Detection of SARS-CoV-2 Antibodies in Immunoglobulin Products.

BACKGROUND: For patients with primary antibody deficiency, the first line of therapy is replacement with immunoglobulin (Ig) products. Prior to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, Ig products did not contain antibodies with specificity for this virus, and there have been limited data on the antibodies present in the Ig products in current use. OBJECTIVE: To quantitatively examine SARS-CoV-2 antibodies in current Ig products. METHODS: We examined 142 unique lots of 11 different Ig products intended for intravenous and/or subcutaneous delivery for IgG-binding activities against recombinant SARS-CoV-2 receptor binding domain, spike, and nucleocapsid proteins by enzyme-linked immunosorbent assays. In addition, to assess functionality, 48 of these unique lots were assessed for their ability to inhibit the variants SARS-CoV-2 Ancestral, Alpha, Beta, Delta, and Omicron spike binding to angiotensin-converting enzyme 2 (ACE2). RESULTS: Significantly increased antibody values were observed for products manufactured after the year 2020 (expiration dates 2023-2024), as compared with Ig products before 2020 (prepandemic). Sixty percent and 85% of the Ig products with expiration dates of 2023 and 2024 were positive for antibody to SARS-CoV-2 proteins, respectively. The area under the curve values were significantly higher in products with later expiration dates. Later dates of expiration were also strongly correlated with inhibition of ACE2-binding activity; however, a decline in inhibition activity was observed with later variants. CONCLUSIONS: Overall, more recent Ig products (expiration dates 2023-2025) contained significantly higher binding and inhibition activities against SARS-CoV-2 proteins, compared with earlier, or prepandemic products. Normal donor SARS-CoV-2 antibodies are capable of inhibiting ACE2-binding activities and may provide a therapeutic benefit for patients who do not make a robust vaccine response.

In silico design of ACE2 mutants for competitive binding of SARS-CoV-2 receptor binding domain with hACE2

The receptor binding motif (RBM) within the S-protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been touted as one of the main targets for vaccine/therapeutic development due to its interaction with the human angiotensin II converting enzyme 2 (hACE2) to facilitate virus entry into the host cell. The mechanism of action is based on the disruption of binding between the RBM and the hACE2 to prevent virus uptake for replication. In this work, we applied in silico approaches to design specific competitive binders for SARS-CoV-2 S-protein receptor binding motif (RBM) by using hACE2 peptidase domain (PD) mutants. Online single point mutation servers were utilised to estimate the effect of PD mutation on the binding affinity with RBM. The PD mutants were then modelled and the binding free energy was calculated. Three PD variants were designed with an increased affinity and interaction with SARS-CoV-2-RBM. It is hope that these designs could serve as the initial work for vaccine/drug development and could eventually interfere the preliminary recognition between SARS-CoV-2 and the host cell. © 2022 Walter de Gruyter GmbH, Berlin/Boston. All rights reserved.

Artificial Lipid Biomembranes for Full-Length SARS-CoV-2 Receptor.

The angiotensin-converting enzyme 2 (ACE2), as a functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for assessing potential hosts and treatments. However, many studies are based on its truncated version but not full-length structure. Indeed, a single transmembrane (TM) helix presents in the full-length ACE2, influencing its interaction with SARS-CoV-2. Therefore, synthesis of the full-length ACE2 is an urgent requirement. Here, cell-free membrane protein synthesis systems (CFMPSs) are constructed for full-length membrane proteins. MscL is screened as a model among ten membrane proteins based on their expression and solubility. Next, CFMPSs are constructed and optimized based on natural vesicles, vesicles with four membrane proteins removed or two chaperonins added, and 37 types of nanodiscs. They all increase membrane protein solubility to over 50%. Finally, the full-length ACE2 of 21 species are successfully expressed with yields between 0.4 and 0.9 mg mL-1 . The definite functional differences from the truncated version suggest that the TM region affects ACE2's structure and function. CFMPSs can be extended to more membrane proteins, paving the way for further applications.

Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein.

