scholarly journals Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One

2021 ◽  
Vol 22 (15) ◽  
pp. 8284
Author(s):  
Soran Mohammed ◽  
Natalie Ferry
Keyword(s):  
Sialic Acid ◽  
Fluorescent Protein ◽  
Protein Docking ◽  
Binding Activity ◽  
Binding Domain ◽  
Mistletoe Lectin ◽  
Binding Domains ◽  
Lectin Family ◽  
Green Fluorescent ◽  
Binding Lectin

Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and β-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.

scholarly journals Porcine Arterivirus Attachment to the Macrophage-Specific Receptor Sialoadhesin Is Dependent on the Sialic Acid-Binding Activity of the N-Terminal Immunoglobulin Domain of Sialoadhesin

2007 ◽  
Vol 81 (17) ◽  
pp. 9546-9550 ◽  
Author(s):  
Peter L. Delputte ◽  
Wander Van Breedam ◽  
Iris Delrue ◽  
Cornelia Oetke ◽  
Paul R. Crocker ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Binding Activity ◽  
Sialic Acids ◽  
Binding Domain ◽  
Respiratory Syndrome Virus ◽  
Specific Receptor ◽  
Terminal Domain ◽  
Sialic Acid Binding ◽  
Immunoglobulin Domain ◽  
Binding Lectin

ABSTRACT The sialic acid-binding lectin sialoadhesin (Sn) is a macrophage-restricted receptor for porcine reproductive and respiratory syndrome virus (PRRSV). To investigate the importance of pSn sialic acid-binding activity for PRRSV infection, an R116-to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSnRE, that could not bind sialic acids. PSn, but not pSnRE, allowed PRRSV binding and internalization. These data show that the sialic acid-binding activity of pSn is essential for PRRSV attachment to pSn and thus identifies the variable, N-terminal domain of Sn as a PRRSV binding domain.

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

2004 ◽  
Vol 286 (3) ◽  
pp. L506-L513 ◽  
Author(s):  
Christopher E. Helt ◽  
Rhonda J. Staversky ◽  
Yi-Jang Lee ◽  
Robert A. Bambara ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Nuclear Antigen ◽  
Cell Cycle Regulators ◽  
Amino Terminal ◽  
Binding Domains ◽  
Green Fluorescent

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1 when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of the other Cip or INK4 G1 kinase inhibitors. To confirm the role of p21 in G1 arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1 arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1 during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1 arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

scholarly journals Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic

2020 ◽  
Vol 8 (7) ◽  
pp. 1051 ◽  
Author(s):  
Aleksandar Božić ◽  
Robin C. Anderson ◽  
Tawni L. Crippen ◽  
Christina L. Swaggerty ◽  
Michael E. Hume ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Saccharomyces Boulardii ◽  
Binding Activity ◽  
Intestinal Cells ◽  
E Coli ◽  
Enteric Infections ◽  
Green Fluorescent

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

scholarly journals Massive Ca-induced Membrane Fusion and Phospholipid Changes Triggered by Reverse Na/Ca Exchange in BHK Fibroblasts

2008 ◽  
Vol 132 (1) ◽  
pp. 29-50 ◽  
Author(s):  
Alp Yaradanakul ◽  
Tzu-Ming Wang ◽  
Vincenzo Lariccia ◽  
Mei-Jung Lin ◽  
Chengcheng Shen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Fluorescent Protein ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Hill Coefficient ◽  
Induced Membrane ◽  
Binding Domains ◽  
Green Fluorescent

Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25–100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to ∼5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient ≈ 2). Capacitance can return to baseline in 1–3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P2 is rapidly cleaved upon activating Ca influx and recovers within 2 min. However, PI(4,5)P2 depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca influx remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P2 is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P2-binding peptides, and PLD modification by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might reflect phosphatidylserine (PS) “scrambling” between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fibroblasts.

scholarly journals The domains of human fibronectin mediating the binding of α antigen, the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterial infection

2000 ◽  
Vol 347 (3) ◽  
pp. 725-731 ◽  
Author(s):  
Mariko NAITO ◽  
Tomohiko FUKUDA ◽  
Kiyotoshi SEKIGUCHI ◽  
Takeshi YAMADA
Keyword(s):  
Recombinant Dna ◽  
Mycobacterial Infection ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Heparin Binding ◽  
Binding Domains ◽  
Immunodominant Antigen ◽  
Heparin Binding Domain ◽  
Recombinant Dna Techniques

We have recently shown that α antigen (α-Ag), the immunodominant antigen of mycobacteria, has a novel fibronectin (FN)-binding motif that is unique among mycobacteria [Naito, Ohara, Matsumoto and Yamada (1998) J. Biol. Chem. 273, 2905-2909]. In this study, we examined the domains of human FN that interacted with α-Ag. Fragments of FN generated by either proteolysis or recombinant DNA techniques were compared for their ability to bind to α-Ag. Fragments containing either the C-terminal heparin-binding domain or the central cell-binding domain consistently bound to α-Ag. The fragment of the C-terminal heparin-binding domain, upon mutation that resulted in the loss of its heparin-binding activity, could not bind with α-Ag. These findings suggested that the mutated site, i.e. the main heparin-binding site of FN, was also the principal site for binding to α-Ag. The α-Ag-binding domains of FN could bind whole mycobacterial bacilli, suggesting that these two domains are important contributors to mycobacterial infection.

