scholarly journals Roles of the clip domains of two protease zymogens in the coagulation cascade in horseshoe crabs

2020 ◽  
Vol 295 (26) ◽  
pp. 8857-8866
Author(s):  
Keisuke Yamashita ◽  
Toshio Shibata ◽  
Toshiaki Takahashi ◽  
Yuki Kobayashi ◽  
Shun-ichiro Kawabata
Keyword(s):  
Binding Activity ◽  
Coagulation Cascade ◽  
Binding Assays ◽  
Peptide Substrates ◽  
Hydrophobic Cavity ◽  
Activation Rate ◽  
Horseshoe Crabs ◽  
Specific Peptide ◽  
Autocatalytic Activation ◽  
Lps Binding

The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs comprises three protease zymogens: prochelicerase C (proC), prochelicerase B (proB), and the proclotting enzyme (proCE). The presence of LPS results in autocatalytic activation of proC to α-chelicerase C, which, in turn, activates proB to chelicerase B, converting proCE to the clotting enzyme (CE). ProB and proCE contain an N-terminal clip domain, but the roles of these domains in this coagulation cascade remain unknown. Here, using recombinant proteins and kinetics and binding assays, we found that five basic residues in the clip domain of proB are required to maintain its LPS-binding activity and activation by α-chelicerase C. Moreover, an amino acid substitution at a potential hydrophobic cavity in proB's clip domain (V55A-proB) reduced both its LPS-binding activity and activation rate. WT proCE exhibited no LPS-binding activity, and the WT chelicerase B-mediated activation of a proCE variant with a substitution at a potential hydrophobic cavity (V53A-proCE) was ∼4-fold slower than that of WT proCE. The kcat/Km value of the interaction of WT chelicerase B with V53A-proCE was 7-fold lower than that of the WT chelicerase B-WT proCE interaction. The enzymatic activities of V55A-chelicerase B and V53A-CE against specific peptide substrates were indistinguishable from those of the corresponding WT proteases. In conclusion, the clip domain of proB recruits it to a reaction center composed of α-chelicerase C and LPS, where α-chelicerase C is ready to activate proB, leading to chelicerase B–mediated activation of proCE via its clip domain.

scholarly journals Implication of Antigenic Conversion of Helicobacter pylori Lipopolysaccharides That Involve Interaction with Surfactant Protein D

2012 ◽  
Vol 80 (8) ◽  
pp. 2956-2962 ◽  
Author(s):  
Shin-ichi Yokota ◽  
Ken-ichi Amano ◽  
Chiaki Nishitani ◽  
Shigeru Ariki ◽  
Yoshio Kuroki ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Biological Activities ◽  
Surfactant Protein ◽  
Binding Activity ◽  
Surfactant Protein D ◽  
Dependent Manner ◽  
Antigenic Epitope ◽  
Binding Assays ◽  
Content Type ◽  
Gastric Cancer Patients

ABSTRACTWe propose two antigenic types ofHelicobacter pylorilipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linkedN-acetyl-d-glucosamine and β-linkedd-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca2+-dependent manner, and this interaction was inhibited by methyl-β-d-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-d-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

scholarly journals E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

1990 ◽  
Vol 10 (10) ◽  
pp. 5138-5149 ◽  
Author(s):  
R J Rooney ◽  
P Raychaudhuri ◽  
J R Nevins
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Stable Complex ◽  
Small Subset ◽  
Adenovirus Infection ◽  
Binding Assays ◽  
Dna Binding Activity ◽  
Sequence Recognition ◽  
Nuclear Factors ◽  
Dna Binding Factors

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

scholarly journals 16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis

2018 ◽  
Vol 7 (5) ◽  
pp. 630-636 ◽  
Author(s):  
Kazunori Morohoshi ◽  
Ryo Mochinaga ◽  
Tsukasa Watanabe ◽  
Ryojun Nakajima ◽  
Toshio Harigaya
Keyword(s):  
Endothelial Cells ◽  
Prolactin Receptor ◽  
Binding Activity ◽  
Binding Assays ◽  
Specific Receptor ◽  
Angiogenic Activity ◽  
Integrin Beta1 ◽  
Induced Apoptosis

