Fig 1: Recombinant human GRP78 binds CRD domain of rfhSP-D/rFLhSP-D. (A) Recombinant human GRP78 showed direct binding to rfhSP-D in presence of CaCl2 (Bar 1) and was not inhibited by (Bar 2). Monoclonal antibodies against the CRD domain of rfhSP-D did not recognize rfhSP-D bound to recombinant human GRP78 (Bar 3). Recombinant human GRP78 did not interact with rfhSP-D bound to monoclonal antibodies specific to the CRD domain of SP-D (Bar 4). The data presented are the mean ± S.D from independent experiments conducted three times. *p < 0.05 vs. Bar 1. (B) Standard curve of the rFLhSP-D (0–10 ng/ml) from Duoset SP-D ELISA kit, R & D Systems. (C) The rFLhSP-D binding to anti-human SP-D capture antibody (Duoset) was significantly inhibited in the presence of GRP78 in a dose-dependent manner. The GRP78 concentration ‘0’ in the graph represents the control where only buffer was coated and the experiments were conducted three times, *p < 0.05 vs. control. **p<0.01 vs. control, ns is non significant.
Fig 2: Work flow and data analysis. PC3 cells were incubated with rfhSP-D for 2 h at 37°C. Cells were then subjected to membrane protein extraction. The isolated PC3 cell membrane proteins were incubated with polyclonal anti-human SP-D antibody for obtaining rfhSP-D-bound membrane proteins (pull-down assay). The rfhSP-D interacting membrane proteins were subjected to immunodepletion. The immunodepleted rfhSP-D-bound PC3 cell membrane proteins were subjected to LC-MS/MS analysis. The identified rfhSP-D interacting membrane proteins were further examined using Network and Ingenuity Pathways Analysis. HSPA5/GRP78 was identified as a potential binding/interacting partner of rfhSP-D and was confirmed by in silico analysis (molecular docking) and in vitro studies (Direct and competitive ELISA).
Fig 3: In silico validation of the interaction of rfhSP-DSP-D with GRP78. (A) Top-ranked docked pose of rfhSP-D (CRD) depicted in cyan and GRP78 (SBD) depicted in pink; (B) N-acetyl galactosamine (GalNAc) modeled at Thr203 of GRP78 (pink) was found to be within 10 Å radius of residues of rfhSP-D (cyan) in the docked complex; (C) residues involved in intermolecular interactions of GRP78 (pink) and rfhSP-D (cyan).
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