key: cord-1017947-2n5skb51 authors: Mamedov, Tarlan; Gürbüzaslan, İrem; Ilgin, Merve; Yuksel, Damla; Mammadova, Gunay; Ozkul, Aykut; Hasanova, Gulnara title: High level production and characterization of truncated human angiotensin converting enzyme 2 in Nicotiana benthamiana plant as a potential therapeutic target in COVID-19 date: 2021-05-17 journal: bioRxiv DOI: 10.1101/2021.05.17.444533 sha: 4530a8297e6dc78a8f3cb9368885cf1cdfe5f5ea doc_id: 1017947 cord_uid: 2n5skb51 The COVID-19 pandemic, which is caused by SARS-CoV-2 has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs safe, a cost-effective SARS-CoV-2 coronavirus vaccine, therapeutic and antiviral drugs to combat the COVID-19. Angiotensin-converting enzyme 2 (ACE2), as a key receptor for SARS-CoV-2 infections, has been proposed as a potential therapeutic target in COVID-19 patients. In this study, we report high level production (about ∼0.75 g /kg leaf biomass) of glycosylated and non-glycosylated forms of recombinant human truncated ACE2 in Nicotiana benthamiana plant. The plant produced recombinant human truncated ACE2s successfully bind to the SARC-CoV-2 spike protein, but deglycosylated ACE2 binds more strongly than the glycosylated counterpart. Importantly, both deglycosylated and glycosylated forms of AEC2 stable at elevated temperatures for prolonged periods and demonstrated strong anti-SARS-CoV-2 activity in vitro. The IC50 values of glycosylated and deglycosylated AEC2 were 0.4 and 24 μg/ml, respectively, for the pre-entry infection, when incubated with 100TCID50 of SARS-CoV-2. Thus, plant produced truncated ACE2s are promising cost-effective and safe candidate as a potential therapeutic targets in the treatment of COVID-19 patients. SARS-CoV-2 is a novel and highly pathogenic coronavirus, which has caused an outbreak in Wuhan city, China in 2019, and then soon spread nationwide and spilled over to other countries and the world, which resulted hundreds of thousands of deaths all over the world. Head of the United Nations has described this humanity's worst crisis since World War II. Although several COVID-19 vaccines are currently available and a number of candidate vaccines are under development, only limited data are available on the effectiveness and safety of these vaccines. The world urgently needs effective and safe SARS-CoV-2 coronavirus vaccines, antiviral and therapeutic drugs to combat the pandemic that kills thousands of people every day. The development of therapeutic drugs can be a useful and alternative approach to suppress the virus entering and spread of the virus. Since angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 (COVID-19) receptor, is a critical molecule in the entry process of the virus into host tissue cells, it could be a potential therapeutic agent. ACE2 is a zinc containing metalloenzyme, present in most organs, attached to the cell membranes of cells in the lungs, heart, kidney, arteries and intestines 1 . ACE2 enzyme has multiple function and its primary function is to cleave the angiotensin I hormone into the vasoconstricting angiotensin II.ACE2 is a transmembrane protein, and serves as receptors for some coronaviruses such as SARS-CoV, SARS-CoV-2 and HCoV-NL63 [2] [3] [4] [5] [6] [7] . Similar to SARS-CoV, SARS-CoV-2 has been shown to bind to its functional receptor ACE2 via receptor binding domain (RBD) of SARS-CoV-2 spike protein as an initial step for entry into the cell 8, 9 . It has been demonstrated that the binding affinity between ACE2 and RBD of SARS-CoV-2 is much stronger than that of SARS-CoV 9 , which can be logically explained the increased infectivity of SARS-CoV-2 versus SARS-CoV. It has been demonstrated that, ACE2 serves not only the entry receptor for SARS-CoV or SARS-CoV-2 but also can protects from lung injury 4,5,10-12 .Therefore, ACE2 has been proposed as potential therapeutic target to be used for SARS-CoV-2 infection 3, 14 . Soluble ACE2 has also been described as a therapeutic candidate, which could neutralize the infection by acting as a decoy 15 . Recombinant human ACE2 is also proposed as a novel treatment to improve pulmonary blood flow and oxygen saturation in piglets 16 . Based on reported pathological findings [17] [18] [19] , it has been shown that SARS-CoV-2 is associated with lung failure and acute respiratory distress syndrome. Pulmonary arterial hypertension (PH) is a devastating lung disease, which is characterized by high blood pressure in the pulmonary circulation 20 . From this point of view, the introduction of soluble recombinant human ACE2 into the human body has been proposed for the treatment of Acute respiratory distress syndrome (ARDS) and pulmonary arterial hypertension 21 . It should be noted that the Phase I (NCT00886353) and Phase II (NCT 01597635) clinical trials for recombinant human ACE2 have been successfully completed. The administration of soluble recombinant ECE2 has demonstrated safety and efficacy for the treatment of ARDS and without clinically significant changes in healthy people, as well as in patients with ARDS [22] [23] . During the period of taking the drug, there were no serious adverse events no antibodies to recombinant human ACE2 detected 22 . Recently, it was shown that