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

This paper describes the application of two novel screening technologies, i.e. Domain Scan™ (24- and 30-mer peptides) and Matrix Scan™ (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™ technology (12-merpeptides), showed selective binding to peptide-constructs corresponding to the β3-loop of FSH in the Domain™ and/or Matrix Scan™. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70,D71, and L73 form the core of the epitope. A DomainScan™ 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 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan™ 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 70ADSL73-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™ 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 (Kd∼ 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 10IAIEKEECRFAI21to the linear β3-peptide*52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2–3 fold increase in Kd, thus conforming participation of the β 1-loop in antibody binding for these mAb's.

[1]  D. Suckau,et al.  Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Jemmerson,et al.  Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes. , 1986, Science.

[3]  G. Wick,et al.  Antigenic features of human follicle stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiomometric assay. , 1988, Endocrinology.

[4]  Yingdong Zhao,et al.  Findings on T cell specificity revealed by synthetic combinatorial libraries. , 2002, Journal of immunological methods.

[5]  H. M. Geysen,et al.  Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Sodroski,et al.  Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. , 1987, Science.

[7]  R. Meloen,et al.  Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides , 1989, European journal of immunology.

[8]  S. Rodda.  Peptide libraries for T cell epitope screening and characterization. , 2002, Journal of immunological methods.

[9]  P. Roey,et al.  HUMAN FOLLICLE STIMULATING HORMONE , 2001 .

[10]  R. Dighe,et al.  Long-term contraceptive efficacy of vaccine of ovine follicle-stimulating hormone in male bonnet monkeys (Macaca radiata). , 1992, Journal of reproduction and fertility.

[11]  K. Tomer,et al.  Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody , 1998, Journal of the American Society for Mass Spectrometry.

[12]  J. Schneider-Mergener,et al.  Mapping of the interleukin‐10/interleukin‐10 receptor combining site , 1998, Protein science : a publication of the Protein Society.

[13]  J. Sodroski,et al.  Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding , 1990, Journal of virology.

[14]  L. Sijtsma,et al.  Synthetic peptides derived from the beta2-beta3 loop of Raphanus sativus antifungal protein 2 that mimic the active site. , 2001, The journal of peptide research : official journal of the American Peptide Society.

[15]  G. Nakamura,et al.  Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor , 1987, Cell.

[16]  S. Mahale,et al.  Identification of bioneutralization epitopes of human follicle stimulating hormone in the regions 31-52 and 66-75 of its beta-subunit. , 1997, Journal of reproductive immunology.

[17]  J. Dias,et al.  Mapping of an assembled epitope of human follicle-stimulating hormone-beta utilizing monoclonal antibodies, synthetic peptides, and hormone-receptor inhibition. , 1990, Endocrinology.

[18]  R. Houghten,et al.  Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: Identification of potent calcium-independent antigens , 1995, Molecular Diversity.

[19]  J. Schneider-Mergener,et al.  A synthetic mimic of a discontinuous binding site on interleukin-10 , 1999, Nature Biotechnology.

[20]  I. Roitt,et al.  Immunochemical mapping of gonadotropins , 1996, Molecular and Cellular Endocrinology.

[21]  A. Balen,et al.  Definition and measurement of follicle stimulating hormone. , 2000, Endocrine reviews.

[22]  J. Slootstra,et al.  In vitro inhibition of the biological activity of follicle-stimulating hormone by anti-peptide antisera representing the human follicle-stimulating hormone beta subunit sequence 33-53. , 1997, Biology of reproduction.