Abstract: The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N-terminal domain (DmI) of b2-glycoprotein I (b2GpI). To possibly block anti-b2GpI Abs activity, we synthesized the entire DmI comprising residues 1–64 of b2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N-DmI) was purified by RP-HPLC, and its chemical identity and correct disulfide pairing (Cys4-Cys47 and Cys32-Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far- and near-UV CD and fluorescence spectroscopy, provided strong evidence for the native-like structure of DmI, which is also quite resistant to both Gdn-HCl and thermal denaturation. However, the thermodynamic stability of N-DmI at 37C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N-DmI in direct binding experiments. From ELISA competition experiments with plate-immobilized b2GpI, a mean IC50 value of 8.8 lM could be estimated for N-DmI, similar to that of the full-length protein, IC50(b2GpI) 5 6.4 lM, whereas the cysteine-reduced and carboxamidomethylated DmI, RC-DmI, failed to bind to anti-b2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N-terminally biotin-(PEG)2-derivative of N-DmI (Biotin- N-DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin-N-DmI loaded onto a streptavidin-coated plate selectively recognized aAbs from APS patients.

Chemical Synthesis and Characterization of Wild-type and Biotinylated N-terminal Domain 1-64 of Beta2-glycoprotein I

POZZI, NICOLA;BANZATO, ALESSANDRA;PENGO, VITTORIO;DE FILIPPIS, VINCENZO
2010

Abstract

Abstract: The antiphospholipid syndrome (APS) is a severe autoimmune disease associated with recurrent thrombosis and fetal loss and characterized by the presence of circulating autoantibodies (aAbs) mainly recognizing the N-terminal domain (DmI) of b2-glycoprotein I (b2GpI). To possibly block anti-b2GpI Abs activity, we synthesized the entire DmI comprising residues 1–64 of b2GpI by chemical methods. Oxidative disulfide renaturation of DmI was achieved in the presence of reduced and oxidized glutathione. The folded DmI (N-DmI) was purified by RP-HPLC, and its chemical identity and correct disulfide pairing (Cys4-Cys47 and Cys32-Cys60) were established by enzymatic peptide mass fingerprint analysis. The results of the conformational characterization, conducted by far- and near-UV CD and fluorescence spectroscopy, provided strong evidence for the native-like structure of DmI, which is also quite resistant to both Gdn-HCl and thermal denaturation. However, the thermodynamic stability of N-DmI at 37C was remarkably low, in agreement with the unfolding energetics of small proteins. Of note, aAbs failed to bind to plates coated with N-DmI in direct binding experiments. From ELISA competition experiments with plate-immobilized b2GpI, a mean IC50 value of 8.8 lM could be estimated for N-DmI, similar to that of the full-length protein, IC50(b2GpI) 5 6.4 lM, whereas the cysteine-reduced and carboxamidomethylated DmI, RC-DmI, failed to bind to anti-b2GpI Abs. The versatility of chemical synthesis was also exploited to produce an N-terminally biotin-(PEG)2-derivative of N-DmI (Biotin- N-DmI) to be possibly used as a new tool in APS diagnosis. Strikingly, Biotin-N-DmI loaded onto a streptavidin-coated plate selectively recognized aAbs from APS patients.
2010
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2470999
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