STRA6 (vitamin A receptor), as a Novel binding receptor of COVID-19

A global pandemic of pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in Wuhan, China, at the end of 2019. Although, the ACE2 receptor has been shown to be the main entry receptor of COVID-19, but here, in our docking analysis , we predicted and discovered a novel receptor called STRA6 that may play a critical role in the pathogenicity of COVID-19 and explain the common pre and post COVID-19 symptoms with uncertain etiology. STRA6 receptor expressed in many organs and immune cells, upregulated by retinoic acid jm6 (STRA6) was the first protein to be identified in a novel category of proteins, cytokine signaling transporters, due to its ability to function as both a cell surface receptor and a membrane protein that binds to retinol binding protein facilitating cellular uptake of retinol. In agreement to our findings, the main ligand of STRA6 (vitamin/retinol) was found to be significantly reduced during COVID-19 infection.


Background
A global pandemic of pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in Wuhan, China, at the end of 2019. Although, the ACE2 receptor has been shown to be the main entry receptor of COVID-19, but here, in our docking analysis , we predicted and discovered a novel receptor called STRA6 that may play a critical role in the pathogenicity of COVID-19 and explain the common pre and post COVID-19 symptoms with uncertain etiology. STRA6 receptor expressed in many organs and immune cells, upregulated by retinoic acid jm6 (STRA6) was the first protein to be identified in a novel category of proteins, cytokine signaling transporters, due to its ability to function as both a cell surface receptor and a membrane protein that binds to retinol binding protein facilitating cellular uptake of retinol. In agreement to our findings, the main ligand of STRA6 (vitamin/retinol) was found to be significantly reduced during COVID-19 infection.

Methods
The STRA6 receptor protein were submitted to the server for functional interaction associated network between partners for the STRING (Research Online of Interacting Genes/Proteins Data Basis version 10.0)13 .Docking study of each Spike -ACE 2 and STRA6 receptor protein were carried out using HDOCK server (http://hdock.phys.hust.edu.cn/). The binding mode of Spike -ACE 2 and STRA6 receptor protein is retrieved form the PDB https://www.rcsb.org/ with accession number (7DMU , 5sy1)

Results
Surprisingly, our molecular docking based analysis showed that spike protein Receptor Binding Domain(RDB) of COVID-19 strongly and efficiently binds to STRA6 receptor, definitely to the RDB vital residues of RBP-binding motif located in STRA6 receptor. STRA6 receptor is a membrane receptor responsible for signaling and transporting of Vitamin A(Retinol) from plasma retinol binding protein (RBP) to our cells. In an outstanding manner, COVID-19 Spike protein exhibited high docking score with human STRA6 with low binding energy . The docking score of COVID-19 spike protein was stronger than the docking score of spike protein with ACE2.The surface view of complex reveals that the binding pocket of STRA6-Spike protein and Spike ACE 2 complexes with RMSD (189.44 Å , 1.00 Å ) representatively and docking score (-341.21 ,-354.68) kcal/mol the quality of the receptor and the ligand are LGscore and MaxSub ( 2.416 , 0.147 ). The spike to bind to RDB of the STRA 6 protein in the ILE 131C  ,  MET 145C , HIS 86A with interface residue( 4.961 , 4.953 and 3.271) representatively

In conclusion
STRA6 mutations results in a broad spectrum of complication related to malformations including congenital heart defects , anophthalmia, alveolar capillary dysplasia, diaphragmatic hernia, lung hypoplasia and mental retardation. Moreover, Retinoic acid metabolism is defective in COVID-19 (cytokine storm), sepsis, ARDS and SIRS. Therefore, We believe that this novel discovery that STRA6 receptor acts as a novel binding receptor for COVID-19 will shed new light on effective drug against COVID-19and explain many previously unexplained pre and post-covid-91 infection symptoms. Therefore, reconstitution of the signaling of retinoid may prove to be a valid strategy for COVID-19