Many functions of vasoinhibins have been reported, but its receptor has not been clarified yet. Vasoinhibins, 11–18 kDa N-terminal fragments of prolactin, have anti-angiogenic activity and act on endothelial cells to induce apoptosis and to inhibit migration and proliferation, which are opposite to the effects of prolactin. Although vasoinhibins bind to the prolactin receptor, its binding activity is very weak compared to prolactin. Therefore, in this study, we evaluated the binding activity between 16 kDa vasoinhibin and integrin beta1, alpha5 beta1, alpha1 beta1 and alphaV beta3 to identify a specific receptor for vasoinhibins. Moreover, we examined whether 16 kDa vasoinhibin induced apoptosis through integrin beta1 and alpha5 beta1 in endothelial cells. In this study, binding assays and co-immunoprecipitation experiments demonstrated that 16 kDa vasoinhibin could bind strongly to integrin beta1 and alpha5 beta1. Moreover, neutralizing with integrin beta1 and alpha5 beta1 antibody could inhibit 16 kDa vasoinhibin-induced apoptosis in endothelial cells. These findings suggest that vasoinhibins can act on endothelial cells through integrin alpha5 beta1 to induce apoptosis.

Interaction Of Folk Medicinal Plant Extracts With Human ɑ2-Adrenoceptor Subtypes

2002 ◽  
Vol 57 (3-4) ◽  
pp. 332-338 ◽  
Author(s):  
Ammar Saleem ◽  
Mia Engström ◽  
Siegfried Wurster ◽  
Juha-Matti Savola ◽  
Kalevi Pihlaja
Keyword(s):  
Medicinal Plant ◽  
Binding Activity ◽  
Binding Assays ◽  
Peganum Harmala ◽  
Plant Materials ◽  
Drug Candidates ◽  
Medicinal Plant Extracts ◽  
Gtpγs Binding ◽  
Competition Binding ◽  
Specific Ligand

Forty-two extracts of folk medicinal plant organs from Pakistan were tested in competition binding assays for their interaction with the specific ligand recognition sites on the human α2-adrenoceptor subtypes α2A, α2B and α2C. Strong binding of the extracts (40 mg/ml) from Acacia nilotica (L.) Delile leaves (88-98% displacement of radiolabel) and Peganum harmala seeds (89-96% displacement) on three subtypes prompted us to extract these plant materials with 40% and 80% methanol, ethanol, and acetone. The extraction results indicated an absence of α2-adrenoceptor binding activity in the stalk of A. nilotica and A. tortils, whereas the leaves of both plants contained activity. The extracts of A. nilotica leaves showed a slight, but consistent, preference for the α2C-adrenoceptor, whereas the leaves of A. tortils were slightly more active on the α2B subtype. The extract of P. harmala stalks was less active than that of its seeds. The binding activities of A. nilotica leaves and P. harmala seeds were mainly concentrated in the water and 30% methanol fractions and further sub-fractions. In a functional activity assay, the active fractions inhibited epinephrine-stimulated 35S-GTPγS binding, thus indicating a predominantly antagonistic nature of the compounds with α2-adrenoceptor affinity in these fractions. Among the known major alkaloids of P. harmala (demissidine, harmaline, harmine, 6-methoxyharmalan, and norharmane), only 6-methoxyharmalan showed moderate affinity (dissociation constant (Ki) of 530 ± 40 nᴍ for α2A subtype). This study is a first systematic attempt towards the discovery of potential drug candidates from these plant materials for treating α2-adrenoceptor related diseases

An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

2015 ◽  
Vol 53 (1) ◽  
pp. 253-264 ◽  
Author(s):  
Zhihao Jia ◽  
Tao Zhang ◽  
Shuai Jiang ◽  
Mengqiang Wang ◽  
Qi Cheng ◽  
...  
Keyword(s):  
Crassostrea Gigas ◽  
Binding Activity ◽  
Vibrio Splendidus ◽  
Lps Binding

scholarly journals Clostridium botulinum Strains Producing BoNT/F4 or BoNT/F5

2014 ◽  
Vol 80 (10) ◽  
pp. 3250-3257 ◽  
Author(s):  
Brian H. Raphael ◽  
Marite Bradshaw ◽  
Suzanne R. Kalb ◽  
Lavin A. Joseph ◽  
Carolina Lúquez ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Southern Hybridization ◽  
Pcr Analysis ◽  
Specific Marker ◽  
Peptide Substrates ◽  
Content Type ◽  
Mass Spectral ◽  
Culture Supernatants ◽  
Specific Peptide

