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Lupine Publishers | A Hmsh2 c.2332 T>A Mutation and Membrane-hMSH2/ TCRγδ/NKG2D-Mediated Cytotoxicity of Human Vγ9vδ2 T Cells in Ovarian Serous Cystadenocarcinoma

 Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

Membrane human MutS homologue 2 (mhMSH2) is a well-characterized endogenous ligand for human Vγ9Vδ2 T cells, but its germline gene expression in mhMSH2-overexpressing ovarian serous cystadenocarcinoma (OSC) cells and tissues is unclear. Herein, we discovered a silent hmsh2 c.2332 T>A mutation in the OSC HO8910 cell line (GenBank accession no.MG674653) and identified serval novel soluble forms of hMSH2 protein from its whole cell protein extracts and culture supernatant. The mhMSH2/ TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells were validated in mhMSH2/6-overexpressing SKOV3 cells. Positive expression of cytoplasmic and/or membrane hMSH2/6, cytoplasmic and/or nuclear hMSH3 and complete loss of nuclear expression of hMSH2 was observed in all examined ovarian cancer tissues. The clinical significance of the novel hmsh2 c.2332 T>A mutation, the soluble full-length and truncated forms of hMSH2 proteins, and the complete loss of nuclear hMSH2 protein expression in OSC development and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further elucidated.

Keywords: Hmsh2; Mutation; Membrane hMSH2; Vγ9Vδ2 T cells; Ovarian serous cystadenocarcinoma.

Abbreviations: hMSH2: Human MutS Homologue 2; MMR: Mismatch Repair; M: membrane; EBV: Epstein-Barr virus; OSC: Ovarian Serous Cystadenocarcinoma; ATCC: American Type Culture Collection; IHC: Immunochemistry; mAb: monoclonal Antibody; FCM: Flow Cytometry; PFA: Paraformaldehyde; FITC: Fluorescein Isothiocyanate; DAPI: 4’,6-diamidino-2-phenylindole; LS: Lynch Syndrome.

Introduction

Human MutS homologue 2 (hMSH2), a critical element of the DNA mismatch repair (MMR) system, is normally localized in the nucleus of host cells and dimerizes with hMSH3 or hMSH6 (hMSH3/6) to form complexes that participate in DNA damage and cell apoptotic signaling. Inherited or acquired defects in the hmsh2 gene or protein lead to dysfunctional correction of errors in DNA synthesis and duplication, the cell cycle and Ig isotype switching of B cells and thus have a close association with the genesis of many types of tumors [1,2]. In our published study, we reported a broad overexpression of membrane hMSH2 (mhMSH2) in a series of epithelial tumor cell lines and Epstein-Barr virus (EBV)- transformed B lymphoid cells [3]. mhMSH2 overexpressed on malignant cells was subsequent characterized as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells, a subset of innate immune cells that play a crucial role in antitumor/antiviral immunity and autoimmunity [3-5]. The induction of ectopic mhMSH2 expression on renal carcinoma cells by oxidative stress via the p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways with interleukin-18 promotion was subsequently revealed, but the systemic expression of hmsh2 gene in mhMSH2- overexpressing carcinomas is still unknown [6].
There are 238,700 estimated new cases of ovarian cancer and 151,900 deaths per year worldwide, and thus, this disease is the fifth most common cause of female cancer-related death in the United States in 2018 [7,8]. Among the 4 subtypes of ovarian cancer (high-grade serous, endometrioid, clear cell ovarian carcinomas and non-epithelial subtypes), Ovarian Serous Cystadenocarcinoma (OSC) is the most common and lethal type in women [9]. Previously, we reported the unusual ectopic mhMSH2 expression on the human OSC cell lines HO8910 and SKOV3 and the specific binding of mutated mhMSH2 on SKOV3 cells to synthesized OT3 peptides of human Vδ2 TCR [3,10]. In this work, we further elucidated the hmsh2 gene mutation and ectopic subcellular protein expression in HO8910 cells and the mhMSH2-mediated recognition and cytolytic effects of Vγ9Vδ2 T cells towards SKOV3 cells. The abnormal subcellular distribution of hMSH2/3/6 in different subtypes of ovarian cancer tissues was demonstrated by immunochemistry (IHC).

