scholarly journals The Effects of Amine-Carboxyborane Related Derivatives on UMR-106 Bone Metabolism

1996 ◽  
Vol 3 (1) ◽  
pp. 31-47 ◽  
Author(s):  
Margaret E. Murphy ◽  
Amy L. Elkins ◽  
Robert P. Shrewsbury ◽  
Anup Sood ◽  
Bernard F. Spielvogel ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Calcium Uptake ◽  
Metabolic Effects ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Cell Monolayers ◽  
High Affinity ◽  
Proline Incorporation ◽  
High Affinity Receptor ◽  
Umr 106

The amine-carboxyboranes and related derivatives have been shown to be potent anti-inflammatory and anti-osteoporosis agents. Their action in part appears to be mediated by the modulation of cytokines, e.g. TNFα or IL-1. Previous studies have demonstrated that LPS induced macrophages release of TNFα maximally at 60 to 90 min. and IL-1 from 5 to 8 hr. The amine-carboxyboranes reduced significantly the release of these cytokines but also blocked TNFα high affinity binding to UMR-106 receptor at 90 min. at 10 μM, and IL-1 high affinity binding at 5 hr. at 12.5 μM. In addition, the agents suppressed IL-8 binding to CHO K1 high affinity receptor at 24 hr. at 50 μM and IL-2 binding to HuT-8 receptors at 25 μM at 90 min. and 5 hr. Correlation of metabolic events associated with osteoporosis showed that at 90 min., when TNFα receptor binding was reduced by the agents, calcium uptake into UMR-106 cells was reduced at 10 μM as well as the acid and alkaline phosphatases, and the prostaglandin cyclo-oxygenase activities and adhesion of leukocytes and macrophages to UMR-106 cell monolayers. At 5hr. when the agents reduced IL-1 binding to UMR-106 receptors, calcitonin and 1,25-dihydrovitamin D3 binding was reduced by the agents as was acid and alkaline phosphatase, and 5′-lipoxygenase activities and white blood cell adhesion. At this time calcium uptake and proline incorporation was increased significantly by the agents. At later times e.g. 18-48 hr. calcium uptake was still increased, and NAG activity was inhibited in the presence of the agents. These effects may be related more to the inhibition of other cytokine receptor binding, e.g. IL-8. Thus, many of the observed metabolic effects of amine-carboxyboranes as antiosteoporosis agents can be correlated with their inhibition of cytokine high affinity binding to target cell receptors.

scholarly journals A broadly neutralizing antibody protects against SARS-CoV, pre-emergent bat CoVs, and SARS-CoV-2 variants in mice

2021 ◽  
Author(s):  
David R. Martinez ◽  
Alexandra Schaefer ◽  
Sophie Gobeil ◽  
Dapeng Li ◽  
Gabriela De la Cruz ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Receptor Binding ◽  
Specific Antibody ◽  
Neutralizing Antibody ◽  
Affinity Binding ◽  
Receptor Binding Domain ◽  
High Affinity Binding ◽  
High Affinity ◽  
Broadly Neutralizing Antibody ◽  
Conserved Epitope

AbstractSARS-CoV in 2003, SARS-CoV-2 in 2019, and SARS-CoV-2 variants of concern (VOC) can cause deadly infections, underlining the importance of developing broadly effective countermeasures against Group 2B Sarbecoviruses, which could be key in the rapid prevention and mitigation of future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV, bat CoVs WIV-1 and RsSHC014, and SARS-CoV-2 variants D614G, B.1.1.7, B.1.429, B1.351 by a receptor-binding domain (RBD)-specific antibody DH1047. Prophylactic and therapeutic treatment with DH1047 demonstrated protection against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B1.351infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among Sarbecoviruses. We conclude that DH1047 is a broadly neutralizing and protective antibody that can prevent infection and mitigate outbreaks caused by SARS-like strains and SARS-CoV-2 variants. Our results argue that the RBD conserved epitope bound by DH1047 is a rational target for pan Group 2B coronavirus vaccines.

scholarly journals Mutations in the DG Loop of Adenovirus Type 5 Fiber Knob Protein Abolish High-Affinity Binding to Its Cellular Receptor CAR

1999 ◽  
Vol 73 (11) ◽  
pp. 9508-9514 ◽  
Author(s):  
Ian Kirby ◽  
Elizabeth Davison ◽  
Andrew J. Beavil ◽  
Cecilia P. C. Soh ◽  
Thomas J. Wickham ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Receptor Binding ◽  
Adenovirus Type ◽  
Cellular Receptor ◽  
Affinity Binding ◽  
Cd Spectra ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Adenovirus Type 5

ABSTRACT The amino acid residues in adenovirus type 5 (Ad5) fiber that interact with its cellular receptor, the coxsackie B virus and Ad receptor (CAR), have not been defined. To investigate this, multiple mutations were constructed in the region between residues 479 and 497 in Ad5 fiber (β-strands E and F and the adjacent region of the DG loop). The effects of these mutations on binding to CAR were determined by use of cell-binding competition experiments, surface plasmon resonance, and direct binding studies. The mutation effects on the overall folding and secondary structure of the protein were assessed by circular dichroism (CD) spectroscopy. Deletions of two consecutive amino acids between residues 485 and 493 abolished high-affinity binding to CAR; the CD spectra indicated that although there was no disruption of the overall folding and secondary structure of the protein, local conformational changes did occur. Moreover, single site mutations in this region of residues with exposed, surface-accessible side chains, such as Thr492, Asn493, and Val495, had no effect on receptor binding, which demonstrates that these residues are not in contact with CAR themselves. This implies the involvement of residues in neighboring loop regions. Replacement of the segment containing the two very short β-strands E and F and the turn between them (residues 479 to 486) with the corresponding sequence from Ad3 (βEFAd3→5 mutation) resulted in the loss of receptor binding. The identical CD spectra for βEFAd3→5 and wild-type proteins suggest that these substitutions caused no conformational rearrangement and that the loss of binding may thus be due to the substitution of one or more critical contact residues. These findings have implications for our understanding of the interaction of Ad5 fiber with CAR and for the construction of targeted recombinant Ad5 vectors for gene therapy purposes.

scholarly journals Comparison of Target Recognition by TRAF1 and TRAF2

2020 ◽  
Vol 21 (8) ◽  
pp. 2895
Author(s):  
Chang Min Kim ◽  
Hyun Ho Park
Keyword(s):  
Receptor Binding ◽  
Target Recognition ◽  
Affinity Binding ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
Critical Determinant ◽  
High Affinity Binding ◽  
High Affinity ◽  
Caspase 2

Although TRAF1 and TRAF2 share common receptors and have extremely conserved amino acid residues, recent studies have shown that key differences in receptor binding preferences with different affinities exist, which might be important for their different functions in TRAF-mediated signal transduction. To better understand TRAF1 and TRAF2 signaling, we analyzed and compared their receptor binding-affinities. Our study revealed that TRADD, TANK, and caspase-2 bind to both TRAF1 and TRAF2 with different affinities in vitro. Sequence and structural analyses revealed that S454 on TRAF2 (corresponding to A369 of TRAF1) is critical for the binding of TRADD, and F347 on TRAF1 (corresponding to L432 of TRAF2) is a critical determinant for high affinity binding of TANK and caspase-2.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

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