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The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation site...
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Context 1
... there was no evidence of a sialic acid component in the N-glycans of the HCoV-19 S protein. A total of 220 N-glycans were found in hACE2; of these, 78.2% were complex (Fig. 3(a)), while hybrid, high-mannose, and pauci-mannose glycan only constituted 10.9%, 5.0%, and 5.9%, respectively (Fig. 2 (b) and Table 3). Based on LC-MS intensity, the most dominant hACE2 glycan was NeuGc1Hex5HexNAc4, which was predicted to be a bi-antennary complex containing a sialic acid in the distal end. ...Context 2
... our data suggest that the glycosylation status does not contribute directly to the binding between HCoV-19 S and hACE2. A detailed summary of the BLI binding kinetics can be found in Table S3 in Appendix A. Fig. 4. Impact of glycosylation on binding between the HCoV-19 spike protein and hACE2. Binding of the HCoV-19 S protein and hACE2 was measured by bio-layer interferometry. ...Citations
... While not being directly encoded in the viral genetic sequence (14), glycans act as a protective barrier, camouflaging the underlying protein structure from the host immune system, thus, promoting the virus infection (12,15,16). Among experimental studies on elucidating the role of spike-glycan interactions (9,10,12,13,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), the work of Huang et al. demonstrated that enzymatic removal of glycans from the SARS-CoV-2 spike protein enhances immune responses and protection in animal models, further highlighting the significance of glycans in viral pathogenesis and vaccine development (22). ...
... Regardless of the conformational state and the protein structural changes along the trajectories, the glycans maintained a stable level of exposure to the surrounding solvent molecules. Such stability suggests the formation of a consistent and robust protective barrier, effectively camouflaging (17)(18)(19)23) the underlying protein structure from the external environment. Prior literature underscores the crucial role of glycan camouflage in CoV spike proteins (i.e, SARS-CoV-2), impacting host attachment, immune responses, and virion assembly (11, 16-19, 35, 36). ...
To develop therapeutic strategies against COVID-19, we introduce a high-resolution all-atom polarizable model capturing many-body effects of protein, glycans, solvent, and membrane components in SARS-CoV-2 spike protein open and closed states. Employing µs-long molecular dynamics simulations powered by high-performance cloud-computing and unsupervised density-driven adaptive sampling, we investigated the differences in bulk-solvent-glycan and protein-solvent-glycan interfaces between these states. We unraveled a sophisticated solvent-glycan polarization interaction network involving the N165/N343 residues that provide structural support for the open state and identified key water molecules that could potentially be targeted to destabilize this configuration. In the closed state, the reduced solvent polarization diminishes the overall N165/N343 dipoles, yet internal interactions and a reorganized sugar coat stabilize this state. Despite variations, our glycan-solvent accessibility analysis reveals the glycan shield capability to conserve constant interactions with the solvent, effectively camouflaging the virus from immune detection in both states. The presented insights advance our comprehension of viral pathogenesis at an atomic level, offering potential to combat COVID-19.
... Considering the proposed persistence of spike protein in long-COVID syndrome, this study aimed to specifically investigate the presence of viral and vaccine spike proteins in the blood serum of long-CO-VID syndrome patients using mass spectrometry analysis 46,47 . Secondly, polymerase chain reaction (PCR) was used for a preliminary study to check for SARS-CoV-2 integration in the long-COVID patients' leukocytes 18 , without further investigation 48,49 . ...
... Official data sustain that the vaccine spike protein remains in the vicinity of the injection site and local lymph nodes and that it may persist in the body up to a few weeks after vaccination [20][21][22][23][24] . Our findings, in alignment with other studies and in contradiction with official data, show the presence of both the vaccine and the viral spike protein in the bloodstream even after infection clearance and several months after vaccination [40][41][42][45][46][47][48][49] . Furthermore, viral integration in patients' leukocytes was assessed with a preliminary study following the protocol of Merchant 18 , without further investigation (Supplementary Data). ...
Objective:
COVID-19 patients experience, in 10-20% of the cases, a prolonged long-COVID syndrome, defined as the persistence of symptoms for at least two months after the infection. The underlying biological mechanisms of this syndrome remain poorly understood. Several hypotheses have been proposed, among which are the potential autoimmunity resulting from molecular mimicry between viral spike protein and human proteins, the reservoir and viral reproduction hypothesis, and the viral integration hypothesis. Although official data state that vaccinal spike protein is harmless and remains at the site of infection, several studies proposed spike protein toxicity and found it in blood circulation several months after the vaccination. To search for the presence of viral and vaccine spike protein in a cohort of long-COVID patients.