Materials and Methods

Cell lines, culture medium and tumor tissues

HO8910 cells (OSC) were maintained in RPMI-1640 complete medium (Invitrogen, Shanghai, China). SKOV3 (OSC) and HK-2 (proximal tubular cell line derived from normal kidney) cells were cultured in 10% FBS DMEM/F12 medium (Niuyin, Beijing, China). All cell lines were purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences and confirmed to have no mycoplasma contamination before use. Expansion and subset separation of effector human Vγ9Vδ2 T cells were carried out as we previously described [3]. Ovarian cancer tissues belonging to different histotype (OV-1, serous adenocarcinoma; OV-2, embryo sinus carcinoma; OV-3, cyst-myxomatous adenoma) were freshly obtained from Peking Union Hospital with informed consent provided by the patients. The use of human tissues for research was also approved by the ethics committee of the Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou Institute of Pediatrics (2015020917).

Gene sequencing of hmsh2 in HO8910 cells

Appropriate numbers of HO8910 cells in the logarithmic growth phase were collected for total mRNA extraction and cDNA reverse transcription. The target hmsh2 gene was amplified using fragment PCR as we described in Subcellular hMSH2 expression is aberrant in membrane-hMSH2-overexpressing cervical, lung and gastric cancer cell lines and tissues (article in progression). Three PCR products with predicted lengths (990, 1250 and 1549bp) were purified and introduced into the pGEM-T easy vector (Promega, USA). Recombinant plasmids were positively selected and characterized by Not I digestion before gene sequencing. Human msh2 variants in HO8910 cells were screened by alignment with the full-length hmsh2 sequence (NM_000251) using Laser gene 7 software.

Soluble full-length and truncated forms of hMSH2 in the whole cell extracts and culture supernatant of HO8910 cells

Soluble forms of hMSH2 protein in the complete protein extracts and the cell culture supernatant of HO8910 cells were simultaneously separated by SDS-PAGE and blotted with antihMSH2 McAb (65021-1 Ig, 1:500, Proteintech Group, USA) and with goat anti-mouse IgG/HRP secondary antibody (1:5000, Zhongshan Jinqiao Company, China). 𝛽-actin was blotted as endogenous reference control (MW 43 kDa).

Growth curves of HO8910 cells

For growth curves of both OSC cell lines, appropriate numbers of trypsin-digested HO8910, SKOV3 and HK-2 cells were planted in 24-well plates in triplicate and cultured for 8 consecutive days in a 5% CO2 atmosphere at 37°C. Growth curves of the examined cell lines were obtained by using the cell counting method. The expansion rates of target OSC cells were compared with that of HK-2 control cells.

Quantitative real-time PCR for hmsh2 mRNA expression in HO8910 cells

The human msh2 gene transcription levels in HO8910, SKOV3 and HK-2 cells were comparatively analyzed by qRT-PCR. The results were processed with Sequence Detector Version 1.2 (Applied Biosystems, USA) and Sigma Plot 11.0 as we previously described [3].

Ectopic membrane, cytoplasmic and nuclear expression of hMSH2 in HO8910 cells

The membrane, cytoplasmic and nuclear hMSH2 expression in HO8910, SKOV3 and HK-2 cells were further investigated by laser confocal microscopy with different fixation reagents. The target cells (2-3×105) were cultured overnight on pre-autoclaved glass cover slips in a 24-well format, properly fixed with 4% paraformaldehyde (PFA) or ice-cold methanol for 10-15 min and blocked with 0.5% BSA for 30 min at 4°C before labeling with specific anti-hMSH2 (N- 20, Santa Cruz Biotechnology, USA) polyclonal antibody or rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (Zhongshan Jinqiao Company, Beijing, China). The nuclei of the examined tumor cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:1000, Sigma, USA) before being mounted on a Leica DMIRE2 inverted microscope (objective, 40×; numerical aperture, 1.25).