BACKGROUND: Mammalian cell lines are frequently used as protein expression hosts because of their ability to correctly fold and assemble complex proteins, produce them at high titers, and confer post-translational modifications (PTMs) critical to proper function. Increasing demand for proteins with human-like PTMs, particularly viral proteins and vectors, have made human embryonic kidney 293 (HEK293) cells an increasingly popular host. The need to engineer more productive HEK293 platforms and the ongoing nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presented an opportunity to study strategies to improve viral protein expression in transient and stable HEK293 platforms. RESULTS: Initial process development was done at 24 deep well plate (DWP) -scale to screen transient processes and stable clonal cell lines for recombinant SARS-CoV-2 receptor binding domain (rRBD) titer. Nine DNA vectors that drove rRBD production under different promoters and optionally contained Epstein-Barr virus (EBV) elements to promote episomal expression were screened for transient rRBD production at 37 °C or 32 °C. Use of the cytomegalovirus (CMV) promoter to drive expression at 32 °C led to the highest transient protein titers, but inclusion of episomal expression elements did not augment titer. In parallel, four clonal cell lines with titers higher than that of the selected stable pool were identified in a batch screen. Flask-scale transient transfection and stable fed-batch processes were then established that produced rRBD up to 100 mg/L and 140 mg/L, respectively. While a bio-layer interferometry (BLI) assay was crucial for efficiently screening DWP batch titers, an enzyme-linked immunosorbent assay (ELISA) was used to compare titers from the flask-scale batches due to varying matrix effects from different cell culture media compositions. CONCLUSION: Comparing yields from the flask-scale batches revealed that stable fed-batch cultures produced up to 2.1x more rRBD than transient processes. The stable cell lines developed in this work are the first reported clonal, HEK293-derived rRBD producers and have titers up to 140 mg/L. As stable production platforms are more economically favorable for long-term protein production at large scales, investigation of strategies to increase the efficiency of high-titer stable cell line generation in Expi293F or other HEK293 hosts is warranted.

How the Competition for Cysteine May Promote Infection of SARS-CoV-2 by Triggering Oxidative Stress.

SARS-CoV-2 induces a broad range of clinical manifestations. Besides the main receptor, ACE2, other putative receptors and co-receptors have been described and could become genuinely relevant to explain the different tropism manifested by new variants. In this study, we propose a biochemical model envisaging the competition for cysteine as a key mechanism promoting the infection and the selection of host receptors. The SARS-CoV-2 infection produces ROS and triggers a massive biosynthesis of proteins rich in cysteine; if this amino acid becomes limiting, glutathione levels are depleted and cannot control oxidative stress. Hence, infection succeeds. A receptor should be recognized as a marker of suitable intracellular conditions, namely the full availability of amino acids except for low cysteine. First, we carried out a comparative investigation of SARS-CoV-2 proteins and human ACE2. Then, using hierarchical cluster protein analysis, we searched for similarities between all human proteins and spike produced by the latest variant, Omicron BA.1. We found 32 human proteins very close to spike in terms of amino acid content. Most of these potential SARS-CoV-2 receptors have less cysteine than spike. We suggest that these proteins could signal an intracellular shortage of cysteine, predicting a burst of oxidative stress when used as viral entry mediators.

Optimization of In Vivo Electroporation Conditions and Delivery of DNA Vaccine Encoding SARS-CoV-2 RBD Using the Determined Protocol.

Vaccination against SARS-CoV-2 and other viral infections requires safe, effective, and inexpensive vaccines that can be rapidly developed. DNA vaccines are candidates that meet these criteria, but one of their drawbacks is their relatively weak immunogenicity. Electroporation (EP) is an effective way to enhance the immunogenicity of DNA vaccines, but because of the different configurations of the devices that are used for EP, it is necessary to carefully select the conditions of the procedure, including characteristics such as voltage, current strength, number of pulses, etc. In this study, we determined the optimal parameters for delivery DNA vaccine by electroporation using the BEX CO device. BALB/c mice were used as a model. Plasmid DNA phMGFP was intramuscular (I/M) injected into the quadriceps muscle of the left hind leg of animals using insulin syringes, followed by EP. As a result of the experiments, the following EP parameters were determined: direct and reverse polarity rectangular DC current in three pulses, 12 V voltage for 30 ms and 950 ms intervals, with a current limit of 45 mA. The selected protocol induced a low level of injury and provided a high level of GFP expression. The chosen protocol was used to evaluate the immunogenicity of the DNA vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 protein (pVAXrbd) injected by EP. It was shown that the delivery of pVAXrbd via EP significantly enhanced both specific humoral and cellular immune responses compared to the intramuscular injection of the DNA vaccine.

Comparative Assessment of the Inhibitory Potential of the Herbicide Glyphosate and Its Structural Analogs on RGD-Specific Integrins Using Enzyme-Linked Immunosorbent Assays.

