Mapping protein-protein contact sites using cellulose-bound peptide scans

Abstract

We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-alpha). In addition, the interaction between TNF-alpha and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-alpha, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-alpha, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF-beta in the TNF-beta-TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.

Mapping of the interleukin‐10/interleukin‐10 receptor combining site

“…Usually the affinities of peptides spanning single ligate binding regions to the ligand are too low for competition studies. One exception is the interaction of IgM antibodies with single binding regions of nonlinear epitopes due to avidity effects ("functional affinity"), caused by multiple binding sites (Reineke et al, 1995). In our case, peptides derived from L l O as well as IL-10R were neither able to compete with this interaction nor the interaction of these two proteins with neutralizing antibodies recognizing discontinuous epitopes.…”

“…In addition, a fourth binding region was found in both peptide scans. However, since it has been shown that peptides of this region interact with constant regions of IgM monoclonal antibodies, such as CB/RS/S (Reineke et al, 1995) this region was considered to represent unspecific binding. The fact that binding regions A, B, and C are in close vicinity on the IL-10 surface (Fig.…”

“…Discontinuous epitopes usually consist of between two and five binding regions which are separated in the antigen sequence, but adjacent on the protein surface (Davies & Cohen, 1996;Jones & Thomton, 1996;Wells, 1996). Only a few examples for the mapping of discontinuous epitopes using overlapping peptide scans supported by structural information have been reported so far (Reineke et al, 1995;Yone et al, 1995;Gao & Esnouf, 1996;Weiergraber et al, 1996;Reineke et al, 1997aReineke et al, , 1997b. Random peptide libraries presented on the surface of filamentous phages have also been applied to the mapping of discontinuous epitopes (Hoess et al, 1994;Chen et al, 1996).…”

Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid: Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection

“…This implies that the epitope sequences are separated by more than 100 aa, as only sequences of up to 100 aa can be presented on the phage surface in this system. Since conformational epitopes can also be identified by peptide scanning (31,32,35,39), this method was employed for mapping of the A20, C24-B, C37-B, and D3 epitopes. For each antibody, specific regions of the VP3 protein sequence based on their reactivity with specific serotypes were synthesized as overlapping 10-mer peptides and probed with A20, C24-B, C37-B, or D3.…”

Localization of type-specific epitopes of herpes simplex virus type 2 glycoprotein G recognized by human and mouse antibodies.

“…Liljeqvist and others J.-A H . Liljeqvist and others (Kramer et al, 1993(Kramer et al, , 1994 as well as discontinuous epitopes (Gao & Esnouf, 1996 ;Korth et al, 1997 ;Reineke et al, 1995).…”