Participation of mhMSH2/TCRγδ/NKG2D in Vγ9Vδ2 T cell-mediated anti-OSC immunity

For further analysis of the role of mhMSH2 in human Vγ9Vδ2 T cell-mediated anti-OSC immunity, effector Vγ9Vδ2 T cells were incubated with SKOV3 cells at different ratios (effector: target [E: T] 20:1, 10:1, 5:1 and 2.5:1). Cytotoxicity was measured with the LDH method, as we previously described [3]. For cytotoxicity blockade assays, target OSC cells were pretreated with anti-hMSH2 (N20, Santa Cruz Biotechnology), anti-NKG2D (149810, R&D Systems) and anti-TCRγδ (B1.1, Immunotech, France) antibodies before incubation with effector Vγ9Vδ2 T cells at different E:T ratios (20:1, 10:1, 5:1 and 2.5:1). The blockade cytotoxicity was measured and compared to that of the rabbit IgG blockade control group. Target SKOV3 cells were stained with anti-hMSH2, anti-hMSH3 and anti-hMSH6 antibodies to confirm the cell surface expression of hMSH2/3/6 antigens before cytotoxicity and antibody-blocking cytotoxicity assays.

Subcellular hMSH2/3/6 Distribution In OSC Tissues

For IHC analyses, freshly collected ovarian cancer tissues were classically made into paraffin sections (3-4 μm in thickness) after proper neutral formalin fixation. The biopsies were heated at 60°C overnight and gradient dehydrated with methanol before antigen retrieval in boiled citrate buffer, followed by overnight incubation with purified mouse anti-hMSH2 monoclonal antibody (mAb) (clone G219-1129,1:200), mouse anti-hMSH3 mAb (clone 52, 1:20), mouse anti-hMSH6 mAb (clone 44, 1:30) (BD Pharmingen, USA) or isotype-matched mIgG1 at 4°C in a moist environment after 3% H2O2 treatment. The coloration was developed with PV9000 reagents (Zhongshan Jinqiao Company, Beijing, China) and captured with a Leica DM3000 imaging system.

Statistical Analysis

GraphPad Prism 7.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis and data plotting. Data are presented as the mean ± SD. Differences between/among groups were compared with Student’s t-test (two-tailed) or one-way ANOVA, P<0.05 was considered statistically significant.

Results

Hmsh2 gene sequencing and soluble full-length and truncated hMSH2 protein blotting

 

The PCR products of hmsh2 in HO8910 cells were 990, 1250 and 1549 bp in length (Figure 1A). By aligning the spliced full gene sequence with hmsh2 (NM_000251), we observed a point mutation (hmsh2 c.2332 T>A) in the HO8910 cell line (Figure 1B). It was a silent mutation that did not alter the primary structure of hMSH2 protein. The gene sequencing data of mutated hmsh2 in HO8910 cells were submitted to GenBank (Accession number: MG674653) for release. No additional gene mutation of hmsh2 was found in other mhMSH2-overexpressing epithelial tumor cell lines (data not shown here). By SDS-PAGE separation and western blots, several soluble forms of hMSH2 proteins were identified from the whole HO8910 cell protein extract [including the full-length form (MW ~105 kDa) and four truncated variants (MW~70, ~62, ~55 and ~40 kDa)] and the cell culture supernatant (MW~88, ~68 kDa) (Figure 1C).

