High-resolution mapping of epitopes on the C2 domain of factor VIII by analysis of point mutants using surface plasmon resonance.

Neutralizing anti-factor VIII (FVIII) antibodies that develop in patients with hemophilia A and in murine hemophilia A models, clinically termed "inhibitors," bind to several distinct surfaces on the FVIII-C2 domain. To map these epitopes at high resolution, 60 recombinant FVIII-C2 proteins were generated, each having a single surface-exposed residue mutated to alanine or a conservative substitution. The binding kinetics of these muteins to 11 monoclonal, inhibitory anti-FVIII-C2 antibodies were evaluated by surface plasmon resonance and the results compared with those obtained for wild-type FVIII-C2. Clusters of residues with significantly altered binding kinetics identified "functional" B-cell epitopes, defined as those residues contributing appreciable antigen-antibody avidity. These antibodies were previously shown to neutralize FVIII activity by interfering with proteolytic activation of FVIII by thrombin or factor Xa, or with its binding to phospholipid surfaces, von Willebrand factor, or other components of the intrinsic tenase complex. Fine mapping of epitopes by surface plasmon resonance also indicated surfaces through which FVIII interacts with proteins and phospholipids as it participates in coagulation. Mutations that significantly altered the dissociation times/half-lives identified functionally important interactions within antigen-antibody interfaces and suggested specific sequence modifications to generate novel, less antigenic FVIII proteins with possible therapeutic potential for treatment of inhibitor patients.

Probing the orientation of surface-immobilized protein G B1 using ToF-SIMS, sum frequency generation, and NEXAFS spectroscopy.

The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0-9.5) and salt concentration (0-1.5 M NaCl). SFG spectral peaks characteristic of ordered α-helix and ß-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the α-helix and ß-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to π* transition of ß-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner ß-strands of 40-50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

Six amino acid residues in a 1200 Å2 interface mediate binding of factor VIII to an IgG4κ inhibitory antibody.

The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies.

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers.

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.