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide's active ingredient on cell adhesion and its αvß3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvß3, α5ß1 and αllbß3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvß3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvß3 binding to a synthetic polymer containing RGD; (ii) αvß3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5ß1 binding to the above polymer containing RGD; (iv) αllbß3 binding to its ECM protein, fibrinogen and (v) αvß3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvß3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5ß1 binding to RGD.

Regulation of Dynamic Cell Adhesion by Integrin-Integrin Crosstalk.

Most cells express several integrins. The integrins are able to respond to various cellular functions and needs by modifying their own activation state, but in addition by their ability to regulate each other by activation or inhibition. This crosstalk or transdominant regulation is strictly controlled. The mechanisms resulting in integrin crosstalk are incompletely understood, but they often involve intracellular signalling routes also used by other cell surface receptors. Several studies show that the integrin cytoplasmic tails bind to a number of cytoskeletal and adaptor molecules in a regulated manner. Recent work has shown that phosphorylations of integrins and key intracellular molecules are of pivotal importance in integrin-cytoplasmic interactions, and these in turn affect integrin activity and crosstalk. The integrin ß-chains play a central role in regulating crosstalk. In addition to Integrin-integrin crosstalk, crosstalk may also occur between integrins and related receptors, including other adhesion receptors, growth factor and SARS-CoV-2 receptors.

Lactoferrin Inhibition of the Complex Formation between ACE2 Receptor and SARS CoV-2 Recognition Binding Domain.

The present investigation focuses on the analysis of the interactions among human lactoferrin (LF), SARS-CoV-2 receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2) receptor in order to assess possible mutual interactions that could provide a molecular basis of the reported preventative effect of lactoferrin against CoV-2 infection. In particular, kinetic and thermodynamic parameters for the pairwise interactions among the three proteins were measured via two independent techniques, biolayer interferometry and latex nanoparticle-enhanced turbidimetry. The results obtained clearly indicate that LF is able to bind the ACE2 receptor ectodomain with significantly high affinity, whereas no binding to the RBD was observed up to the maximum "physiological" lactoferrin concentration range. Lactoferrin, above 1 µM concentration, thus appears to directly interfere with RBD-ACE2 binding, bringing about a measurable, up to 300-fold increase of the KD value relative to RBD-ACE2 complex formation.

In silico design of ACE2 mutants for competitive binding of SARS-CoV-2 receptor binding domain with hACE2

The receptor binding motif (RBM) within the S-protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been touted as one of the main targets for vaccine/therapeutic development due to its interaction with the human angiotensin II converting enzyme 2 (hACE2) to facilitate virus entry into the host cell. The mechanism of action is based on the disruption of binding between the RBM and the hACE2 to prevent virus uptake for replication. In this work, we applied in silico approaches to design specific competitive binders for SARS-CoV-2 S-protein receptor binding motif (RBM) by using hACE2 peptidase domain (PD) mutants. Online single point mutation servers were utilised to estimate the effect of PD mutation on the binding affinity with RBM. The PD mutants were then modelled and the binding free energy was calculated. Three PD variants were designed with an increased affinity and interaction with SARS-CoV-2-RBM. It is hope that these designs could serve as the initial work for vaccine/drug development and could eventually interfere the preliminary recognition between SARS-CoV-2 and the host cell.

The underlying mechanisms for severe COVID-19 progression in people with diabetes mellitus: a critical review.

Diabetes mellitus (DM) has a high incidence of comorbidities among patients with severe coronavirus disease 2019 (COVID-19). The elevated prevalence of DM in the world population makes it a significant risk factor because diabetic individuals appear to be prone to clinical complications and have increased mortality rates. Here, we review the possible underlying mechanisms involved in DM that led to worse outcomes in COVID-19. The impacts of hyperglycemia side effects, secondary comorbidities, weakened innate and adaptive immunity, chronic inflammation, and poor nutritional status, commonly present in DM, are discussed. The role of the SARS-CoV-2 receptor and its polymorphic variations on higher binding affinity to facilitate viral uptake in people with DM were also considered. Clinical differences between individuals with type 1 DM and type 2 DM affected by COVID-19 and the potential diabetogenic effect of SARS-CoV-2 infection were addressed.

Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry.

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

Identification of the SARS-CoV-2 Entry Receptor ACE2 as a Direct Target for Transcriptional Repression by Miz1.