Hmsh2 gene transcription and ectopic cytoplasm/ membrane expression

The growth and proliferation of both OSC cell lines were depicted with growth curves determined by cell counting assays. Both HO8910 cells and SKOV3 cells achieved rapid growth on day 5 and expanded much faster than the normal control HK-2 cells (Figure 2A). The mRNA expression of hmsh2 was slightly decreased in SKOV3 cells but substantially elevated in HO8910 cells compared with HK-2 cells (Figure 2B). Further detection of the ectopic membrane overexpression of hMSH2 on target OSC cells with laser confocal microscopy showed that both HO8910 and SKOV3 displayed different degrees of green fluorescence on the cell surface after labelling with a specific anti-hMSH2 antibody. The cell surface green fluorescence was much stronger on SKOV3 cells than on HO8910 cells. No green fluorescence was observed on normal control HK-2 cells (Figure 2C, the upper panels of pictures). The subcellular distribution of hMSH2 in HO8910 and SKOV3 cells was further determined by confocal microscopy with ice-cold methanol fixation. The membranes of both cells displayed different degrees of green fluorescence. The density of green fluorescence in the cytoplasm and the nuclei of SKOV3 cells was much stronger than that of HO8910 cells. There was no green fluorescence observed in HK-2 cells (Figure 2C, the lower panels of pictures).

 

Previously, we identified the membrane-overexpressed hMSH2 on several epithelial tumor cell lines as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells [3,6]. Herein, mhMSH2-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells towards target OSC cells were confirmed by cytotoxicity and independent specific antibody blockade cytotoxicity assays. mhMSH2 overexpression on OSC targets was validated with flow cytometry (FCM) before cytotoxicity and cytotoxicity blockade assays (Figure 3A). At E:T ratios of 20:1, 10:1, 5:1 and 2.5:1, the cytotoxic efficiency of effector human Vγ9Vδ2 T cells against target SKOV3 cells was 53%, 26%, 23% and 12%, respectively (Figure 3B). The ectopic mhMSH2-mediated recognition and cytotoxicity could be strongly blocked by specific anti-hMSH2, anti-NKG2D or anti-TCRγδ antibodies at different E:T ratios, indicating the participation of mhMSH2 in Vγ9Vδ2 T cell-mediated anti-OSC immunity by NKG2D/γδ TCR recognition (Figure 3C).

 

Subcellular distribution of hMSH2/3/6 in OSC tissues

As shown in Table 1, all ovarian cancer cells displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, while loss of nuclear expression of hMSH2 was observed in all 3 categories of ovarian cancer tissues. Strong and clear cytoplasmic and/or membrane hMSH2/3/6 expression was strikingly observed in ovary cyst-myxomatous adenoma nests (Figure 4A). Total loss of nuclear expression of hMSH6 was found in 66.67% (2/3) of the examined ovarian cancer tissues (Figure 4A). hMSH2/3/6 expression showed substantial heterogeneity among individual ovarian cancer patients (Figure 4B).

 

Discussion

Human msh2 is one of the most crucial genes involved in the DNA MMR pathway and was strongly associated with increased tumor mutational burden in a multivariate analysis [11]. Recent studies have shown that high hMSH2 expression is significantly associated with smoking, while low hMSH2 expression is an indicator of MMR deficiency in lung adenocarcinoma [11]. The high expression of hMSH2 accompanied by increased PD-L1 expression and CD8+ T cell infiltration therefore leads to the development of a prominent immunotherapy-responsive microenvironment for lung adenocarcinoma and acts as a potential surrogate biomarker of tumor mutational burden to identify immune checkpoint blockade responders in this disease [11]. Moreover, recent studies revealed that MSH2-MSH6 played a crucial role in activation-induced deaminase-initiated antibody diversity by recognizing uracil(s) in the Ig gene loci to generate DNA breaks [12]. Many studies have identified mutations in hmsh2 as diagnostic and/or prognostic factors in carcinomas not only in the US and Canada but also in the Middle East and China [13-15]. Human msh gene mutations also have strong potential as novel candidate triple-negative breast cancer predisposition genes and are closely associated with acute adverse events and survival in rectal cancer patients receiving postoperative chemoradiotherapy [16-19]. Pathogenic or likely pathogenic germline mutations in pms2, msh2 or msh6 were detected in 0.5% (6/1,179) of lung cancer patients [18]. In this study, we screened a novel mutation in the gene sequence of hmsh2 at c.2332 T>A in the OSC cell line HO8910. This is a silent mutation that theoretically does not result in a change to the amino acid sequence of hMSH2 protein or to the phenotype of HO8910 cells. However, a rapid expansion and a strong increase in hmsh2 mRNA expression were observed in this cell line compared with the control cell line.
Moreover, several soluble full-length or truncated variants of hMSH2 proteins were observed in the whole cell protein extract and the culture supernatant of HO8910. We deduce that these altered biological characteristics of HO8910 cells and the genesis of OSC might be linked to the hmsh2 c.2332 T>A variation, as evidence showed that germline mutations of hmsh2 and its family members were closely associated with the pathogenesis of Lynch syndrome (LS). For example, hmsh2 c.2152 C>T alteration has been recently reported as a founder mutation in Portugal; its high proportion implies combined screening for this mutation and some other previously reported founder mutations will be helpful in the genetic testing of Portuguese families with suspected LS [19]. The occurrence and clinical significance of hmsh2 c.2332 T>A and soluble full-length and truncated forms of hMSH2 proteins in clinical OSC patients remain to be clarified in the future.

