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Random phage-epitope library based identification of a peptide antagonist of Mac-1 β2 integrin ligand binding
Mehdi Houimel1, Luca Mazzucchelli
1Laboratoire d'Immunopathologie Vaccinologie et Génétique Moléculaire, Institut Pasteur de Tunis, Tunisia. mehdi.houimel@pasteur.rns.tn
Insights
Researchers identified a novel peptide that inhibits the function of leukocyte Mac-1 (CD11b/CD18) integrin. This peptide blocks key interactions, offering potential for new anti-inflammatory drugs targeting neutrophil-mediated inflammation.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Leukocyte β2 integrin Mac-1 (CD11b/CD18) is crucial for inflammation and host defense.
- Targeting Mac-1 function is a strategy for developing anti-inflammatory therapeutics.
Purpose of the Study:
- To map structural features of CD11b and identify peptide antagonists of Mac-1.
- To determine the epitope of the neutralizing monoclonal antibody mAb 44a.
Main Methods:
- Utilized a random constrained 6-mer peptide library and phage display.
- Performed direct biopanning against purified mAb 44a.
- Conducted competitive ELISA and functional adhesion assays.
Main Results:
- Identified a disulfide-constrained peptide (C-RLKEKH-C) mimicking a discontinuous epitope on the CD11b-I-domain.
- The peptide binds to fibrinogen, iC3b, and C1q.
- C-RLKEKH-C effectively blocked Mac-1 interaction with fibrinogen and inhibited neutrophil adhesion.
Conclusions:
- The identified mimotope provides insights into CD11b I-domain surface structure.
- This peptide represents a potential therapeutic agent for designing anti-inflammatory drugs targeting PMN-mediated inflammation.
Abstract:
The leukocyte β2 integrin Mac-1 (CD11b/CD18) plays a pivotal role in inflammation and host defense. To develop peptide antagonists selectively inhibiting the function of Mac-1, we used a random constrained 6-mer (cys-6aa-cys) peptide library to map the structural features of CD11b, by determining the epitope of neutralizing monoclonal antibody mAb 44a (anti-CD11b). We have used a stringent phage display strategy, which resulted in the identification of one disulfide C-RLKEKH-C constrained peptide by direct biopanning of library on decreasing amounts of purified mAb 44a. The selected peptide mimics a discontinuous epitope, a peculiar shape on the CD11b-I-domain surface. Competitive ELISA experiments with different Mac-1 ligands showed that C-RLKEKH-C is able to bind to fibrinogen, iC3b, and C1q. Furthermore, the monomeric circular peptide C-RLKEKH-C, was effective in blocking the interaction between (125)I-fibrinogen and Mac-1 (IC(50)=3.35±0.1×10(-6)M), and inhibited the adhesion of human neutrophils to fibrinogen and iC3b. These data provide information about the relative location of amino acids on the I-domain surface using mAb 44a imprint of the CD11b protein. The derived mimotope may help in the design of future anti-inflammatory therapeutic agents that can act as specific therapeutic agents targeting PMNs mediated inflammation.
