scholarly journals Association of CD47 with Integrin Mac-1 (αMβ2, CD11b/CD18) Regulates Macrophage Responses

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1109-1109
Author(s):  
Nataly Podolnikova ◽  
Arnat Balabiyev ◽  
Tatiana P. Ugarova
Keyword(s):  
Conflicts Of Interest ◽  
Integrin Αvβ3 ◽  
The Body ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Hek293 Cells ◽  
Proximity Ligation Assay ◽  
Physical Interaction ◽  
Wild Type ◽  
Surface Receptor

Abstract CD47 is a cell surface receptor, which is expressed by virtually all cells in the body, including immune cells. CD47 has originally been identified as an integrin-associated protein (IAP) and shown to associate with several integrins that belong to the β1 and β3 subfamilies. In addition, association of CD47 with a member of the β2 subfamily, integrin αLβ2, has also been reported. In neutrophils, CD47 mediates a number of integrin αvβ3-dependent functions, including adhesion, migration and phagocytosis. Surprisingly, the association of CD47 with integrin αMβ2 (Mac-1, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, has not been documented. Furthermore, while the major focus of recent studies was the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 expressed on the surface of macrophages influences the responses of these cells has not been addressed. In the present study, we demonstrated that an association of CD47 with Mac-1 regulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. In addition, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional association of CD47 with Mac-1 implied that similar to other integrins, Mac-1 might form a complex with CD47. Indeed, co-immunoprecipitation experiments using peritoneal mouse macrophages, the IC21 murine macrophage cell line and Mac-1-expressing HEK293 cells revealed that Mac-1 forms a complex with CD47. The cis interaction between Mac-1 and CD47 was also detected using the proximity ligation assay. Together, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions. Studies to determine the structural requirements for the physical interaction between Mac-1 and CD47 are in progress. Disclosures No relevant conflicts of interest to declare.

Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 541-541
Author(s):  
Giancarlo Castaman ◽  
Sofia Helene Giacomelli ◽  
Paula M. Jacobi ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Negative Impact ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Wild Type ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 541 Background. Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study. We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules. Methods. Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results. The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression. In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions. Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Critical Sequence Hot-spots for Binding of nCOV-2019 to ACE2 as Evaluated by Molecular Simulations

2020 ◽  
Author(s):  
Mahdi Ghorbani ◽  
Bernard R. Brooks ◽  
Jeffery B. Klauda
Keyword(s):  
Md Simulations ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Receptor Protein ◽  
Structural Mechanism ◽  
The World ◽  
Surface Receptor ◽  
Escape Mutants ◽  
The Stability ◽  
Novel Coronavirus

AbstractThe novel coronavirus (nCOV-2019) outbreak has put the world on edge, causing millions of cases and hundreds of thousands of deaths all around the world, as of June 2020, let alone the societal and economic impacts of the crisis. The spike protein of nCOV-2019 resides on the virion’s surface mediating coronavirus entry into host cells by binding its receptor binding domain (RBD) to the host cell surface receptor protein, angiotensin converter enzyme (ACE2). Our goal is to provide a detailed structural mechanism of how nCOV-2019 recognizes and establishes contacts with ACE2 and its difference with an earlier coronavirus SARS-COV in 2002 via extensive molecular dynamics (MD) simulations. Numerous mutations have been identified in the RBD of nCOV-2019 strains isolated from humans in different parts of the world. In this study, we investigated the effect of these mutations as well as other Ala-scanning mutations on the stability of RBD/ACE2 complex. It is found that most of the naturally-occurring mutations to the RBD either strengthen or have the same binding affinity to ACE2 as the wild-type nCOV-2019. This may have implications for high human-to-human transmission of coronavirus in regions where these mutations have been found as well as any vaccine design endeavors since these mutations could act as antibody escape mutants. Furthermore, in-silico Ala-scanning and long-timescale MD simulations, highlight the crucial role of the residues at the interface of RBD and ACE2 that may be used as potential pharmacophores for any drug development endeavors. From an evolutional perspective, this study also identifies how the virus has evolved from its predecessor SARS-COV and how it could further evolve to become more infectious.

scholarly journals Hydroxyzine inhibits SARS-CoV-2 Spike protein binding to ACE2 in a qualitative in vitro assay

2021 ◽  
Author(s):  
Maria Dolores Rivas ◽  
Jose Maria Rafael Saponi-Cortes ◽  
Jose Zamorano
Keyword(s):  
Public Health Problem ◽  
In Vitro Assay ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
Major Public Health Problem ◽  
Receptor Binding Domain ◽  
Vitro Assay ◽  
Surface Receptor

