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J. Biol. Chem., Vol. 278, Issue 39, 37815-37821, September 26, 2003

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A Consensus Tetrapeptide Selected by Phage Display Adopts the Conformation of a Dominant Discontinuous Epitope of a Monoclonal Anti-VWF Antibody That Inhibits the von Willebrand Factor-Collagen Interaction^*

Karen Vanhoorelbeke , Hilde Depraetere , Roland A. P. Romijn , Eric G. Huizinga  ¶, Marc De Maeyer || and Hans Deckmyn  **

From the Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, E. Sabbelaan 53, 8500 Kortrijk, Belgium, the Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center, HP G03.647, P.O. Box 85500, 3508 GA Utrecht, The Netherlands and the ^||Laboratory for Bio-Molecular Modelling KU Leuven Campus Heverlee, Celestijnenlaan 200D, 3001 Heverlee, Belgium

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg⁹⁶³, Pro⁹⁸¹, Asp¹⁰⁰⁹, Arg¹⁰¹⁶, Ser¹⁰²⁰, Met¹⁰²², and His¹⁰²³ are part of the epitope of mAb 82D6A3. Inspection of residues Ser¹⁰²⁰, Arg¹⁰¹⁶, Pro⁹⁸¹, and Trp⁹⁸² in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser¹⁰²⁰ and Arg¹⁰¹⁶ in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp⁹⁸². In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.

Received for publication, April 24, 2003 , and in revised form, July 10, 2003.

^* This work was supported by EU Biomed BMH4-CT98-3517. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Dept. of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

^** To whom correspondence should be addressed. Tel.: 32-59-24-64-22; Fax: 32-56-24-69-97; E-mail: Hans.Deckmyn{at}kulak.ac.be.

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