A Sema7a derived RTS-disintegrin peptide binds to integrin alpha1beta1 and promotes migration of myeloid-derived suppressor cells (MDSCs)

Abstract

Purpose : We have previously shown that Semaphorin 7a (Sema7a) increases the presence of YFP-MDSCs in the cornea after nerve transection. Sema7a contains integrin binding sites (RGD-integrin and RTS-disintegrin motifs). In this study, we investigated the binding of a peptide spanning RTS disintegrin motif with integrin α1β1 and determined its effect on migration of MDSCs.

Methods : Surface plasmon resonance (SPR) analyses of Sema7a peptides and integrin α1β1 complex formation were performed. YFP-MDSCs were sorted from bone marrow of Thy1-YFP mice (8-10 week old) and were used for all assays. Sema7a and integrin α1β1 expression were analyzed by flow cytometry. The migration of YFP-MDSCs in response to RGD-integrin and RTS-disintegrin peptides was evaluated in a Boyden Chamber using the real-time xCELLigence system. Both peptides were used to perform adhesion inhibition test on MDSCs. In vivo intracorneal injection was used to confirm YFP-MDSCs migration in Thy1-YFP mice (n=5/group).

Results : Sema7a showed the binding affinity (the equilibrium dissociation constant (K_D)) at 63.6 ± 17.4 nM to the active integrin α1β1 dimer. The binding affinity of Sema7a RGD peptide toward integrin α1β1 dimer was slightly tighter at K_D value of 12.6 ± 6.1 nM than that of RTS peptide at K_D value of 34.4 ± 10.0 nM, both of which showed comparable binding to the full-length Sema7a protein. There was no binding between integrin β1 and Sema7a peptides, indicating that the binding site might be located in α1. FACS analysis showed that integrin α1 and integrin β1 were expressed in YFP-MDSCs. RTS-disintegrin peptide increased migration of YFP-MDSCs by 3.5-folds as compared to scrambled control peptide. The increased YFP-MDSCs migration in response to RTS-disintegrin peptide was abrogated by integrin α1 blocking antibodies but not by integrin β1 blocking antibodies. RTS-disintegrin peptide significantly inhibited adhesion of YFP-MDSCs. In vivo intracorneal injection of RTS significantly increased migration of YFP-MDSCs.

Conclusions : Sema7a derived RTS-disintegrin peptide binds to integrin α1β1 and promotes migration of YFP-MDSCs, possibly by decreasing their adherence to the substrate.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.