AbstractCOVID-19 currently represents a major public health problem. Multiple efforts are being performed to control this disease. Vaccinations are already in progress. However, no effective treatments have been found so far. The disease is caused by the SARS-CoV-2 coronavirus that through the Spike protein interacts with its cell surface receptor ACE2 to enter into the host cells. Therefore, compounds able to block this interaction may help to stop disease progression. In this study, we have analyzed the effect of compounds reported to interact and modify the activity of ACE2 on the binding of the Spike protein. Among the compounds tested, we found that hydroxyzine could inhibit the binding of the receptor-binding domain of Spike protein to ACE2 in a qualitative in vitro assay. This finding supports the reported clinical data showing the benefits of hydroxyzine on COVID-19 patients, raising the need for further investigation into its effectiveness in the treatment of COVID-19 given its well-characterized medical properties and affordable cost.

scholarly journals Mechanism of Borna Disease Virus Entry into Cells

1998 ◽  
Vol 72 (1) ◽  
pp. 783-788 ◽  
Author(s):  
Daniel Gonzalez-Dunia ◽  
Beatrice Cubitt ◽  
Juan Carlos de la Torre
Keyword(s):  
Disease Virus ◽  
Virus Entry ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Animal Cells ◽  
Borna Disease Virus ◽  
C Terminus ◽  
Surface Receptor ◽  
Infected Cells ◽  
Borna Disease

ABSTRACT We have investigated the entry pathway of Borna disease virus (BDV). Virus entry was assessed by detecting early viral replication and transcription. Lysosomotropic agents (ammonium chloride, chloroquine, and amantadine), as well as energy depletion, prevented BDV infection, indicating that BDV enters host cells by endocytosis and requires an acidic intracellular compartment to allow membrane fusion and initiate infection. Consistent with this hypothesis, we observed that BDV-infected cells form extensive syncytia upon low-pH treatment. Entry of enveloped viruses into animal cells usually requires the membrane-fusing activity of viral surface glycoproteins (GPs). BDV GP is expressed as two products of 84 and 43 kDa (GP-84 and GP-43, respectively). We show here that only GP-43 is present at the surface of BDV-infected cells and therefore is likely the viral polypeptide responsible for triggering fusion events. We also present evidence that GP-43, which corresponds to the C terminus of GP-84, is generated by cleavage of GP-84 by the cellular protease furin. Hence, we propose that BDV GP-84 is involved in attachment to the cell surface receptor whereas its furin-cleaved product, GP-43, is involved in pH-dependent fusion after internalization of the virion by endocytosis.

scholarly journals Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor

2020 ◽  
Vol 21 (19) ◽  
pp. 7075
Author(s):  
Munkhzaya Byambaragchaa ◽  
Jeong-Soo Kim ◽  
Hong-Kyu Park ◽  
Dae-Jung Kim ◽  
Sun-Mee Hong ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Surface ◽  
Hormone Receptor ◽  
Follicle Stimulating Hormone ◽  
Chinese Hamster ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Surface Receptor ◽  
Stimulating Hormone

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

SARS-CoV-2 Entry inhibitors targeting virus-ACE2 or virus-TMPRSS2 interactions

2021 ◽  
Vol 28 ◽  
Author(s):  
Hao Lin ◽  
Srinivasulu Cherukupalli ◽  
Da Feng ◽  
Shenghua Gao ◽  
Dongwei Kang ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Entry Inhibitors ◽  
Concise Report ◽  
Surface Receptor ◽  
Transmembrane Serine Protease

: COVID-19 is an infectious disease caused by SARS-CoV-2. The life cycle of SARS-CoV-2 includes the entry into the target cells, replicase translation, replicating and transcribing genomes, translating structural proteins, assembling and releasing new virions. Entering host cells is a crucial stage in the early life cycle of the virus, and blocking this stage can effectively prevent virus infection. SARS enters the target cells mediated by the interaction between the viral S protein and the target cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the cleavage effect of type-II transmembrane serine protease (TMPRSS2) on the S protein. Therefore, the ACE2 receptor and TMPRSS2 are important targets for SARS-CoV-2 entry inhibitors. Herein, we provide a concise report/information on drugs with potential therapeutic value targeting virus-ACE2 or virus-TMPRSS2 interactions, to provide a reference for the design and discovery of potential entry inhibitors against SARS-CoV-2.

scholarly journals CD16b: Primary Structure of Human Neutrophil Antigen Epitopes and Their Functional Consequences

