Chemical Synthesis of 7 kDa RHAMM As Receptor for Discovery of Novel Peptide Ligands
Receptor for Hyaluronan Mediated Motility (RHAMM) is one of the receptors for hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic properties when fragmented, and is associated with the progression of a number of cancers, such as prostate and breast cancer.1,2 RHAMM has two binding domains that are selective for HA, both of which are located near the carboxy-terminus of the protein. Synthesis and purification of full native RHAMM (85 kDa) using recombinant DNA methods is difficult and produces very low yields. We propose synthesizing a shortened version of the protein (7 kDa) by automated solid phase peptide synthesis (SPPS). This truncated receptor, whose sequence is derived from that of native RHAMM, is 62 amino acids in length and contains both HA-binding domains. Thus, we hypothesize that this 7 kDa RHAMM can be used as the target receptor in the screening of new RHAMM-binding peptides in order to develop peptide-based therapeutic agents for treating RHAMM-related disease.
Truncated RHAMM (7 kDa) was synthesized by fluorenyl-9-methoxycarbonyl (Fmoc) SPPS using microwave irradiation at 75°C for coupling reactions. It was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and analyzed by ESI-MS and circular dichroism (CD) spectroscopy. Analysis by LC-MS shows that the final compound has a purity of >95%, and all charged states up to [M+13H]13+ are observed.
7 kDa RHAMM is believed to be a suitable replacement receptor for the full native protein in our studies. Full RHAMM has been identified as being a highly coiled protein, with its HA-binding domains contained within two distinct alpha helices. Structural analysis of 7 kDa RHAMM using CD spectroscopy demonstrated that the mini-protein has alpha-helical character, which is similar to the predicted structure for that segment of the native protein. In addition, 7 kDa RHAMM continues to bind HA by enzyme-linked immunosorbent assay (ELISA), in which varying concentrations of biotinylated RHAMM are added to HA-coated plates and the binding is detected by streptavidin-AP.
HA-RHAMM binding is governed by ionic interactions, which occurs over a short sequence of amino acids. Peptide analogues derived from tubulin were shown to compete with HA for RHAMM binding, potentially impeding downstream signaling pathways that lead to cancer metastasis. Interaction studies using surface plasmon resonance (SPR) have demonstrated that these peptides bind with high affinity and specificity to truncated 7 kDa RHAMM. For this reason, we are actively developing RHAMM-binding peptide ligands as candidates for peptide-based therapeutics.
Acknowledgements: Translational Breast Cancer Research Unit, funded in part by the Breast Cancer Society of Canada; Novare Pharmaceuticals; Canada Foundation for Innovation.
(1) Veiseh, M.; Breadner, D.; Ma, J.; Akentieva, N.; Savani, R. C.; Harrison, R.; Mikilus, D.; Collis, L.; Gustafson, S.; Lee, T.-Y.; Koropatnick, J.; Luyt, L. G.; Bissell, M. J.; Turley, E. A. Biomacromolecules 2012, 13, 12.
(2) Veiseh, M.; Kwon, D. H.; Borowsky, A. D.; Tolg, C.; Leong, H. S.; Lewis, J. D.; Turley, E. A.; Bissell, M. J. Proceedings of the National Academy of Sciences 2014, 111, E1731.