Introduction
Normal cellular function depends on vitamin A homeostasis. Plasma retinol-binding protein (RBP) is the only specialized transporter of retinol, the most common form of vitamin A, in the plasma. By recognizing RBP-retinol and triggering retinol release and internalization, the integral membrane receptor STRA6 initiates and controls cellular uptake of vitamin A( (1). STRA6 is a particularly important receptor. because it was found to be the first protein to be recognized as a cytokine signaling transporter, owing to its ability to work as both a membrane protein and a cell surface receptor that facilitates cellular uptake of retinol by attaching to retinol binding protein (2,3). Because of the critical function of vitamin A in the the immune cell development , STRA6 was found to be expressed on all subsets of peripheral blood mononuclear cells at varying amounts. A recent study showed that all T cell, monocyte, natural killer cell and dendritic cell subsets expressed the retinol binding receptor (STRA6)(3). STRA6 initiates cellular retinol uptake , in immune cells for improving the immune system homeostasis in various populations (3).Many independent studies confirmed that vitamin A uptake mediated by STRA6 from holo-retinol/retinol binding protein complex (holo-RBP) is joined to intracellular proteins (4,5) and the mechanism by which it joins to specific intracellular proteins has been explained (6).It was recently showed that single nucleotide polymorphisms or mutations (SNPs) in STRA6 are connected with the recurrence of type 2 diabetes in humans (7). Moreover, Pasutto et al. (7)reported that STRA6 mutations associated with lung malformations and many heart , eye diaphragm as well as retardation in mentality as in syndrome of Matthew-Wood in humans, validating its reported functions in vitamin A uptake by cells as vitamin A/retinoic acid is very critical in the process of organogenesis. STRA6 mutations results in a broad spectrum of complication related to malformations counting congenital heart defects , lung hypoplasia , anophthalmia, alveolar capillary dysplasia, diaphragmatic hernia, and mental retardation (8).
Recent findings showed that mutations in the gene of STRA6 are connected to the congenital microphthalmia of eye malformations, coloboma and anophthalmia(7), (9) (10). STRA6 genetic null mutation in mice model leads to significant reduction of retinoid in the neurosensory retina and retinal pigment epithelium , diminished eye morphology and visual responses , despite the fact the last-mentioned complication is not as serious as in individuals with mutant STRA6 (11) .According to a recent publication, STRA6 is not only a receptor of vitamin A transporter, but it can also act as a cytokine receptor. Upon attaching to holo-RBP, STRA6 is directly phosphorylated at its region of tyrosine residue 643, which, in turn, triggers and recruits activation of STAT5 and the Janus Kinase 2 gene,( JAK2) (12). STRA6 seems to be very important receptor and transporter of vitamin A which critically participate in synthesis of retinoic acid which is the active and the main vitamin A metabolite . Nutrients that contains Vitamin A is needed by all mammals. It is required for the proper process of vision in its form of retinaldehyde (retinal). (13) ; as retinoic acids (RAs), it provides ligands for RAR (retinoic acid receptor) and RXR (retinoid X receptor) nuclear receptor transcription factors (14). Consequently, retinoid metabolism affects numerous biological processes (15) , with many disease implications from viral infection and cancer to blindness (16,17) . In the world Vitamin A deficiency is the third most popular nutritional deficiency, affecting of millions of children and pregnant women life (18) .

Retinoic acid(RA) is a morphogen
and important metabolite synthesized from vitamin A (retinol) (19). Two dehydrogenasecatalyzed enzymatic reactions are essential for the production of RA from retinol . Vitamin A( Retinol) is converted to retinal, which is then converted to RA. The RA interacts with retinoic acid X receptor (RXR) and retinoic acid receptor (RAR) which then regulate the expression of targeted gene (19).Based on the investigations and previous researches it is clear that RA play a major modulatory function in the immune system. Actually , retinol is an important hormone and immune system regulator.it participates with Zinc for improving the function of the immune system(20) . Retinoids are a molecules that possess qualitative activity relative to all-trans retinol (vitamin A), that includes all-transretinoic acid (RA) retinyl-esters and all-trans retinal (20) . RA is the biologically active retinoid metabolite that, works through its receptors RA receptors (RAR β, α and γ), regulates the generation of various genes involved many biological pathways including both innate and adaptive immune responses (21). Retinoids act as enhancers of the Tcell mediated innate immune responses and adaptive immunity via induction of antigen presenting dendritic cells (DCs), NK cells and innate lymphoid cells (ILCs) (21,22).
It has been established that retinoic acid induce gut-homing receptors on B cells , T cells and ILCs. A mounting body of evidence indicates that RA exert far-reaching impact on fate and functional differentiation of these lymphocytes(23).. Retinoids can directly stimulate the Messenger RNA (mRNA) expression of Interferon-stimulated gene (ISG) , including IFN regulatory factor 1 (IRF-1) and retinoic acid-inducible gene I(RIG-I) ( 24,25,26) .Furthermore, retinoic acid plays critical physiological roles in synaptic plasticity, learning and memory (27), hormone production(27,28) and adult neurogenesis(27). Retinoic acid insufficiency in the olfactory epithelium, both in mouse and chick models, causes progenitor cell maintenance failure and, consequently, olfactory neurons differentiation is not maintained . An explant system, showed that renewal of olfactory neurons is inhibited if retinoic acid synthesis was failed in the olfactory epithelium (29) . In the immune system, retinoic acid (RA), metabolite of vitamin A is known for its critical function in increasing gut-homing molecules in B and T lymphocyte cells, boosting tolerance and regulatory T cells (Tregs) (30,31). Synthetic and natural retinoids also have potent inhibitory effects on replication of many viruses, such as MeV, cytomegalovirus, influenza, norovirus and hepatitis B virus (HBV) (32,33) (27-30). There is additional evidence that retinoid signaling activation can effectively suppress coronaviruses. (34).
Our docking study reveals that COVID-19 spike protein binds directly to The integral membrane receptor (STRA6). STRA6 mediates cellular uptake of retinol (vitamin A) by recognizing a molecule of RBP-retinol to trigger release and internalization of retinol (1). Therefore COVID-19 may leads to downregulation of STRA6 receptor leading to inhibition the regulatory function of retinoic acid and helps in existing of pre and post-covid-91 infection symptoms and complications such as immune suppression (lymhopenia), Nuerogical disorders, ineffective RIG-I pathway, interferon inhibition, Cytokine storm, Diabetes, Hormonal imbalance, thrombosis, and smell loss.