recombinant human ACE2 was significantly inhibited SARS-CoV-2 infection of Vero E6 cells 24 and can neutralize SARS-CoV-2 infectivity in human kidney organoids 25 , Human Capillary and Kidney Organoids 24 . It was previously proposed that SARS-CoV may deregulate a lung protective pathway 4, 10 . Thus, recombinant AEC2 might not only reduce lung injury, but also could block early the entry of SARS-CoV-2 infections in target cells. Therefore, the development of a cost-effective, safe, and functionally active recombinant ACE2 could be very We engineered and produced a truncated version of human ACE2 in N. benthamiana plant as described in Materials and Methods. To understand the role of glycosylation, we produced both glycosylated and non-glycosylated variants of ACE2 protein in N. benthamiana plant. Figure 1 demonstrates the confirmation of the production of glycosylated and non-glycosylated variants of ACE2 in N. benthamiana by western blot analysis. N. benthamiana leaf samples were harvested at different post infiltration days (dpi) and expression levels of glycosylated and nonglycosylated variants of ACE2 reached the maximum level at 6 dpi. For purification, a vacuum infiltration was used for large-scale production of glycosylated and non-glycosylated variants of ACE2. Glycosylated and deglycosylated variants of ACE2 were purified using HisPur™ Ni-NTA resin. The purification yields of recombinant plant produced glycosylated or deglycosylated forms were ~0.4 or 0.5 g/kg leaf, respectively. The purity of glycosylated and deglycosylated variants of ACE2 enzyme was higher than 90% or 95%, for glycosylated or deglycosylated, respectively, as estimated based on SDS-PAGE and western blot analysis ( Figure 2 ). Molecular masses were 80 or 90 kDa for deglycosylated or glycosylated AEC2, respectively ( Figure 2 ). The binding activity of plant produced recombinant ACE2 protein was confirmed by measuring the binding activity of ACE2 with spike protein of SARC-CoV-2 as described in Materials and Methods. The results presented at Figure3 demonstrate that plant produced, glycosylated and non-glycosylated ACE2s successfully bind to commercial or plant produced spike proteins of SARC-CoV-2. Deglycosylated ACE2 variant binds to the deglycosylated plant-produced Sprotein more strongly than the glycosylated counterparts. The stability of plant produced glycosylated and in vivo deglycosylated forms of AEC2 were examined after incubation at 37°C for a prolonged time period: 24, 48, 96 and 144 hours ( Figure 4 ). Analysis by SDS-PAGE showed that plant produced glycosylated ACE2 had almost no degradation at 37 o C for 144 h and degradation of in vivo Endo H glycosylated ACE2 at the same condition was less than 5 %. These results demonstrate that plant produced, both glycosylated and deglycosylated AEC2s are stable at an elevated temperature for prolonged periods. Anti -SARS-CoV2 activity of plant produced glycosylated and deglycosylated forms were evaluated as described in Materials and Methods. Figure 5 demonstrate apparent activities of plant produced recombinant truncated glycosylated and deglycosylated ACE2 variants to RBDs plotted against IC50 of authentic SARS-CoV-2 neutralizationat the pre-infection phase. The half maximal inhibitory concentration (IC 50 ) values for glycosylated and deglycosylated AEC2 were 0.4 and 24 g/ml, respectively, when they mixed with 100TCID 50 of SARS-CoV-2 ( Figure 5 ). The new coronavirus COVID-19 disease is currently responsible for the pandemic, created huge global human health crisis with significant negative impacts on health and economy worldwide. The sequences of ACE2 was optimized for expression in N. benthamiana plants and synthesized de-novo. The constructed ACE2 gene was inserted into the pEAQ binary expression vector 38 to obtain pEAQ-ACE2. pEAQ-ACE2 plasmid was introduced into the Agrobacterium tumefaciens strain AGL1. AGL1 carrying the pEAQ-ACE2 plasmid was then infiltrated into 6-7-week old N. benthamiana plants. To produce deglycosylated ACE2, ACE2 gene was in vivo co-expressed with Endo H 28 . To confirm the expression of His6 tagged ACE2 protein variants, a leaf tissue was harvested at 6 dpi (day post infiltration) and homogenized in three volumes of extraction buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) as described previously 29 . To produce the AEC-2 protein (both glycosylated and deglycosylated variants) in N. benthamiana, plants were infiltrated with ACE2 (glycosylated) or ACE2 + Endo H Stability assessments of different variants of ACE2 were performed using similar procedure as described previously 28, 30 . Plant produced glycosylated and deglycosylated variants of ACE2 and ImageJsoftware (https://imagej.nih.gov/ij). SDS-PAGE and western blot analysis of plant produced glycosylated and non-glycosylated variants of ACE2 were performed as described previously 30 . The His tagged ACE2 variants were detected using anti-His mAb (Cat. No. 652505, BioLegend). Anti SARS-CoV2 activity of plant produced ACE2s was monitored in vitro. kDa) proteins were used as a standard. The image was taken using a highly sensitive GeneGnome XRQ Chemiluminescence imaging system. 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