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Disha Varma, Eunjae Kim, Ilangovan Raju, Zhiqiang Liu, Anubhav Pradeep, Christine Mun, Joy Sarkar, Sandeep Jain; Sema7a peptide that spans RTS disintegrin motif binds alpha1beta1 integrin and affects neuronal adherence.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4915.
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© ARVO (1962-2015); The Authors (2016-present)
Semaphorin 7a (Sema7a) sequence contains RTS disintegrin tripeptide motif in addition to RGD motif. While RGD tripeptide motif promotes nerve regeneration, the biological effects of RTS disintegrin motif are unknown. We determined whether a peptide spanning RTS disintegrin motif in Sema7a protein binds to α1β1 integrins and whether it can block the adhesive interaction of α1β1 integrin (on neurons) with collagen type I.
N-acetylated, C-amidated RGD- or RTS-containing 15-mer peptides derived from the Sema7a protein sequence were synthesized at >95% purity in the UIC Protein Research Laboratory. The binding affinity between integrin α1β1 dimer and Sema7a and the peptides (RGD and RTS) were analyzed by surface plasmon resonance (SPR). Trigeminal ganglions (TG) were harvested from thy1-YFP mice, dissociated into single cell suspensions and cultured on collagen I coated tissue culture dishes. Prior to coating the tissue culture dishes, collagen I was mixed with either Sema7a protein or its peptides (RGD, RTS, RGD plus RTS). After 48 hours of incubation, adherent neurons were imaged, and cell adherence was determined under different conditions, and compared for differences.
The binding affinity between integrin α1β1 dimer and Sema7a protein is not too strong or too weak. The equilibrium dissociation constant (KD) was 0.23 µM. The binding affinity between integrin α1β1 dimer and Sema7a peptide RGD was higher as compared to RTS (KD = 12.6 ± 6.1 nM and 34.4 ± 10.0 nM, respectively). There was no binding affinity between integrin β1 and Sema7a peptides. The adherence of neurons in culture dish coated with RGD peptide was similar as compared to Sema7a protein (7.6 ± 1.76 and 9.6 ± 1.5 cells/field, respectively; P=0.19). However, the adherence of neurons in culture dish coated with RTS peptide was significantly less as compared to Sema7a protein (4.3 ± 0.6 and 9.6 ± 1.5 cells/field, respectively; P=0.004).
RGD and RTS peptides bind with integrin α1β1 integrin. The binding affinity is neither too strong nor too weak. Our findings suggest that RGD integrin-binding peptide facilitates neuron adherence, like Sema7a, whereas RTS dysintegrin peptide reduces adherence of the neurons to collagen. RTS-disintegrin peptide may block the adhesive interaction of α1β1 integrin with collagens type I, and cause detachment of the neurites to facilitate extension of their processes.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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