Patients and methods:
In this study, we employed a proteomic-based approach utilizing mass spectrometry to analyze the serum of 81 patients with long-COVID syndrome. Moreover, viral integration in patients' leukocytes was assessed with a preliminary study, without further investigation.
Results:
We identified the presence of the viral spike protein in one patient after infection clearance and negativity of COVID-19 test and the vaccine spike protein in two patients two months after the vaccination.
Conclusions:
This study, in agreement with other published investigations, demonstrates that both natural and vaccine spike protein may still be present in long-COVID patients, thus supporting the existence of a possible mechanism that causes the persistence of spike protein in the human body for much longer than predicted by early studies. According to these results, all patients with long-COVID syndrome should be analyzed for the presence of vaccinal and viral spike protein.
... The best option for determining nAbs are the plaque reduction neutralization tests (PRNTs) and pseudo-typed virus neutralization tests (PVNTs), which require high-cost infrastructure and are time-consuming, further increasing the gap in testing capacity, especially in low-income countries. Despite limitations, surrogate virus neutralization tests (sVNTs) have emerged as affordable and fast alternatives for studying humoral neutralizing responses (Tan et al., 2020). By its simple design based on the blockade of RBD-ACE2 binding in multiwell plates, sVNTs can be produced in-house and achieved in standard laboratories, giving sovereignty in vaccine decision-making, notably when lacking PRNTs or PVNTs capabilities. ...
... However, in this case, it allowed us to detect differences in binding accurately (if any) using virtually the same analyte injection, thus avoiding protein quantification biases. Furthermore, despite showing differences with ITC and SPR (using RBD clv as an analyte), binding affinities remained invariable despite deglycosylation of captured RBD-ST (Figure 5b,c; Supporting Information Table S10), thus showing that N-glycosylation does not affect its binding, as previously shown (Sun et al., 2020). ...
The interaction between the receptor‐binding domain (RBD) of the spike glycoprotein of SARS‐CoV‐2 and the peptidase domain of the human angiotensin‐converting enzyme 2 (ACE2) allows the first specific contact at the virus–cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost‐effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep‐tag with yields >40 mg L⁻¹ in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep‐tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep‐Tactin‐HRP as a proof‐of‐concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in‐house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations.
... Furthermore, an investigation of N protein ubiquitination in cells observed lysine 169, 374, and 388 to be ubiquitin-modified. However, there was no follow-up functional study [19]. ...
Viruses, such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect hosts and take advantage of host cellular machinery for genome replication and new virion production. Identifying and elucidating host pathways for viral infection is critical for understanding the development of the viral life cycle and novel therapeutics. The SARS-CoV-2 N protein is critical for viral RNA (vRNA) genome packaging in new virion formation. Using our quantitative Förster energy transfer/Mass spectrometry (qFRET/MS) coupled method and immunofluorescence imaging, we identified three SUMOylation sites of the SARS-CoV-2 N protein. We found that (1) Small Ubiquitin-like modifier (SUMO) modification in Nucleocapsid (N) protein interaction affinity increased, leading to enhanced oligomerization of the N protein; (2) one of the identified SUMOylation sites, K65, is critical for its nuclear translocation. These results suggest that the host human SUMOylation pathway may be critical for N protein functions in viral replication and pathology in vivo. Thus, blocking essential host pathways could provide a novel strategy for future anti-viral therapeutics development, such as for SARS-CoV-2 and other viruses.
... Advanced mass spectrometry (MS) techniques have greatly facilitated the analysis of N-and O-glycoforms of SARS-CoV-2 S proteins by directly characterizing glycancontaining peptides derived from proteins [6,23,24,[37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56]. The glycosylation profiles of S have been determined mostly with various constructs of recombinant proteins, including 2-(or 6-) proline stabilized ectodomain, and separate subunits of S1, S2, and RBD, expressed in human, monkey, or insect cells. ...
N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.
... It is worth remarking that although MD simulations have highlighted the importance of N-linked glycosylation of the receptor in the interaction with the RBD, 13,44-51 experiments showed that ablating the ACE2 glycans has a minimal impact on the dissociation constant. [50][51][52][53][54][55][56] In addition, N-linked glycosylation is not required for ACE2 activity either. 55 Thus, given the dynamical nature of the polysaccharides, as vividly illustrated by Casalino et al., 11 at least to a first approximation, it is consistent with the ENM, which accounts for small fluctuations around the experimental structure, to ignore the glycans. ...
SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.