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3680-3680
Author(s):  
Ulrich J Sachs ◽  
Angelika Reil ◽  
Piyapong Simtong ◽  
Sentot Santoso
Keyword(s):  
Cell Lines ◽  
Human Neutrophil ◽  
Conflicts Of Interest ◽  
Solid Phase ◽  
Hek293 Cells ◽  
Adhesion Assay ◽  
Wild Type ◽  
Capture Assay ◽  
Diagnostic Work ◽  
And Function

Abstract Neutrophil specific Fcg receptor IIIb (CD16b) is a glycosylphosphatidylinositol-anchored low-affinity glycoprotein that plays a significant role in phagocytosis and the clearance of immune complexes. CD16b has numerous polymorphic variants; the most relevant variants are associated with human neutrophil antigens (HNA) -1a, -1b, and -1c. HNA-1a and HNA-1b differ in four amino acids (aa) (positions 36, 65, 82 and 106). One additional aa mutation on HNA-1b at position 78 results in HNA-1c. Immunization against CD16b variants can lead to the production of alloantibodies (aabs) responsible for neonatal alloimmune neutropenia (NIN), autoimmune neutropenia of infancy (AIN) and transfusion-related acute lung injury (TRALI). The exact contribution of five aa mutations for the formation of HNA-1a, -1b and -1c and function of CD16b is currently unknown. In this study, permutation of each polymorphic aa from wild-type CD16b cDNA constructs was performed and stably expressed on HEK293 cells. In total, 3 cell lines expressing wild-type (HNA-1a, -1b and -1c) and 18 cell lines expressing mutant variants were produced; 14 derived from HNA-1a or HNA-1b (panel 1) and 4 derived from HNA-1c (panel 2). When panel 1 was tested with well-defined aabs by antigen capture assay, 4 cell lines reacted with anti-HNA-1a only, 4 with anti-HNA-1b only, 4 with both aabs, and 4 did not react with both aabs. These results indicate that aa36 and aa65 are responsible for the formation of the HNA-1a and HPA-1b epitope(s), respectively. Analysis of panel 2 showed that 2/4 mutant variants reacted with anti-HNA-1c exclusively, suggesting that the HNA-1c epitope is formed by Ala78Asp irrespective of the other polymorphic aa. To analyze the binding affinity of CD16b variants, adhesion assay onto immobilized IgG was performed. In comparison to HNA-1a, HNA-1b cells bound slightly weaker to IgG (p<0.002). In contrast, HNA-1c cells interacted significantly stronger with IgG in comparison to both, HNA-1a and -1b cells (p<0.0001). Similar results were obtained with HNA-1a, -1b and -1c recombinant proteins by direct protein-binding analysis (solid phase ELISA and Surface Plasmon Resonance). Interestingly, all mutant variants carrying HNA-1c bound also stronger to IgG than HNA-1a and -1b transfected cells. Similar results were observed with HNA-1c phenotyped neutrophils. In conclusion, although HNA-1a and HNA-1b differ in four aa positions, only two (aa36, aa65) are necessary for the complete formation of the respective epitopes. In contrast, the HNA-1c epitope is created by only one aa mutation (Ala78Asp). Furthermore, our results show that HNA-1c exhibits higher affinity to IgG compared to HNA-1a and HNA-1b forms. Accordingly, the Ala78Asp mutation is not only responsible for epitope formation, but is also involved in the regulation of CD16 affinity. Characterization of HNA-1 epitopes by tailored recombinant tools will help us to improve our diagnostic work-up in patients with suspected NIN, AIN and TRALI and our understanding on the role of CD16 polymorphism in other diseases such as, immune complex-mediated disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression levels of functional folate receptors α and β are related to the number of N-glycosylated sites

1997 ◽  
Vol 327 (3) ◽  
pp. 759-764 ◽  
Author(s):  
Feng SHEN ◽  
Huiquan WANG ◽  
Xuan ZHENG ◽  
Manohar RATNAM
Keyword(s):  
Folic Acid ◽  
Cell Surface ◽  
Folate Receptor ◽  
Glycosylation Site ◽  
Cell Surface Receptor ◽  
Wild Type ◽  
Mutant Proteins ◽  
Surface Receptor ◽  
And Function

In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type α and type β have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn → Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-α and FR-β are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-α and FR-β respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-β the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-α and FR-β.

scholarly journals Identification of a Putative Coreceptor on Vero Cells That Participates in Dengue 4 Virus Infection

2001 ◽  
Vol 75 (17) ◽  
pp. 7818-7827 ◽  
Author(s):  
José de Jesús Martı́nez-Barragán ◽  
Rosa M. del Angel
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
Vero Cells ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Virus Binding ◽  
Dengue Virus Infection ◽  
Surface Receptor ◽  
A Cell

ABSTRACT Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (α-mannose orN-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.

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