Binding affinity of full-length and extracellular domains of recombinant human (pro)renin receptor to human renin when expressed in the fat body and hemolymph of silkworm larvae

Abstract

Transmembrane domains of some receptors have been found to be very important in the process of constitutive oligomerization, and in the stability and functioning of the receptor. In this study, full-length of human (pro)renin receptor (hPRR) and hPRR lacking cytoplasmic domain (hPRR-DeltaCD) were expressed in fat body of silkworm larvae, and the extracellular domain of hPRR (hPRR-DeltaTMDeltaCD) in hemolymph. Three forms of hPRR were used for investigation of the interaction between receptor and ligand using surface plasmon resonance (SPR). As a result, the cytoplasmic domain was not an essential requirement for binding affinity, but the transmembrane domain of hPRR was indispensable in the formation of functional hPRR. The dissociation equilibrium constants (K(D)) of purified hPRR and hPRR-DeltaCD were estimated to be 46 nM and 330 nM, respectively. No evidence of binding by the extracellular domain of hPRR located in hemolymph was found. However, the solubilized microsomal fraction of the extracellular domain of hPRR expressed in the fat body showed specific affinity, but lost its binding affinity after purification. Its binding affinity was recovered by mixing microsomal fraction of mock-injected fat body to the purified extracellular domain. It is probable that an artificial transmembrane domain stabilizes the extracellular domain of hPRR and native conformation may be structurally recovered. To our knowledge, these are the first findings describing the interaction of transmembrane and extracellular domains of hPRR with ligand and this may help towards the understanding of binding affinity of hPRR to ligand.

Silkworm expression system as a platform technology in life science

“…Many membrane proteins have been produced in the hemolymph and pupae of silkworms as secretory proteins. Recently, human (pro)renin receptor (hPRR) and its complex with human prorenin were expressed in silkworm larvae, with the same expression level as some secretory proteins, and were purified from the fat bodies of larvae (Du et al 2008, 2009a). This hPRR may be helpful for the development of an hPRR blocker.…”

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

“…However, as the concentration of Triton X-100 was increased, more amounts of GP64 and other proteins were solubilized into the supernatant fraction. It was previously reported that GFP uv -hPRR was not solubilized from the microsome fraction of silkworm fat body efficiently by Triton X-100 [23]. Considering the result of Table 2 0.01% TritonX-100 treatment are milder to baculovirus envelope than 0.1% or 1% Triton X-100 treatment.…”

“…Also, an understanding of the functional properties of hPRR through detailed biochemical and biophysical analysis is required. In a previous study, hPRR fused with GFP uv at its N-terminus (GFP uv -hPRR) was expressed and purified from the fat body of silkworm larvae infected with recombinant baculovirus [22,23]. However, the binding capacity of purified GFP uv -hPRR to human prorenin was reduced compared with that before purification.…”

Expression of Protein Complex Comprising the Human Prorenin and (Pro)Renin Receptor in Silkworm Larvae Using Bombyx mori Nucleopolyhedrovirus (BmNPV) Bacmids for Improving Biological Function

“…We investigated the binding affinity of full length of hPRR, hPRR lacking cytoplasmic domain, and the extracellular domain of hPRR. Interestingly, the transmembrane domain of hPRR is indispensable in the formation of functional hPRR [18]. The extracellular domain in the microsomal fraction of the fat body was observed to be bound with human renin while no affinity was observed after purification.…”