Multiple lines of evidence have demonstrated that cigarette smoke or Chronic Obstructive Pulmonary Disease upregulates angiotensin-converting enzyme 2, the cellular receptor for the entry of the severe acute respiratory syndrome coronavirus 2, which predisposes individuals to develop severe Coronavirus disease 2019. The reason for this observation is unknown. We recently reported that the loss of function of Miz1 in the lung epithelium in mice leads to a spontaneous COPD-like phenotype, associated with upregulation of angiotensin-converting enzyme 2. We also reported that cigarette smoke exposure downregulates Miz1 in lung epithelial cells and in mice, and Miz1 is also downregulated in the lungs of COPD patients. Here, we provide further evidence that Miz1 directly binds to and represses the promoter of angiotensin-converting enzyme 2 in mouse and human lung epithelial cells. Our data provide a potential molecular mechanism for the upregulation of angiotensin-converting enzyme 2 observed in smokers and COPD patients, with implication in severe Coronavirus disease 2019.

Role of Extracellular Vimentin in Cancer-Cell Functionality and Its Influence on Cell Monolayer Permeability Changes Induced by SARS-CoV-2 Receptor Binding Domain.

The cytoskeletal protein vimentin is secreted under various physiological conditions. Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted into the extracellular space. While surface vimentin is involved in processes such as viral infections and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer cells, and its activation pathway has significant roles in general cellular functions. In this study, we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion, and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7 monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown that the receptor binding domain of SARS-CoV-2 spike protein can alter blood-brain barrier integrity. Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This defines an influence of extracellular vimentin in SARS-CoV-2 infections.

SARS-CoV-2 Receptor Binding Domain as a Stable-Potential Target for SARS-CoV-2 Detection by Surface-Enhanced Raman Spectroscopy.

In this work, we report a new approach for detecting SARS-CoV-2 RBD protein (RBD) using the surface-enhanced Raman spectroscopy (SERS) technique. The optical enhancement was obtained thanks to the preparation of nanostructured Ag/Au substrates. Fabricated Au/Ag nanostructures were used in the SERS experiment for RBD protein detection. SERS substrates show higher capabilities and sensitivity to detect RBD protein in a short time (3 s) and with very low power. We were able to push the detection limit of proteins to a single protein detection level of 1 pM. The latter is equivalent to 1 fM as a detection limit of viruses. Additionally, we have shown that the SERS technique was useful to figure out the presence of RBD protein on antibody functionalized substrates. In this case, the SERS detection was based on protein-antibody recognition, which led to shifts in the Raman peaks and allowed signal discrimination between RBD and other targets such as Bovine serum albumin (BSA) protein. A perfect agreement between a 3D simulated model based on finite element method and experiment was reported confirming the SERS frequency shift potential for trace proteins detection. Our results could open the way to develop a new prototype based on SERS sensitivity and selectivity for rapid detection at a very low concentration of virus and even at a single protein level.

Implications of cardiac markers in risk-stratification and management for COVID-19 patients.

BACKGROUND: COVID-19 has resulted in high mortality worldwide. Information regarding cardiac markers for precise risk-stratification is limited. We aim to discover sensitive and reliable early-warning biomarkers for optimizing management and improving the prognosis of COVID-19 patients. METHODS: A total of 2954 consecutive COVID-19 patients who were receiving treatment from the Wuhan Huoshenshan Hospital in China from February 4 to April 10 were included in this retrospective cohort. Serum levels of cardiac markers were collected after admission. Coronary artery disease diagnosis and survival status were recorded. Single-cell RNA-sequencing and bulk RNA-sequencing from different cohorts of non-COVID-19 were performed to analyze SARS-CoV-2 receptor expression. RESULTS: Among 2954 COVID-19 patients in the analysis, the median age was 60 years (50-68 years), 1461 (49.5%) were female, and 1515 (51.3%) were severe/critical. Compared to mild/moderate (1439, 48.7%) patients, severe/critical patients showed significantly higher levels of cardiac markers within the first week after admission. In severe/critical COVID-19 patients, those with abnormal serum levels of BNP (42 [24.6%] vs 7 [1.1%]), hs-TNI (38 [48.1%] vs 6 [1.0%]), α- HBDH (55 [10.4%] vs 2 [0.2%]), CK-MB (45 [36.3%] vs 12 [0.9%]), and LDH (56 [12.5%] vs 1 [0.1%]) had a significantly higher mortality rate compared to patients with normal levels. The same trend was observed in the ICU admission rate. Severe/critical COVID-19 patients with pre-existing coronary artery disease (165/1,155 [10.9%]) had more cases of BNP (52 [46.5%] vs 119 [16.5%]), hs-TNI (24 [26.7%] vs 9.6 [%], α- HBDH (86 [55.5%] vs 443 [34.4%]), CK-MB (27 [17.4%] vs 97 [7.5%]), and LDH (65 [41.9%] vs 382 [29.7%]), when compared with those without coronary artery disease. There was enhanced SARS-CoV-2 receptor expression in coronary artery disease compared with healthy controls. From regression analysis, patients with five elevated cardiac markers were at a higher risk of death (hazards ratio 3.4 [95% CI 2.4-4.8]). CONCLUSIONS: COVID-19 patients with pre-existing coronary artery disease represented a higher abnormal percentage of cardiac markers, accompanied by high mortality and ICU admission rate. BNP together with hs-TNI, α- HBDH, CK-MB and LDH act as a prognostic biomarker in COVID-19 patients with or without pre-existing coronary artery disease.

