Isolation of a Tripeptide from a Random Phage Peptide Library That Inhibits P¹,P⁴-Diadenosine 5‘-Tetraphosphate Binding to Its Receptor

Abstract

Extracellular P1,P4-diadenosine 5'-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have previously demonstrated specific receptors for Ap4A at the surface of cardiac myocytes (Walker et al., 1993a). In addition, we have isolated a monoclonal antibody (mAb TL4) that recognized the Ap4A receptor and inhibited binding of Ap4A to its receptor (Walker & Hilderman, 1993). As part of our effort to characterize the Ap4A receptor building domain, we screened a random phage peptide library with mAb TL4. After affinity purification of specifically bound phage, we isolated 38 individual phage clones. Twenty-eight of these clones bound mAb TL4 in ELISA and dot blot analyses. Twenty-two of the twenty-eight individual clones contained inserts with an RGS tripeptide sequence. Synthetic RGS peptide specifically inhibits the binding of mAb TL4 to its membrane receptor. Furthermore, the RGS peptide also inhibits [3H]Ap4A binding to its receptor. These data are consistent with the RGS peptide mimicking part of the mAb TL4 recognition site on the Ap4A receptor. The The RGS peptide may be used to help characterize the Ap4A receptor binding domain and to help determine the physiological significance of the interaction between Ap4A and its receptor.

A peptide isolated from a random phage peptide library is a structural mimic to the P1, P4‐diadenosine 5′‐tetraphosphate binding site on its receptor

“…Also, 20 of the 24 phage clones isolated by screening the 15-residue library contained the RGSSS pentapeptide (clones A, B, D, and E in Table 1). The RGSSS pentapeptide insert was one of the two consensus sequences found by screening a hexapeptide phage library [23]. These data are consistent with the RGSSS pentapeptide being part of the epitope recognized by mAb TL4.…”

“…Therefore, mAb TL4 was used to screen a random phage hexapeptide library and to identify a set of sequences with a common tripeptide motif, RGS. Synthetic RGS peptide interfered with both mAb TL4 and [ 3 H]Ap 4 A binding to its receptor [23]. These data are consistent with the RGS motif interfering with Ap 4A binding to its membrane receptor.…”

“…In a previous communication we reported that phage selected from screening a random hexapeptide phage library with mAb TL4 contained a consensus RGS sequence. Synthetic RGS peptide inhibited both mAb TL4 and Ap 4 A binding to its membrane receptor [23]. However, we did not determine whether RGS inhibits Ap 4A binding to its receptor by interacting with Ap 4 A or with the receptor.…”

“…To further identify the epitope recognized by mAb TL4, a 15-residue random peptide phage library was screened with mAb TL4 as described [23]. After three rounds of panning and phage amplification, 30 individual bacterial clones were grown and their phages were assayed for antibody binding.…”

“…The 15-residue peptide epitope library used in this study was provided by George P. Smith (University of Missouri, Columbia). Panning of the library and DNA sequencing of the inserts of the purified phage were performed as described [23].…”