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The J-elongated conformation of β₂-glycoprotein I predominates in solution: implications for our understanding of antiphospholipid syndrome.

ABSTRACT: β₂-Glycoprotein I (β₂GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a life-threatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound β₂GPI to that in solution. β₂GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, β₂GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and O-circular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant β₂GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular or twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound β₂GPI arises from the ability of its preexisting J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of β₂GPI provides a strategy to block pathogenic aPLs in APS.

SUBMITTER: Ruben E

PROVIDER: S-EPMC7397106 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bovine leukemia virus (BLV) is a deltaretrovirus that infects domestic cattle. The structural protein Gag, found in all retroviruses, is a polyprotein comprising three major functional domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Previous studies have shown that both mature BLV MA and NC are able to bind to nucleic acids; however, the viral assembly process and packaging of viral genomic RNA requires full-length Gag to produce infectious particles. Compared to lentiviruses, little is known about the structure of the Gag polyprotein of deltaretroviruses. In this work, structural models of full-length BLV Gag and Gag lacking the MA domain were generated based on previous structural data of individual domains, homology modeling, and flexible fitting to SAXS data using molecular dynamics. The models were used in molecular dynamic simulations to determine the relative mobility of the protein backbone. Functional annealing assays revealed the role of MA in the nucleic acid chaperone activity of BLV Gag. Our results show that full-length BLV Gag has an elongated rod-shaped structure that is relatively rigid, with the exception of the linker between the MA and CA domains. Deletion of the MA domain maintains the elongated structure but alters the rate of BLV Gag-facilitated annealing of two complementary nucleic acids. These data are consistent with a role for the MA domain of retroviral Gag proteins in modulating nucleic acid binding and chaperone activity. IMPORTANCE: BLV is a retrovirus that is found worldwide in domestic cattle. Since BLV infection has serious implications for agriculture, and given its similarities to human retroviruses such as HTLV-1, the development of an effective treatment would have numerous benefits. The Gag polyprotein exists in all retroviruses and is a key player in viral assembly. However, the full-length structure of Gag from any virus has yet to be elucidated at high resolution. This study provides structural data for BLV Gag and could be a starting point for modeling Gag-small molecule interactions with the ultimate goal of developing of a new class of pharmaceuticals.

Project description:Antiphospholipid antibodies (aPL), the serological hallmark of antiphospholipid syndrome (APS), are a heterogeneous group of autoantibodies raised against circulating blood proteins. Of these proteins, the phospholipid-binding b2-glycoprotein I (β2GPI) is considered to be the main autoantigen in APS. Indeed, IgG antibodies targeting b2GPI (ab2GPI) directly cause both thrombosis and pregnancy morbidity in several mouse models. While antibodies raised against all five domains of b2GPI have been reported, a subgroup of IgG ab2GPI raised against the first domain (DI) of b2GPI (aDI), strongly correlate with thrombotic APS, and drive thrombosis and pregnancy loss in vivo. Few studies have focused on determining the type of IgG subclass(es) for aPL. The subclass of an antibody is important as this dictates the potential activity of an antibody; for example, IgG1 and IgG3 can fix complement better and are able to cross the placenta compared to IgG2 and IgG4. It is unknown what subclass IgG aDI are, and whether they are the same as ab2GPI. To determine IgG subclass distribution for ab2GPI and aDI, we purified total IgG from the serum of 19 APS patients with known ab2GPI and aDI activity. Using subclass-specific conjugated antibodies, we modified our established in-house ab2GPI and aDI ELISAs to individually measure IgG1, IgG2, IgG3, and IgG4. We found that while IgG1, IgG2, and IgG3 ab2GPI levels were similar, a marked difference was seen in IgG subclass aDI levels. Specifically, significantly higher levels of IgG3 aDI were detected compared to IgG1, IgG2, or IgG4 (p < 0.05 for all comparisons). Correlation analysis of subclass-specific ab2GPI vs. aDI demonstrated that IgG3 showed the weakest correlation (r = 0.45, p = 0.0023) compared to IgG1 (r = 0.61, p = 0.0001) and IgG2 (r = 0.81, p = 0.0001). Importantly, total subclass levels in IgG purified from APS and healthy serum (n = 10 HC n = 12 APS) did not differ, suggesting that the increased IgG3 aDI signal seen in APS-derived IgG is antigen-specific. To conclude, our data suggests that aDI show a different IgG subclass distribution to ab2GPI. Our results highlight the importance of aDI testing for patient stratification and may point toward differential underlying aPL-driven pathogenic processes that may be subclass restricted.

Project description:ObjectivePatients with coronavirus disease 2019 (COVID-19) present coagulation abnormalities and thromboembolic events that resemble antiphospholipid syndrome (APS). This work has aimed to study the prevalence of APS-related antigens, antibodies, and immune complexes in patients with COVID-19 and their association with clinical events.MethodsA prospective study was conducted on 474 adults with severe acute respiratory syndrome coronavirus 2 infection hospitalized in two Spanish university hospitals. Patients were evaluated for classic and extra-criteria antiphospholipid antibodies (aPLs), immunoglobulin G (IgG)/immunoglobulin M (IgM) anticardiolipin, IgG/IgM/immunoglobulin A (IgA) anti-β2-glicoprotein-I (aβ2GPI), IgG/IgM antiphosphatidylserine/prothrombin (aPS/PT), the immune complex of IgA aβ2GPI (IgA-aβ2GPI), bounded to β2-glicoprotein-1 (β2GPI) and β2GPI levels soon after COVID-19 diagnosis and were followed-up until medical discharge or death.ResultsPrevalence of aPLs in patients with COVID-19 was as follows: classic aPLs, 5.8%; aPS/PT, 4.6%; IgA-aβ2GPI, 15%; and any aPL, 21%. When patients were compared with individuals of a control group of a similar age, the only significant difference found was the higher prevalence of IgA-aβ2GPI (odds ratio: 2.31; 95% confidence interval: 1.16-4.09). No significant differences were observed in survival, thrombosis, or ventilatory failure in aPL-positive versus aPL-negative patients. β2GPI median levels were much lower in patients with COVID-19 (15.9 mg/l) than in blood donors (168.8 mg/l; P < 0.001). Only 3.5% of patients with COVID-19 had normal levels of β2GPI (>85 mg/l). Low levels of β2GPI were significantly associated with ventilatory failure (P = 0.026).Conclusionβ2GPI levels were much lower in patients with COVID-19 than in healthy people. Low β2GPI-levels were associated with ventilatory failure. No differences were observed in the COVID-19 evolution between aPL-positive and aPL-negative patients. Functional β2GPI deficiency could trigger a clinical process similar to that seen in APS but in the absence of aPLs.

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