Compared with HO8910 cells, SKOV3 cells displayed stronger ectopic membrane and nuclear expression of hMSH2 in laser confocal microscopy and FCM analyses, suggesting better membrane anchoring and a more efficient nuclear importing system in SKOV3 cells. The ability of the notably overexpressed mhMSH2 on SKOV3 cells to promote human Vγ9Vδ2 T cell-mediated recognition and cytotoxicity via TCRγδ/NKG2D receptors towards target OSC cells was later validated by independent cytotoxicity and specific antibody blockade cytotoxicity assays, providing further evidence for mhMSH2 functioning as an OSC-associated self-antigen (ligand) for human Vγ9Vδ2 T cells in anti-ovarian cancer immunity [10]. In the absence of information/data demonstrating that the observed mutation impacts function, one cannot determine if the mutation is physiologically relevant. In further investigations on the subcellular distribution of hMSH2 and its companion proteins in ovarian cancer tissues, we found that all examined ovarian cancer specimens (serous adenocarcinoma, embryo sinus carcinoma, cystmyxomatous adenoma) displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, and total loss of nuclear expression of hMSH2/6 commonly occurred in almost all of the ovarian cancer tissues. Considering the high hmsh2 gene transcription and the loss of nuclear distribution of hMSH2 protein in HO8910 cells, we hypothesized that the nuclear importing system of hMSH2 and/ or hMSH6 protein was dysfunctional. By contrast, recently, it has been reported that a high overall rate (16.2%) of MMR deficiency was surprisingly observed in ovarian endometrioid carcinoma and was significantly associated with increased IFOG (International Federation of Obstetrics and Gynecology) grade and CD8+ intraepithelial lymphocyte infiltration but not with cancer-specific death [19]. These findings suggest a promising future in which loss of nuclear expression of hMSH2 and/or hMSH6 protein may function as effective screening/diagnostic markers or therapeutic targets for different subtypes of ovarian cancers and as endogenous immune ligands not only for Vγ9Vδ2 T cells [20]. We expected that a human cell-based assay system for functional testing of hMSH2 and its family members will facilitate the identification of highrisk ovarian carcinoma patients and the generation of individual autoantigen-targeted immunotherapies for OSC.

Conclusion

In this study, we identified a silent hmsh2 c.2332 T>A mutation (GenBank accession no. MG674653) along with serval novel soluble truncated forms of hMSH2 variants in HO8910 cells and validated the mhMSH2/TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells against mhMSH2/6-overexpressing SKOV3 cells. We also demonstrated the abnormal subcellular distribution of hMSH2/3/6 in the examined ovarian cancer tissues. The clinical significance of the critical findings in OSC genesis and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further investigated.