Sulfoglycodendrimer Therapeutics for HIV-1 and SARS-CoV-2.

Hexavalent sulfoglycodendrimers (SGDs) are synthesized as mimics of host cell heparan sulfate proteoglycans (HSPGs) to inhibit the early stages in viral binding/entry of HIV-1 and SARS-CoV-2. Using an HIV neutralization assay, the most promising of the seven candidates are found to have sub-micromolar anti-HIV activities. Molecular dynamics simulations are separately implemented to investigate how/where the SGDs interacted with both pathogens. The simulations revealed that the SGDs: 1) develop multivalent binding with polybasic regions within and outside of the V3 loop on glycoprotein 120 (gp120) for HIV-1, and consecutively bind with multiple gp120 subunits, and 2) interact with basic amino acids in both the angiotensin-converting enzyme 2 (ACE2) and HSPG binding regions of the Receptor Binding Domain (RBD) from SARS-CoV-2. These results illustrate the considerable potential of SGDs as inhibitors in viral binding/entry of both HIV-1 and SARS-CoV-2 pathogens, leading the way for further development of this class of molecules as broad-spectrum antiviral agents.

Asymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in a Rehabilitation Facility: Evolution of the Presence of Nasopharyngeal SARS-CoV-2 and Serological Antibody Responses.

At the start of the UK coronavirus disease 2019 epidemic, this rare point prevalence study revealed that one-third of patients (15 of 45) in a London inpatient rehabilitation unit were found to be infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but asymptomatic. We report on 8 patients in detail, including their clinical stability, the evolution of their nasopharyngeal viral reverse-transcription polymerase chain reaction (RT-PCR) burden, and their antibody levels over time, revealing the infection dynamics by RT-PCR and serology during the acute phase. Notably, a novel serological test for antibodies against the receptor binding domain of SARS-CoV-2 showed that 100% of our asymptomatic cohort remained seropositive 3-6 weeks after diagnosis.

Functional Complexes of Angiotensin-Converting Enzyme 2 and Renin-Angiotensin System Receptors: Expression in Adult but Not Fetal Lung Tissue.

Angiotensin-converting enzyme 2 (ACE2) is a membrane peptidase and a component of the renin-angiotensin system (RAS) that has been found in cells of all organs, including the lungs. While ACE2 has been identified as the receptor for severe acute respiratory syndrome (SARS) coronaviruses, the mechanism underlying cell entry remains unknown. Human immunodeficiency virus infects target cells via CXC chemokine receptor 4 (CXCR4)-mediated endocytosis. Furthermore, CXCR4 interacts with dipeptidyl peptidase-4 (CD26/DPPIV), an enzyme that cleaves CXCL12/SDF-1, which is the chemokine that activates this receptor. By analogy, we hypothesized that ACE2 might also be capable of interactions with RAS-associated G-protein coupled receptors. Using resonance energy transfer and cAMP and mitogen-activated protein kinase signaling assays, we found that human ACE2 interacts with RAS-related receptors, namely the angiotensin II type 1 receptor (AT1R), the angiotensin II type 2 receptor (AT2R), and the MAS1 oncogene receptor (MasR). Although these interactions lead to minor alterations of signal transduction, ligand binding to AT1R and AT2R, but not to MasR, resulted in the upregulation of ACE2 cell surface expression. Proximity ligation assays performed in situ revealed macromolecular complexes containing ACE2 and AT1R, AT2R or MasR in adult but not fetal mouse lung tissue. These findings highlight the relevance of RAS in SARS-CoV-2 infection and the role of ACE2-containing complexes as potential therapeutic targets.

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients.