Journal Article

Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-beta (FSH-beta) using domain Scan(TM) and Matrix Scan(TM) technology

Perry, Paul, Coley, John, Puijk, Wouter, Slootstra, Jerry, Meloen, Rob H, Hunt, Tim, Schaaper, Wim, Piron, Gerry, Eida, Steve, Timmerman, Peter, Gani, M, Van Dijk, Evert, Carlisle, Stephen J

2004

Overview

Issue Title: Peptide Libraries and Peptide Drugs This paper describes the application of two novel screening technologies, i.e. Domain Scan(TM) (24- and 30-mer peptides) and Matrix Scan(TM) (24-mer peptides)technology, in the mapping of a discontinuous epitope on FSH-β for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan(TM) technology (12-merpeptides), showed selective binding to peptide-constructs corresponding to the β3-loop of FSH in the Domain(TM) and/or Matrix Scan(TM). Systematic replacement analysis studies with peptide-construct ^sub 57^VYETVRVPGCAC-SAc-ADSLYTYPVATQ^sub 81^ revealed that for most mAb's the amino acids R^sub 62^, A^sub 70^,D^sub 71^, and L^sub 73^ form the core of the epitope. A DomainScan(TM) performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three β1-β3 peptide-constructs covering the residues ^sub 60^TVRVPGCAHHADSLY^sub 74^ in combination with ^sub 10^IAIEKEECRFAI^sub 21^, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues^sub 97^RGLGPSYCSFGEMKE^sub 114^ in combination with the residues ^sub 10^IAIEKEECRFAI^sub 21^. A Matrix Scan(TM) of mAb 17 showed that peptides from four different regions on FSH (1st strand β3-loop, α1-loop, longα2-loop, det. loop) showed enhanced binding in combination with several ^sub 70^ADSL^sub 73^-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and MatrixScan(TM) screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the β3-loop showed measurable binding. Cyclic β3-loop peptide mimics were found to bind significantly stronger (K^sub d^ 5 μM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair β1-peptide ^sub 10^IAIEKEECRFAI^sub 21^to the linear β3-peptide*^sub 52^TFKELVYETVRVPGCAHHADSLYTYPVATQAH^sub 83^# via disulfide bond formation showed a 2-3 fold increase in K^sub d^, thus conforming participation of the β 1-loop in antibody binding for these mAb's.[PUBLICATION ABSTRACT]

• Publisher: Springer Nature B.V
• Subject: Binding sites / Peptides