key: cord-0259791-eav04oih authors: Marashi, Hasan; Beihaghi, Maria; Khaksar, Samad title: In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity date: 2021-11-28 journal: bioRxiv DOI: 10.1101/2021.11.25.470018 sha: ac4058a3f8095e59afd361ea33fc5e6fa90923c8 doc_id: 259791 cord_uid: eav04oih CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize it’s in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7CCR7+ cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests. Since the three dimensional (3D) structure of the recombinant protein is not available, comparative 124 modeling method was initially used to create the 3D structure of the recombinant protein. 125 Comparative modeling is one of the best approaches to prediction of the 3D structure of the target 126 protein, in which 3D structures with sequences very similar to the target sequence are used as 127 templates. One of the best software for comparative modeling is MODELLER. The authors used 128 Modeller 9.24 for 3D modeling of the target . Ten models were produced, and the one with the 129 highest interaction and connectivity between the aforementioned epitopes was selected as the best After three dimensional modeling of the target recombinant protein sequence, the molecular 134 dynamics simulation method was used to optimize the modeled structure. Molecular dynamics 135 simulations allow researchers to observe changes in the orientation of different amino acids over 136 time. In this method, each atom has its dynamics and motion and this causes movement in the 137 structure of the whole protein, and one can optimize the 3D structure of the recombinant protein. Motion is the main factor in the function of biological macromolecules and this has led to 139 development of the "dynamic relationship between activities" approach. Dynamics is the key to the The correct hydrogen bonding patterns of histidines was defined for all proteins. The surface charge 148 of the structure was neutralized by adding chlorine ions. The protein was placed in a layer of 8 149 angstrom-thick TIP3P water molecules inside an octahedron box using gmx solvate software. Reduction of energy on the structures was achieved with 50,000 steps by the steepest descent 151 method to eliminate van der Waals interactions and hydrogen bonds between water molecules and 152 the complex. In the next step, temperature of the system was gradually increased from 0 to 310 K 153 for 200 ps in constant volume, and then the system was equilibrated at constant pressure for 200 pc. Molecular dynamics simulations were performed at 37°C for 100 nanoseconds. Non-bonded 155 interactions with 10-angstrom intervals were calculated by the PME method. The SHAKE method 156 was used to restrict the hydrogen atoms involved in bonding in order to enhance computing 157 performance. Finally, simulation data were stored at 0.4 pc intervals for analysis [28] . Two of the 158 best methods for such analyses are root mean square deviation (RMSD) and Radius of Gyration This project aimed to investigate the interaction between chemokine ligands and CCR7 receptors. The CCR7 protein is intermembrane and, therefore, to simulate the CCR7-CCL21 complex, this 168 complex must first be located inside the plasma membrane. For this purpose, the CHARMM-GUI 169 server was used to place the complex inside the plasma membrane. POPC phospholipid was 170 selected to generate the lipid membrane. After placing the protein complex inside the POPC 171 membrane according to the protocol in the CHARMM-GUI server, the desired output was selected 172 for molecular dynamics simulation with GROMACS 2019.6 software. At this stage, after energy 173 minimization using the steepest descent algorithm, the system was balanced in NVT conditions in a 174 time step of 1 femtosecond for 1 nanosecond. Equilibration was then performed under NPT 175 conditions in a time step of 2 femtoseconds for 4 nanoseconds. The Berendsen algorithm was used 176 to keep the temperature constant at 310 K and the pressure at 1 atmosphere in NVT and NPT 177 conditions. Also, during the equilibration process, a series of restrictions according to the protocols 178 available on the CHARMM-GUI server was used to limit proteins, water, and phospholipid 179 molecules. After the equilibration steps were completed, molecular dynamics simulations were 180 performed in the production phase for 100 nanoseconds. At production stage, Nose-Hoover and (Table 1) . Epitopes with HLA coverage above 90% and IC50 374 below 50 were selected as the best epitopes for binding to MHCI and MHCII molecules. In Figure 1C . The slope of RMSD increased rapidly in the first 10 ns of MD simulation. After approximately 385 10000 ps, RMSD reached 1 nm, which means that RMSD is equal to 1.25 nm at 60000 ps. As mentioned, the purpose of this project was to investigate the interactions between ligands and 400 CCR7 receptors. As shown in Table 2 , molecular docking was performed and the best cluster had a 401 score of -27, with a size of 34 complexes. In addition, Z-score was equal to -2.3. The image of this 402 complex is shown in Figure 1E . The RMSD parameter was used to study the stability of the The CCR7 protein is intermembrane and therefore, the CCR7-CCL21/IL1β complex must be were observed for DNA samples of transgenic lines ( Figure 2B ). Results indicated that the foreign gene was transcribed in the transgenic lines ( Figure 2C ). for two transgenic lines, and these lines were named H2 and E1 respectively. Total Soluble Protein 463 (%TSP) of recombinant protein was measured to be 2.14% and 1.81% for H2 and E1 transgenic 464 lines, respectively. In contrast, no significant differences were observed for non-transgenic plants. Figure 7 , the number of monocytes moving toward 552 the adsorbent substance was higher than that toward the non-adsorbent material, and the chemotaxis 553 of the recombinant protein was estimated to be about 91% (Table 3) Probiotics and prebiotics associated with aquaculture: a review. Fish & shellfish 670 immunology The role of chemoattraction in cancer metastases Biased signaling of CCL21 and CCL19 does not rely on N-terminal 673 differences, but markedly on the chemokine core domains and extracellular loop 2 of CCR7. 674 Frontiers in immunology CCL21/CCR7 interaction promotes EMT and enhances the stemness of OSCC via a 676 JAK2/STAT3 signaling pathway Marked T cell activation, senescence, exhaustion and skewing towards TH17 in 678 patients with COVID-19 pneumonia CCL21/CCR7 signaling in macrophages promotes joint inflammation 680 and Th17-mediated osteoclast formation in rheumatoid arthritis. 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