Protein-Protein Interaction Network (STRA6 receptor and five interacting partners from both shells of interactions)
The STRA6 receptor protein were submitted to the server for functional interaction associated network between partners for the STRING (Research HDOCK server (http://hdock.phys.hust.edu.cn/) is a highly integrated suite of homology search, template-based modeling, structure prediction, macromolecular docking, biological information incorporation and job management for robust and fast protein-protein docking the server automatically predicts their interaction through a hybrid algorithm of template-based and template-free docking. The HDOCK server distinguishes itself from similar docking servers in its ability to support amino acid sequences as input and a hybrid docking strategy in which experimental information about the protein-protein binding site and small-angle X-ray scattering can be incorporated during the docking and post-docking processes also it has detailed important information about the Complex Template Information in protein protein docking like the ID of the protein which is receptor and ligand protein ID of the chain in each protein which its used in docking complex then the alien length and this important information can allowed us to know where exactly the receptor protein are bind to ligand protein then the quarry coverage and sequence identity to know if the protein are realable and good or its un realable . Correspondingly, new challenges have been presented during the development process of molecular docking approaches, as seen by the community-wide experiment, Critical Assessment of Prediction of Interactions (CAPRI: https://www.ebi.ac.uk/pdbe/complexpred/capri/)14,15. First, with the rapid development of structural genomics16, more and more protein-protein complex structures are being experimentally determined. As such, more information about the binding interfaces of involved proteins is becoming available in the PDB1.In addition, information about The docking energy scores , The ligand RMSDs from the input structures or modeled structures by homology modeling and The interface residues within 5.0 Å from their interacting partner or each other, and the corresponding distances about the residue contacts between proteins may also be derived through an evolutionary analysis in sequences17 or deep learning18. However, how to efficiently incorporate such binding interface information into docking

Molecular docking of proteins
The molecular docking of protein protein docking is a computational approach in which the binding mode of protein with other protein is assessed. The binding mode of Spike -ACE 2 and STRA6 receptor protein which its retrieved form the PDB https://www.rcsb.org/ with accession number (7DMU , 5sy1) representatively . the Spike -ACE 2 and STRA6 receptor protein was investigated to determine the conservative residues of binding of Spike protein with the ACE to be a control results also Spike with STRA6 receptor to know and discover if the spike protein of the virus are bind with STRA6 receptor in a good binding affinity to declare the mechanism of the interaction. protein .Docking study of each Spike -ACE 2 and STRA6 receptor protein were carried out using HDOCK server There are two working modes in the server :one is the default hybrid docking mode, and the other is the template-free docking mode. First, we put the spike protein after we download it from pdb in accession 6MOJ we enter it on SAMSON software to separate the ACE2 and Spike protein to make docking between them to get the result as a control result to test and know how the complex of spike , STRA6 receptor are how much its efficient and submit into the server , one for receptor (STRA6 receptor) and the other for ligand (spike), in which both amino acid sequences and PDB structures are supported. Then, the server do the template-based modeling of the receptor and ligand molecules by searching thePDB for putative homologous templates based on the sequences of proteins. Then it's found the PDB ID of the two protein then the HDOCK server perform global docking to sample putative binding modes through an FFT-based search method and then evaluate them without intrinsic scoring function for protein-protein interactions. Biological information, such as experimental data on the protein-protein binding site or SAXS profile, can be incorporated during the docking and/or post-docking processes. To provide information about The docking energy scores , The ligand RMSDs from the input structures or modeled structures of the interface residues within 5.0 Å from their interacting partner or each other, to get the corresponding distances about the residue contacts between proteins to assist in an evolutionary analysis in sequences17 or deep learning18.