... Spike (S) glycoprotein forms the crown-like structure at the outer surface of the virus that consists of two subunits, S1 which facilitates the binding of human angiotensin-converting enzyme-2 (hACE2) receptors and S2 which facilitates fusion between the host and the viral cell membrane. Interestingly, mutational behavior of receptor-binding domain (RBD) of S protein and the presence of polybasic furin cleavage sites (that facilitates the strong binding of S-glycoprotein to hACE2) and O-linked glycans are genomic features of SARS-CoV-2 that play an important role for binding efficacy to the host [38][39][40][41][42]. ...
The global anxiety and economic crisis causes the deadly pandemic coronavirus disease of 2019 (COVID 19) affect millions of people right now. Subsequently, this life threatened viral disease is caused due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, morbidity and mortality of infected patients are due to cytokines storm syndrome associated with lung injury and multiorgan failure caused by COVID 19. Thereafter, several methodological advances have been approved by WHO and US-FDA for the detection, diagnosis and control of this wide spreadable communicable disease but still facing multi-challenges to control. Herein, we majorly emphasize the current trends and future perspectives of nano-medicinal based approaches for the delivery of anti-COVID 19 therapeutic moieties. Interestingly, Nanoparticles (NPs) loaded with drug molecules or vaccines resemble morphological features of SARS-CoV-2 in their size (60-140 nm) and shape (circular or spherical) that particularly mimics the virus facilitating strong interaction between them. Indeed, the delivery of anti-COVID 19 cargos via a nanoparticle such as Lipidic nanoparticles, Polymeric nanoparticles, Metallic nanoparticles, and Multi-functionalized nanoparticles to overcome the drawbacks of conventional approaches, specifying the site-specific targeting with reduced drug loading and toxicities, exhibit their immense potential. Additionally, nano-technological based drug delivery with their peculiar characteristics of having low immunogenicity, tunable drug release, multidrug delivery, higher selectivity and specificity, higher efficacy and tolerability switch on the novel pathway for the prevention and treatment of COVID 19.
... They demonstrated that the S-protein is densely glycosylated due to extensive N-linked glycosylation and the high content of carbohydrate fragments. It was calculated that carbohydrate chains cover two-thirds of the SARS-CoV-2 virion surface [40]. In the present study, the measurement of HbA1c was performed on a Bio-Rad D10 HPLC analyzer certified by NGSP and currently considered a gold standard HbA1c test. ...
Introduction
One of the stages of reproduction of SARS-CoV-2 is the S-protein glycosylation to facilitate penetration into target cells. It has been suggested that SARS-CoV-2 is able to enter erythrocytes, interact with heme and porphyrin, which could influence HbA1c levels. Assessment of HbA1c levels in individuals with acute COVID-19 and after recovery may show clinical relevance of this hypothesis.
Aim
To assess HbA1c levels in patients with COVID-19 in the acute phase and in early (6–8 weeks) and late (52±2 weeks) periods after recovery.
Materials and methods
We conducted a multicenter prospective study, which included patients hospitalized in Endocrinology Research Centre and the City Clinical Hospital № 52" diagnosed with COVID-19, virus identified/ not identified. Patients were divided into three groups according to baseline HbA1c level and the presence or absence of previous history of diabetes previous history of diabetes mellitus (DM): HbA1c ≤ 6.0%, HbA1c > 6.0% and patients with DM. Patients were examined during the acute COVID-19 phase and in early (6–8 weeks) and late (52±2 weeks) periods after recovery. Oral glucose tolerance test was performed in the group with initial HbA1c > 6.0% to clarify the diagnosis.
Results
We included 194 patients in the study. During the follow-up, 52 patients were examined in 6–8 week period: 7 with HbA1c ≤ 6.0%, 34 with HbA1c > 6.0%, 11—with previously diagnosed DM. Carbohydrate metabolism assessment in the later stages (52±2 weeks) after recovery was performed in 78 patients: 33 patients with HbA1c ≤ 6.0%, 36 patients with HbA1c > 6.0% and 9 patients with previously established diabetes. HbA1c median in patients with HbA1c ≤ 6.0% was 5.7% [5.3;5.8], with HbA1c>6.0% -6.4% [6.2; 6.6], with previously diagnosed DM—7.7% [7.2; 8.9]. Statistically significant decrease in HbA1c over time 6–8 weeks after extracts were obtained in both groups of individuals without a history of DM (Wilcoxon test, p<0.05). After 52±2 weeks we observed HbA1c decrease in all three groups (Fridman test, p<0.05): in patients with HbA1c ≤ 6.0% median HbA1c was 5.5[5.3;5.7], with HbA1c>6.0% - 6.1[6.15;6.54], with previously diagnosed DM—7.8 [5.83; 8.08]. Development of DM after 52±2 weeks was recorded in 7.24% of all examined patients without a history of DM, which is 16.6% of the total number of patients examined in dynamics with HbA1c > 6.0%.
