Structure function relationship of recombinant sRAGE
thesisposted on 02.03.2017, 03:17 by Premaratne, Dinamithra Gedara Sujeewani Rasika
The receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin super family of cell surface receptors. It acts as the direct mediator of physiological and pathological responses such as inflammation, chemotaxis, neurite outgrowth, angiogenesis, apoptosis and proliferation. RAGE is known to bind with structurally and functionally diverse ligands, such as advanced glycation end-products (AGEs), high mobility group family proteins including HMGB-1/amphoterin, matrix proteins, such as amyloid β peptides, and members of the S100/calgranulin protein family. The up-regulation of RAGE via its ligands and thereby its role in development of disease pathologies in humans is well understood. The blockage of RAGE is considered a therapeutic approach to overcome RAGE-related disease development. One major isoform of RAGE, known as soluble RAGE (sRAGE) acts as a decoy receptor by scavenging RAGE ligands, and prevents them from binding to RAGE or other cell surface receptors. Recent studies have revealed that sRAGE might be useful in treating pathologies caused through RAGE-ligand interactions. Therefore, sRAGE has potential therapeutic properties. In the present study, sRAGE was successfully expressed in hairy root cultures to obtain a yield of ~300 µg purified protein from ~100 g of hairy roots. Plant expression systems, including tobacco hairy root cultures, have the advantage of producing glycosylated proteins similar to mammalian expression system. Although the complex modification of glycans differs between plants and mammals, the core glycans and the frequency and sites of glycosylation are identical. Purified plant-made sRAGE predominantly existed in its monomeric form (as determined by SDS-PAGE analysis) with a molecular weight of ~45 kDa. Enzymatic N-linked deglycosylation assays using PNGase F suggests that the sRAGE produced was N-linked glycosylated. sRAGE was also successfully expressed in E. coli-Rosetta-gami strain as a HaloTag® fusion protein, yielding ~0.3 g Lˉ¹ (monomeric form), with a purity of 90 %. In contrast to plant-made sRAGE, E. coli-made sRAGE produced putative protein aggregates along with the monomeric form. As expected the E. coli expression system produced non-glycosylated sRAGE. The purified monomeric form of plant and E. coli derived sRAGE was used to perform in vitro ligand binding studies with HMGB-1 and S100A8/A9 complex to determine the affinities of these glycosylated and non-glycosylated sRAGE proteins to the ligands. It was shown that glycosylated plant-made sRAGE has a higher affinity to both HMGB-1 and S100A8/A9 than E. coli-made or deglycosylated plant-made sRAGE. The observed higher affinity was statistically significant for HMGB-1. However, the observed higher affinity of plant-made sRAGE to S100A8/A9 complex was not statistically significant compared to E. coli-made sRAGE or the deglycosylated (non-denatured) plant-made sRAGE. The higher affinity of plant-made sRAGE to ligands was related to the N-linked glycosylation of sRAGE. The effects of extracellular components and conditions on sRAGE-ligand binding was tested by the addition of EDTA and several cations. In our study, Mg²⁺/Zn²⁺ enhanced in vitro binding of sRAGE-S100A8/A9 complex with a similar capacity to Ca²⁺/Zn²⁺. The importance of Ca²⁺/Zn²⁺ cations for RAGE binding to S100A9 has been reported previously. The inhibitory effect of EDTA (2.0 mM) observed for HMGB-1 binding could be tested in vivo studies in future, as a potential treatment for RAGE-mediated inflammatory diseases. This is the first report of the expression of biologically active recombinant sRAGE in a plant expression system. Our research has found that if sRAGE is to be used as a therapeutic protein in the future, plant expression system could be used as a potential production platform. The enhanced binding potential of the N-linked glycosylated plant-made sRAGE showed the importance of glycosylation on the activity of the protein. Therefore, in future studies, glycoengineering could be investigated to introduce important glycans and then to enhance the activity of sRAGE. Thus, a bio-better potential therapeutic protein could be produced in a plant expression system.