Results and discussion
In this study, the Spike and STRA6 receptor protein was investigated for declare the mechanism of the interacting of the viral protein with the deferent symptoms of the patients and see the properties of the reaction for the first time, which could be used as alternative for therapeutic purposes. Here, the proteins of the Spike and STRA6 receptor were prepared and docked using HDOCK sever where the reaction are made between the spike protien and the STRA6 receptor for detection and declare the mode of action of the mechanism Complex Template Information between (STRA6 -SPIKE) Docking Score Ligand rmsd (Å) LGscore: MaxSub: LGscore: MaxSub: The interactive residues of target protein STRA6 and ligand spike protein that will help the researchers to design and develop the drugs that are more efficient and specific for their target protein. The docking of STRA6 target protein with spike viral protein which revealed the involvement of the spike protein into the extracellular and membrane part of the STRA6 receptor and amino acids residues of STRA6 along with spike protein which make interactions and play an important role in formation of complexes. STRA6-Spike protein complex with PDB ID (5SY1 , 6LZG) representatively ,the chain A ,B with Alignlength ( 582 ,194 ) then the quarry coverage of proteins 0.793 and 1.000 with sequence identity 96.2 % and 100,0 % representatively. The surface view of complex reveals that the binding pocket of STRA6-Spike protein and Spike ACE 2 complexes with RMSD (189.44 Å , 1.00 Å ) representatively and docking score (-341.21 ,-354.68) kcal/mol the quality of the receptor and the ligand are LGscore and MaxSub ( 2.416 , 0.147 ) where the structure are correct representatively for the STRA6 receptor protein, and LGscore and MaxSub ,(5.056 ,0.217) where the structure are very good and correct representatively for the Spike ligand protein see (table 1) .

Protein-Protein Interaction Network(STRA6 receptor and five interacting partners from both shells of interactions)
The protein-protein interaction associative network for the STRA6 receptor through STRING server. The active interaction sources were set based on the seven parameters including experiments, co-expression, gene fusion, cooccurrence, databases, text mining, and neighborhood. with a maximum of five interacting partners from both shells of interactions. The color nodes describe query proteins and the first shell of interactions, whereas white nodes are the second shell of interactors. The large node size represents characterized proteins and smaller nodes for characterized proteins which the number of nodes: 7, number of edges 10 , average node degree 2.86 , avg. local clustering coefficient 0.895 , expected number of edges 6 and also PPI enrichment p-value 0.0973 . The functional interactive network formed by STRA6 receptor protein was analyzed at the medium confidence level (0.40) has been shown in Figure(1).
The protein was shown to have an interaction with RBP4 Retinol-binding protein 4; Retinol-binding protein that mediates retinol transport in blood plasma , TTR ransthyretin; Thyroid hormonebinding protein. Probably transports thyroxine from the bloodstream to the brain , RBP1 Retinol-binding protein 1; Cytoplasmic retinol-binding protein. Accepts retinol from the transport protein STRA6 , CALM3 Calmodulin 3 (phosphorylase kinase, delta); Calmodulin mediates the control of a large number of enzymes, ion channels, , CALM2 Calmodulin 2 (phosphorylase kinase, delta); EF-hand domain containing and CALM1 Calmodulin-1; Calmodulin mediates the control of a large number of enzymes, ion channels, aquaporins and other proteins with score ( 0.983 , 0.881 , 0.733 , 0.551 , 0.421 and 0.421) representatively . also its interact with deferent processes biological processes ,molecular function , cellular compounds , reactome pathways , disease gen associations Tissue expression and also the subcellular localization

Analysis of binding energy in complex
The interactive residues of target protein STRA 6 and ligand spike protein that will help the researchers to design and develop the drugs that are more efficient and specific for their target protein. The docking of STRA6 target protein with spike viral protein which revealed the involvement of the spike protein into the extracellular and membrane part of the STRA6 receptor and amino acids residues of STRA6 along with spike protein which make interactions and play an important role in formation of complexes the corresponding distances about the residue contacts between proteins STRA6-Spike protein complex are in