ABSTRACTBotulinum neurotoxin type F (BoNT/F) may be produced byClostridium botulinumalone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain ofC. botulinum(Af84) was shown to contain three neurotoxin genes (bont/F4,bont/F5, andbont/A2). In this study, eight strains containingbont/F4and seven strains containingbont/F5were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containingbont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboringbont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring eitherbont/F4orbont/F5are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of abont/F5-carrying plasmid among strains of diverse genomic backgrounds.

scholarly journals Interaction of nuclear proteins with a positive cis-acting element of rat embryonic myosin heavy-chain promoter: identification of a new transcriptional factor.

1989 ◽  
Vol 9 (5) ◽  
pp. 1839-1849 ◽  
Author(s):  
Y T Yu ◽  
B Nadal-Ginard
Keyword(s):  
Binding Site ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Hela Cells ◽  
Promoter Activity ◽  
Point Mutations ◽  
Binding Activity ◽  
Transcriptional Factor ◽  
Binding Assays ◽  
Cis Acting

A DNA fragment of the rat embryonic myosin heavy-chain promoter (MHCemb) has been found to specifically bind a nuclear factor (NFe) present in extracts prepared from mouse C2 myoblasts, myotubes, and HeLa cells. The nucleotide sequence of the binding site (BSe) has been identified as 5'-GTGTCAGTCA-3' and was located between -93 and -84. Transient expression studies on MHCemb promoter deletion constructs in C2 myoblasts and C2 myotubes suggested that NFe is a transcriptional factor. Deletion of the NFe-binding site resulted in four- to sixfold and twofold reduction of promoter activity in C2 myotubes and C2 myoblasts, respectively. Furthermore, point mutations at the BSe not only abolished the NFe-binding activity of the MHCemb promoter but also resulted in reduction of the promoter activity to levels similar to those of the deletion constructs in C2 myotubes, myoblasts, and Hela cells (four- to sixfold). Although BSe and the binding site of the recently identified transcriptional factors AP-1 and ATF share significant homology, the results from competition binding assays indicated that NFe is different from both AP-1 and ATF.

scholarly journals Localization and synthesis of the acetylcholine-binding site in the α-chain of the Torpedo californica acetylcholine receptor

1984 ◽  
Vol 224 (3) ◽  
pp. 995-1000 ◽  
Author(s):  
D J McCormick ◽  
M Z Atassi
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Synthetic Peptide ◽  
Solid Phase ◽  
Essential Elements ◽  
Binding Activity ◽  
Binding Assays ◽  
Free Peptide ◽  
Loop Region ◽  
Alpha Bungarotoxin

The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.

scholarly journals Neurofascin induces neurites by heterophilic interactions with axonal NrCAM while NrCAM requires F11 on the axonal surface to extend neurites.

1996 ◽  
Vol 135 (4) ◽  
pp. 1059-1069 ◽  
Author(s):  
H Volkmer ◽  
R Leuschner ◽  
U Zacharias ◽  
F G Rathjen
Keyword(s):  
Direct Interaction ◽  
Binding Activity ◽  
Soluble Form ◽  
Binding Assays ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Neurite Extension ◽  
Ig Superfamily ◽  
Inert Surface ◽  
Axonal Extension

Neurofascin and NrCAM are two axon-associated transmembrane glycoproteins belonging to the L1 subgroup of the Ig superfamily. In this study, we have analyzed the interaction of both proteins using neurite outgrowth and binding assays. A neurofascin-Fc chimera was found to stimulate the outgrowth of tectal cells when immobilized on an inert surface but not as a soluble form using polylysine as substrate. Antibody blocking experiments demonstrate that neurite extension on immobilized neurofascin is mediated by NrCAM on the axonal surface. Under the reverse experimental conditions where NrCAM induces neurite extension, F11, and not neurofascin, serves as axonal receptor. Binding studies using transfected COS7 cells and immunoprecipitations reveal a direct interaction between neurofascin and NrCAM. This binding activity was mapped to the Ig domains within neurofascin. The neurofascin-NrCAM binding can be modulated by alternative splicing of specific stretches within neurofascin. These studies indicate that heterophilic interactions between Ig-like proteins implicated in axonal extension underlie a regulation by the neuron.

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