Conclusion
HbA1c elevation during the acute phase of COVID-19 may be false due to the effect of SARS-CoV-2 on hemoglobin kinetics and/or detection on the surface of the SARS-CoV-2 virion highly glycosylated S-proteins by high performance liquid chromatography determinations. Upon detection HbA1c > 6.0% in patients with COVID-19 in the active phase of the disease without concomitant hyperglycemia re-determine the level of HbA1c after recovery is recommended.
... It is well known that the spike protein of coronaviruses is highly glycosylated and that these glycosylations play a key role in protein folding and antibody recognition [52]. The N-glycosylations in the spike protein of the SARS-CoV-2 virus have been reported independently in different recent publications [31][32][33][34][35][36]. There is one conserved N-glycosylation site in the NTD, and the other four highly conserved N-glycosylation sites are located downstream of the functional domains. ...
... There are five methylation sites in the S protein ( Figure 1D). Methylations in the spike protein were detected for the first time by Sun et al. [35], and their functional roles are unknown. The methylated residue E340 located in the CTD was selected as a site of interest, suggesting that this modification could be important for the S protein. ...
Post-translational regulation of proteins has emerged as a central topic of research in the field of functional proteomics. Post-translational modifications (PTMs) dynamically control the activities of proteins and are involved in a wide range of biological processes. Crosstalk between different types of PTMs represents a key mechanism of regulation and signaling. Due to the current pandemic of the novel and dangerous SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) virus, here we present an in silico analysis of different types of PTMs in structural proteins of coronaviruses. A dataset of PTM sites was studied at three levels: conservation analysis, mutational analysis and crosstalk analysis. We identified two sets of PTMs which could have important functional roles in the regulation of the structural proteins of coronaviruses. Additionally, we found seven interesting signals of potential crosstalk events. These results reveal a higher level of complexity in the mechanisms of post-translational regulation of coronaviral proteins and provide new insights into the adaptation process of the SARS-CoV-2 virus.
... Another major tropism determinant of SARS-CoV-2 spread is its interaction with the glycosylated ACE2 receptor commonly found on human host cells (Hoffmann et al., 2020;Mehdipour & Hummer, 2021;Oz et al., 2021;Shang et al., 2020). The N-terminal domain of ACE2 has 7 putative N-glycosylation sites: Asn53, Asn90, Asn103, Asn322, Asn432, Asn546, and Asn690 and several potential O-glycosylation sites (Casalino et al., 2020;Gong et al., 2021;Shajahan, Archer-Hartmann, et al., 2021;Sun, Ren, et al., 2021;Zhao et al., 2020) that potentially play vital roles in virus binding. Investigations have already elucidated that inhibition of glycosylation on specific sites (Asn122, Asn331, Asn334, Asn717, Asn801, and Asn1074) significantly reduces the viral infectivity rate . ...
... Interestingly, Sun, Ren, et al. (2021) reported that the binding of recombinant S-protein ectodomain expressed in insect cells and the recombinant extracellular domain of ACE2 receptor expressed in HEK 293 cells did not only depend on the N-glycosylation. Using binding kinetics, the authors deduced that the ACE2 receptor bound to the deglycosylated S-protein. ...
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the on‐going global pandemic of coronavirus disease 2019 (COVID‐19) that continues to pose a significant threat to public health worldwide. SARS‐CoV‐2 encodes four structural proteins namely membrane, nucleocapsid, spike, and envelope proteins that play essential roles in viral entry, fusion, and attachment to the host cell. Extensively glycosylated spike protein efficiently binds to the host angiotensin‐converting enzyme 2 initiating viral entry and pathogenesis. Reverse transcriptase polymerase chain reaction on nasopharyngeal swab is the preferred method of sample collection and viral detection because it is a rapid, specific, and high‐throughput technique. Alternate strategies such as proteomics and glycoproteomics‐based mass spectrometry enable a more detailed and holistic view of the viral proteins and host–pathogen interactions and help in detection of potential disease markers. In this review, we highlight the use of mass spectrometry methods to profile the SARS‐CoV‐2 proteome from clinical nasopharyngeal swab samples. We also highlight the necessity for a comprehensive glycoproteomics mapping of SARS‐CoV‐2 from biological complex matrices to identify potential COVID‐19 markers.
