Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-beta (FSH-beta) 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-beta for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan technology (12-mer peptides), showed selective binding to peptide-constructs corresponding to the beta3-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 Domain Scan performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three beta1-beta3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues 97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan of mAb 17 showed that peptides from four different regions on FSH (1st strand beta3-loop, alpha 1-loop, long alpha2-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 Matrix Scan screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the beta3-loop showed measurable binding. Cyclic beta3-loop peptide mimics were found to bind significantly stronger (Kd approximately 5 microM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair beta1-peptide 1oIAIEKEECRFAI21 to the linear beta3-peptide *52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2-3 fold increase in Kd, thus conforming participation of the beta 1-loop in antibody binding for these mAb's.
Timmerman
Peter
P
Pepscan Systems B.V., Lelystad, The Netherlands. p.timmerman@pepscan.nl
Van Dijk
Evert
E
Puijk
Wouter
W
Schaaper
Wim
W
Slootstra
Jerry
J
Carlisle
Stephen J
SJ
Coley
John
J
Eida
Steve
S
Gani
M
M
Hunt
Tim
T
Perry
Paul
P
Piron
Gerry
G
Meloen
Rob H
RH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D037201:Follicle Stimulating Hormone, beta Subunit / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D040081:Protein Array Analysis / Q000379:methods*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D037201:Follicle Stimulating Hormone, beta Subunit