scholarly journals Engineered Microbes to Sense and Respond to Enterotoxigenic Escherichia coli

2018 ◽  
Author(s):  
A. Bete ◽  
J. Carter ◽  
C. Davis ◽  
J. Dong ◽  
M. Herrmann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Genetically Engineered ◽  
Surface Proteins ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Autoinducer 2 ◽  
Binding Domains ◽  
Sense And Respond ◽  
Green Fluorescent

AbstractEvery year, Enterotoxigenic Escherichia coli (ETEC), the most common form of traveler’s diarrhea, affects thousands of military personnel deployed overseas. The goal of this research was to engineer non-pathogenic E. coli to sense ETEC, respond to its presence, and package the non-pathogenic E. coli in a cellulose matrix to enable environmental detection of ETEC. Two plasmids were created: ‘sense-respond’; and ‘packaging’. The sense-respond plasmid detected autoinducer 2 (AI-2), a quorum sensing molecule created by most ETEC strains, by expressing LsrR which switches on the Lsr promoter. Activation of the Lsr promoter expresses superfolder green fluorescent protein (sfGFP), indicating the presence of ETEC. The packaging plasmid expresses a fusion protein consisting of curli fibers and cellulose binding domains. These modified surface proteins permit the bacteria to bind to cellulose, encapsulating the sense-response module. This genetically engineered machine could be deployed in both the internal and external environment to detect ETEC.

scholarly journals Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

2014 ◽  
Vol 80 (14) ◽  
pp. 4126-4137 ◽  
Author(s):  
Rongzhi Wang ◽  
Shuangshuang Xiang ◽  
Yonghui Zhang ◽  
Qiuyu Chen ◽  
Yanfang Zhong ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Specific Antigen ◽  
Disease Diagnosis ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Single Chain ◽  
High Affinity ◽  
Green Fluorescent ◽  
Complementarity Determining Region

ABSTRACTSingle-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

scholarly journals Glucocorticoid Signaling Is Perturbed by the Atypical Orphan Receptor and Corepressor SHP

2002 ◽  
Vol 277 (51) ◽  
pp. 49761-49766 ◽  
Author(s):  
Lotta Johansson Borgius ◽  
Knut R. Steffensen ◽  
Jan-Åke Gustafsson ◽  
Eckardt Treuter
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Binding Domain ◽  
Orphan Receptor ◽  
Orphan Nuclear Receptor ◽  
Downstream Target ◽  
Glucocorticoid Signaling ◽  
Relevant Role ◽  
Novel Target ◽  
Green Fluorescent

SHP (NROB2) is an atypical orphan nuclear receptor that lacks a DNA-binding domain but contains a putative ligand-binding domain. Previous studies have revealed that SHP interacts with a variety of nuclear receptors and inhibits their transcriptional activity, thereby acting as a corepressor. In this report we identify the glucocorticoid receptor (GR) as a novel downstream target receptor for SHP inhibition. SHP potently inhibits dexamethasone-induced transcriptional GR activity in mammalian cells, and the inhibition involves a functional second NR-box within SHP. Interestingly, this motif shows a high homology with the NR-box in the glucocorticoid and cAMP-inducible GR coactivator PGC-1, indicating similar binding specificity and shared target receptors. We show that SHP antagonizes PGC-1 coactivation and, in addition, we identify the PGC- 1-regulated phospho(enol)pyruvate carboxykinase (PEPCK) promoter as a novel target promoter for SHP inhibition. This implies a physiologically relevant role for SHP in modulating hepatic glucocorticoid action. Furthermore, when coexpressing green fluorescent protein-tagged GR together with SHP, an intranuclear redistribution of GR was observed. As inhibition-deficient SHP mutants were unable to induce this redistribution, intranuclear tethering of target receptors may represent yet another, previously uncovered, aspect of SHP inhibition.

scholarly journals Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation

2010 ◽  
Vol 30 (14) ◽  
pp. 3553-3568 ◽  
Author(s):  
Ru-Feng Wu ◽  
Zhenyi Ma ◽  
Zhe Liu ◽  
Lance S. Terada
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Cell Fractionation ◽  
Protective Mechanisms ◽  
3 Dimensional ◽  
Binding Domains ◽  
Pathway Activation ◽  
Oxidant Production ◽  
Green Fluorescent ◽  
The Unfolded Protein Response

ABSTRACT The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H2O2 sensor targeted to different subcellular compartments, we demonstrate specific production of H2O2 by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H2O2 production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.

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