxLDL/β2GPI complexes significantly increased the oxLDL uptake by macrophages strongly suggests that such IgG autoantibodies are pro-atherogenic. In this article, we review the recent progress in our understanding of LDL oxidation, oxLDL/β2GPI complex formation, and immune regulation of atherogenesis.     

Post-translational oxidative modification of β2-glycoprotein I and its role in the pathophysiology of the antiphospholipid syndrome

Vascular thrombosis and/or recurrent miscarriages are the main characteristics defining Antiphospholipid Syndrome (APS). Currently there is no well-defined clinical features and/or laboratory tests that predicts the risk of adverse prognostic outcomes in APS. In this short review, we report the importance of posttranslational modification of beta2 glycoprotein I, the major autoantigen in the APS beta2 glycoprotein I that may, in part, explain possible mechanisms for the generation of auto antibodies to beta2 glycoprotein I. A specific ELISA measuring the level of oxidised beta2 glycoprotein I could be used as a potential new laboratory test - along with other laboratory tests - to more accurately predict the risk of having a clinical event in patients with APS.     

Critical role of cPLA(2) in Aβ oligomer-induced neurodegeneration and memory deficit

Soluble beta-amyloid (Aβ) oligomers are considered to putatively play a critical role in the early synapse loss and cognitive impairment observed in Alzheimer's disease. We previously demonstrated that Aβ oligomers activate cytosolic phospholipase A(2) (cPLA(2)), which specifically releases arachidonic acid from membrane phospholipids. We here observed that cPLA(2) gene inactivation prevented the alterations of cognitive abilities and the reduction of hippocampal synaptic markers levels noticed upon a single intracerebroventricular injection of Aβ oligomers in wild type mice. We further demonstrated that the Aβ oligomer-induced sphingomyelinase activation was suppressed and that phosphorylation of Akt/protein kinase B (PKB) was preserved in neuronal cells isolated from cPLA(2)(-/-) mice. Interestingly, expression of the Aβ precursor protein (APP) was reduced in hippocampus homogenates and neuronal cells from cPLA(2)(-/-) mice, but the relationship with the resistance of these mice to the Aβ oligomer toxicity requires further investigation. These results therefore show that cPLA(2) plays a key role in the Aβ oligomer-associated neurodegeneration, and as such represents a potential therapeutic target for the treatment of Alzheimer's disease.     

TNF-β +252 A>G (rs909253) polymorphism is independently associated with presence of autoantibodies in rheumatoid arthritis patients

Clinical and Experimental Medicine - The TNF-β +252 A>G (rs909253) polymorphism has been associated with a risk of development of rheumatoid arthritis (RA) and could influence plasma...     

An AciI polymorphism in the 3′ untranslated region of the human phosphomannomutase 2 (PMM2) gene

We found an AciI polymorphism in the 3′ untranslated region of the phosphomannomutase 2 (PMM2) gene located at 16p13. A G-to-C transition at nucleotide position 96 bp downstream from the PMM2 stop codon was detected in polymerase chain reaction (PCR) products after AciI digestion. The heterozygosity of the polymorphic alleles was 0.375 in a Japanese population. This polymorphism is useful for genetic analysis in patients with carbohydrate-deficient glycoprotein syndromes, of which there are four subtypes. Received: March 17, 1999 / Accepted: April 3, 1999     

Association of the Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene and serum lipid levels

The objective of the present study was to detect the association of the Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene and serum lipid levels in the Mulao and Han populations. A total of 879 subjects of Mulao and 844 subjects of Han Chinese were included. The levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and ApoA1 in Mulao, and triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), ApoA1 and the ratio of ApoA1/ApoB in Han were different among the three genotypes of the rs1801690 SNP (P < 0.05-0.01). Subgroup analyses showed that the levels of TC, TG, LDL-C, and ApoA1 in Mulao males; ApoA1 in Mulao females; TC, TG, HDL-C and ApoB and the ApoA1/ApoB ratio in Han males; and HDL-C, ApoA1 and the ApoA1/ApoB ratio in Han females were associated with the genotypes of rs1801690 (P < 0.05-0.001). Serum lipid parameters were also associated with several environmental factors (P < 0.05-0.001). The Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene was associated with some serum lipid parameters in the two ethnic groups, but the trends of association suggest that the Trp316Ser variant (rs1801690) near the apolipoprotein H (β2-glycoprotein-I) gene might have racial/ethnic-and/or gender-specificity.     

Are Oxidized LDL/β2-glycoprotein I Complexes Pathogenic Antigens in Autoimmune-mediated Atherosclerosis?