 

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Lupine Publishers | Comparative in Silico Analysis of Antigenic Proteins “Nucleocapsid Protein “of SARS-Cov-2 from Different Geographical Locations

 Lupine Publishers | LOJ Pharmacology & Clinical Research

Abstract

SARS-CoV-2 Nucleocapsid protein considered as a vaccine target. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 in different geographical locations considered as an important factor in order to produce a global vaccine for the pandemic. The present investigation relies on the analysis of the similarities and differences of the SARS-CoV-2 Nucleocapsid protein from various geographical locations, considering certain protein parameters, as well as the study of the alignment of the protein sequence of amino acids.

Keywords: Nucleocapsid protein; SARS-CoV-2; geographical locations; bioinformatics

Introduction

SARS-CoV-2 is a single-stranded, positive-sense RNA virus with a 30 kb genome, one of the largest among RNA viruses [1]. The viral envelop of SARS-CoV-2, contains many proteins such as spike protein, and glycoprotein [2]. Another protein incorporated with the viral genome is nucleocapsid protein which is responsible for protection the virus from the host cell environment [3]. The SARSCoV- 2 spike protein is being used as the principal antigen target in vaccine progress. However, the multifaceted molecular details of viral entrance may lead to obstacles with the vaccine reaction [4]. The nucleocapsid (N) protein is a significant antigen for coronavirus, which contribute to RNA package and virus particle discharge [5]. After infection, the N protein enters the host cell along with the viral RNA to facilitate its replication and to process the assembly and release of the virus. SARS-CoV N comprises two distinct RNA-binding domains (N-terminal domain and C-terminal domain) connected by a poorly structured linkage region. Due to positive amino acids, SARS-CoV N-terminal domain and C-terminal domain have been documented to bind to the viral RNA genome [6]. Serological diagnosis has shown that the unique antibodies to the N protein in the serum of SARS patients have a higher sensitivity and longer persistence than those of other structural SARS-CoV proteins. In addition, anti-N antibodies have been observed at an early stage of infection with a high specificity. Thus, any information obtained from the study of this protein, whether in vivo or in vitro, will improve our understanding of COVID-19 and enable us to develop better biological agents for the treatment or diagnosis of diseases [7]. It is becoming clearer how important this protein is, to the multiple stages of the viral life cycle. This studie provide valuable and timely insights specific to the SARS-CoV-2 N protein, a vaccine target that has some distinct advantages over other possible SARSCoV- 2 antigens according to geographical location. Because of the conservation of the N-protein sequence, the increased awareness of its genetics and biochemistry regarding different geographical location, the N-protein SARS-CoV-2 should be considered as a global vaccine candidate for SARS-CoV-2. The present investigation relies on study the similarities and difference of Nucleocapsid protein “of SARS-CoV-2 from different geographical locations considering some protein parameters as well as study the alignment of amino acid sequence of protein.

Materials and Methods

Sequences, alignment, and construction of phylogenetic tree

Amino acids sequences for the Nucleocapsid protein of SARSCoV- 2 from different geographical locations were obtained from the National Center for Biotechnology Information database. The accession numbers of the corresponding database entries and isolation source are listed in Table 1. Multiple sequence alignment of Nucleocapsid protein of SARS-CoV-2 was performed in order to find the evolutionary relationships between sequences from different geographical location to identify shared patterns. Sequences were multiply aligned Jalview software version 2.8 [8]. Phylogenetic trees were constructed with neighbour-joining using MEGA [9]. A distance matrix was generated using the model building Jones-Taylor-Thornton [10]. Graphical way of representing and visualizing consensus data developed of amino acid multiple sequence alignment developed were displayed according to method described by Tom Schneider and Mike Stephens [11].

Computation of amino acid composition and molecular weight of protein sequences

Estimation of the amino acid composition and molecular weight was determined using Isoelectric Point Calculator (IPC), a web service and a standalone program for the accurate estimation of protein and peptide characteristic [12].

