Chemical synthesis and characterization of wild-type and biotinylated N-terminal domain 1-64 of beta2-glycoprotein I.
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 beta2-glycoprotein I (beta2GpI). To possibly block anti-beta2GpI Abs activity, we synthesized the entire DmI comprising residues 1-64 of beta2GpI 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 37 degrees C 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 beta2GpI, a mean IC(50) value of 8.8 microM could be estimated for N-DmI, similar to that of the full-length protein, IC(50)(beta2GpI) = 6.4 microM, whereas the cysteine-reduced and carboxamidomethylated DmI, RC-DmI, failed to bind to anti-beta2GpI 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.
Pozzi
Nicola
N
Department of Pharmaceutical Sciences, University of Padova, 35131 Padova, Italy.
Banzato
Alessandra
A
Bettin
Samuele
S
Bison
Elisa
E
Pengo
Vittorio
V
De Filippis
Vincenzo
V
D000595:Amino Acid Sequence; D016736:Antiphospholipid Syndrome; D001323:Autoantibodies / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D053482:beta 2-Glycoprotein I / Q000097:blood / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D001323:Autoantibodies; D053482:beta 2-Glycoprotein I; D001710:Biotin