The oxidative modification of low-density lipoprotein (LDL) in the intima of arteries contributes to the initiation and progression of atherosclerotic lesions. We have previously reported that oxidized LDL (oxLDL) interacts with an endogenous plasma protein, beta2-glycoprotein I (beta2GPI), to form complexes and that the interaction is mediated by oxLDL specific ligands. We have also demonstrated the presence of oxLDL/beta2GPI complexes in the blood stream of patients with systemic inflammatory and autoimmune diseases. These findings implicate that oxLDL/beta2GPI complexes are possible atherogenic autoantigens. Autoantibodies to oxLDL/beta2GPI complexes have been associated with arterial thrombosis. Further, circulating IgG immune complexes containing oxLDL and beta2GPI were also detected in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS). Although many unanswered questions remain about the exact pathogenic mechanism(s) involved, oxLDL/beta2GPI complexes may be described as metabolic products relevant in atherogenesis and as significant participants in autoimmune-mediated atherosclerosis.     

Emergence of influenza A (H1N1) PDM09 in the remote Islands of India – A molecular approach

Background: A disease outbreak of A (H1N1) PDM09 was reported in Andaman and Nicobar islands in 2009 with an attack rate of 33.5% among settler population and 26.3% among the aboriginal Nicobarese tribe. During the ongoing outbreak of A (H1N1) PDM09 disease in different parts of the world, a subject working in Dubai city of Saudi Arabia, came to Port Blair, following which the pandemic triggered for the first time in these Islands. Materials and Methods: During the period August 2009 to January 2011, 30 confirmed cases of Influenza A (H1N1) PDM09 virus infection was detected. To understand the genetic relationship, the NA gene sequences of the viruses were phylogenetically analysed together along with the virus sequence isolated from other parts of the world. Result: Formation of multiple clusters were observed, with the sequences of Andaman Islands, mainland India, Mexico, Saudi Arabia and few other counties clustering together. The sequence analysis data revealed that there was no specific mutation conferring resistance to oseltamivir among the Andaman A (H1N1) PDM09 virus isolates. The result of phylogenetic analysis have also revealed that the A (H1N1) PDM09 virus might have spread in these remote Islands of India via the subject from Saudi Arabia/Dubai. Conclusion: A (H1N1) PDM09 Influenza outbreak have highlighted the need to strengthen the region-specific pandemic preparedness plans and surveillance strategies.     

MDA5 is SUMOylated by PIAS2β in the upregulation of type I interferon signaling

Retinoic acid-inducible protein I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytosolic viral RNA sensors that induce type I interferon production (IFN). In this study, we found that MDA5 undergoes inducible SUMOylation by small ubiquitin-like modifier-1 (SUMO-1) in response to polyI:C stimulation. Enhanced SUMOylation of MDA5 by exogenously expressed SUMO-conjugating enzyme Ubc9 correlated with upregulation of IFN expression and repressed virus replication. Conversely, overexpression of a SUMOylation-deficient mutant of Ubc9 or knockdown of endogenous Ubc9 reduced IFN production. Furthermore, we showed that PIAS2β, a SUMOylation E3 ligase, could specifically interact with and enhance the SUMOylation of MDA5. Consequently, PIAS2β knockdown reduced the SUMOylation of MDA5 and the IFN production. Collectively, these findings suggest that SUMO-1 modification of MDA5 possibly via PIAS2β may play a role in the MDA5-mediated IFN response to viral infections.     

Effects of prostaglandin F2α(PGF2α) and prostaglandin I2 (PGI2) on nerve-mediated secretion in guinea-pig colon

We have applied conventional flux-chamber and intracellular recording methods to investigate the effects of the prostaglandins PGF₂ and PGI₂ upon epithelial ion transport and on the electrical behaviour of submucosal neurones in guinea-pig colon. In flux-chamber experiments on segments of colon, both prostaglandins evoked a dose-dependent increase in short-circuit current that was reduced in chloridedepleted Krebs solution and by serosal addition of tetrodotoxin or atropine, but was unaffected by hexamethonium. These results indicate activation of chloride secretion via submucosal neurones. The response to PGF₂ was decreased by piroxicam. Application of PGF₂ or PGI₂ to submucosal neurones evoked depolarization of the membrane potential associated with an enhanced spike discharge. The depolarizing response was tetrodotoxin insensitive, indicating a direct effect of the prostaglandins on the impaled neurones. Membrane depolarization was frequently associated with the occurrence of fast excitatory postsynaptic potentials, suggesting in addition that part of the excitatory effect is mediated by the activation of neural circuits that drive the impaled neurone synaptically. The results of this study indicate that the secretory effects of prostaglandins are mediated in part by submucosal neurones and further suggest that the colonic submucosal plexus may function as an amplifier to enhance the epithelial response to inflammatory mediators.     