Solvent content of protein crystals

From the currently available sequence, of the solvent content of Nucleocapsid protein. The fraction of the crystal volume occupied by solvent was calculated according to Matthews [13].

Results

Table 2 is displayed the chemical composition of the Nucleocapsid protein “of SARS-CoV-2 from different geographic location. The preset data revealed that amino acid composition of the tested 13 sequences showed high similarity except for the protein sequence from Spain. Figure 1 represented the overall height of the stack indicates the amino acid sequence conservation at each position of protein, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. In general, sequence logo of Nucleocapsid protein provides that almost the sequence from different location did not differ from each other and revealed high similarity. Regarding Phylogeny estimation for Nucleocapsid protein of SARS-CoV-2 from different geographical locations. Neighbour-joining tree constructed using Mega 10.1.8. The multiple sequence alignments are depicted in Figure 2. Constructed phylogenetic tree is depicted in Figure 3. The current results indicated that; Neighbour-joining tree of Nucleocapsid protein of SARS-CoV-2 displays the comparison of amino acid sequences of Nucleocapsid protein from different location. The results shows that the phylogenetic analysis rooted by two clusters (Figure 3). Cluster 1 represents France, Australia, China, India, South Africa, Morocco, United Kingdom and Spain. In the Cluster 1 Spain considered as the main root. Cluster 2 represents USA, Canada, Argentina, Saudi Arabia and Nigeria. The main root of the cluster 2 is Canada. Figure 4 shows Matthews coefficient , the current results revealed that the parameter value equal to 0.68 for all locations except for Spain its value reaches 0.85. Figure 5 shows the results for Solvent content of protein crystals were near 80 % for all geographical locations but has been observed that the value calculate for Spain was the lowest equal to 44%. As showed in Table 3 molecular weight of Nucleocapsid protein “of SARS-CoV-2 from different geographic location ranges from 36759.93 to 45696.7 Dalton, exhibited low variability in molecular weight.

The current study demonstrated that the Nucleocapsid protein analysis from different location shared a common primary amino acid composition which resulted in a uniform sequence alignment. Previously reports declared that alignments are a powerful way to compare related protein sequences. They can be used to capture various facts about the sequences aligned, such as communal evolutionary descent or shared structural function [14]. The obtain composition information of SARS-CoV-2 Nucleocapsid protein revealed high similarity in different geographic location with mild variability in amino acid concentration. The study compared aminoacid distributions between different locations. When looking at overall amino acid frequencies it could be recognized that byand- large, amino acid frequencies in all investigated sequences mirrored to each other except for the sequence obtained from Spain. The biggest differences arose in all amino acids except for Histidine, Isoleucine, Tryptophan and valine. According to Jackson et al. [15] there are several patterns of sequence variation that are consistently seen in natural proteins.
The first steps in a macromolecular structure determination of protein are to detect the count of molecules in the crystallographic asymmetric unit. The crystal volume per unit of protein molecular weight, known as Matthews coefficient. A substantial percentage of the protein crystals volume is employed by solvent ranged from 27% to 78%, with the most common value being about 43% [16]. The Matthews Coefficient and solvent content are calculated for Nucleocapsid protein “of SARS-CoV-2 from different geographic location which revealed almost the same results or all locations except for Spain. Water plays an important role in the structure of biomolecules and often influences protein function [17]. Water molecules not only affect protein folding, but also mediate biological processes such as enzymatic reactions and molecular recognition. Information about the fraction of water (solvent) plays a significant role in the X-ray structure determination process [18]. The present results revealed that solvent content for Nucleocapsid protein of SARS-CoV-2 from different geographical locations were almost similar except for Spain.

Conclusion

In the current study, the variation of Nucleocapsid protein of SARS-CoV-2 composition and its alignment were investigated for different location, it revealed high similarity for each other and showed a uniform sequence alignment with mild variability in amino acid concentration. The crystal volume per unit of protein molecular weight of Nucleocapsid protein “of SARS-CoV-2 revealed almost the same results or all locations except for Spain

 

 

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