Cognitive impairment and increased Aβ levels induced by paraquat exposure are attenuated by enhanced removal of mitochondrial H(2)O(2)

Pesticide exposure is a risk factor of Alzheimer's disease (AD). However, little is known about how pesticide exposure may promote AD pathogenesis. In this study, we investigated the effects of paraquat pesticide exposure on β-amyloid (Aβ) levels and cognition using wild-type (WT) mice and β-amyloid precursor protein (APP) transgenic mice. Our results showed that wild-type mice and APP transgenic mice after paraquat exposure had increased oxidative damage specifically in mitochondria of cerebral cortex and exhibited mitochondrial dysfunction. Moreover, the elevated mitochondrial damage was directly correlated with impaired associative learning and memory and increased Aβ levels in APP transgenic mice exposed to paraquat. Furthermore, overexpression of peroxiredoxin 3, a mitochondrial antioxidant defense enzyme important for H₂O₂ removal, protected against paraquat-induced mitochondrial damage and concomitantly improved cognition and decreased Aβ levels in APP transgenic mice. Therefore, our results demonstrate that mitochondrial damage is a key mechanism underlying cognitive impairment and elevated amyloidogenesis induced by paraquat and that enhanced removal of mitochondrial H₂O₂ could be an effective strategy to ameliorate AD pathogenesis induced by pesticide exposure.     

Molecular diversity of the DNA-β satellites associated with tomato leaf curl disease in India

DNA-β satellites, referred to here as betasatellites, were found associated with tomato leaf curl disease (ToLCD) in India. The size of eight betasatellites isolated from different geographical locations in India varied from 1353 to 1424 nt; these molecules had an ORF βC1, an adenine-rich region, and a satellite conserved region. Their nucleotide sequence identity varied from 45 to 93%. In phylogenetic analysis, these betasatellites grouped according to their geographic locations rather than the host species. Two new betasatellites, tomato leaf curl Bangalore betasatellite and tomato leaf curl Maharashtra betasatellite, were identified.     

Association of estrogen receptor β (ESR2) gene polymorphism with blood pressure

We investigated the association between a dinucleotide (cytosine-adenine; CA) repeat polymorphism located in the flanking region of the human estrogen receptor β (ESR2) gene and systemic blood pressure in 187 healthy postmenopausal Japanese women. The genotype was classified as "A" through "O" according to the number of these repeats from 18 to 32. When we separated the subjects into two groups — bearing at least one “I” allele (26 CA repeats) and those who did not — we found that the former subjects had significantly higher systolic blood pressure than the latter (mean ± SD, 146.0 ± 25.0 vs 136.6 ± 23.4; P = 0.032). These data suggest that genetic variation at the ESR2 locus may be associated with some determinants of blood pressure, and that there is a possible involvement of this polymorphism in causing hypertension in Japanese women. Received: June 26, 2000 / Accepted: August 7, 2000     

Control of Type I Collagen Systhesis: Evidence for Pretranslational Coordination of Proα1 (I) and Proα2 (I) Chain Synthesis in Embryonic Chick Bone

The normal type I collagen molecule contains two αl (I) chains and one α2 (I) chain. In embryonic chick calvaria, the two chains are synthesized in a 2: 1 ratio, and total polysomes from this tissue contain twice as much mRNA for proal (I) as for proα2 (I).⁹ To further investigate the mechanism by which synthesis may be coordinated, RNA isolated from various cell fractions of embryonic chick calvaria was translated in a rabbit reticulocyte lysate cell-free system. The procollagen chain products were separated by gel-electrophoresis and densitometrically quantitated from autoradiograms of the gels. Total cellular RNA, total cytoplasmic RNA, and polysomal RNA each directed the synthesis of proal (I) and proa2 (I) in a proportion of 2: 1, whereas no procollagen mRNA activity was found in nonpolysomal cytoplasmic RNA. These results indicate that in the chick bone cells, all compartments contain twice as much proal (I) mRNA as proa2 (I) mRNA, and that virtually all procollagen mRNA in the cytoplasm is poly some-bound. The coordination of procollagen chain synthesis thus presumably occurs at a pretranslational level, through differential rates of formation and/or degradation of the two mRNAs.