Characterization of plasminogen as an adhesive ligand for integrins αMβ2 (Mac-1) and α5β1 (VLA-5)

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 719-726 ◽  
Author(s):  
Valeryi K. Lishko ◽  
Valery V. Novokhatny ◽  
Valentin P. Yakubenko ◽  
Helen V. Skomorovska-Prokvolit ◽  
Tatiana P. Ugarova
Keyword(s):  
Cell Adhesion ◽  
Aminocaproic Acid ◽  
Genetically Engineered ◽  
Plasmin Activity ◽  
Hek 293 ◽  
293 Cells ◽  
Blood Neutrophils ◽  
Α5β1 Integrin ◽  
Extracellular Proteolysis

AbstractPlasminogen (Pg) has been implicated in many biologic processes involving extracellular proteolysis. We investigated whether Pg, by virtue of its capacity to be deposited within the extracellular matrix, can serve as a ligand for cell surface integrins. We report here that Pg supports cell adhesion by engaging integrins αMβ2 and α5β1. The immobilized Glu-Pg, but not its derivatives with the N-terminal peptide lacking, plasmin and Lys-Pg, supported efficient adhesion that was abolished by anti-αMβ2 and anti-α5β1 integrin-specific monoclonal antibodies (mAbs). In addition, lysine binding sites of Glu-Pg contributed to cell adhesion inasmuch as tranexamic acid and ϵ-aminocaproic acid inhibited cell adhesion. The involvement of αMβ2 and α5β1 in adhesion to Glu-Pg was demonstrable with blood neutrophils, U937 monocytoid cells, and genetically engineered αMβ2-transfected human embryonic kidney (HEK) 293 cells. In αMβ2, the αMI-domain is the binding site for Glu-Pg because the “I-less” form of αMβ2 did not support cell adhesion and the recombinant αMI-domain bound Glu-Pg directly. In comparison with cell adhesion, the binding of soluble Glu-Pg to cells and the concomitant generation of plasmin activity was inhibited by anti-α5β1 but not by anti-αMβ2. These findings identify Glu-Pg as an adhesive ligand for integrins αMβ2 and α5β1 and suggest that α5β1 may participate in the binding of soluble Glu-Pg and assist in its activation.

Functional Characterization of the Regulatory Region of Human CD2-Associated Protein Promoter in HEK 293 Cells

2009 ◽  
Vol 29 (3) ◽  
pp. 203-212 ◽  
Author(s):  
Xin-Ming Su ◽  
Wei Ren ◽  
Chao Lu ◽  
Ji-Qing Chen ◽  
Sheng-Hua Wu ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Regulatory Region ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Characterization of glycine release mediated by glycine transporter 1 stably expressed in HEK-293 cells

1997 ◽  
Vol 49 (1-2) ◽  
pp. 89-94 ◽  
Author(s):  
Kazuko Sakata ◽  
Kohji Sato ◽  
Patrick Schloss ◽  
Heinrich Betz ◽  
Shoichi Shimada ◽  
...  
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
Glycine Release ◽  
Glycine Transporter 1 ◽  
Glycine Transporter ◽  
293 Cells

Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3626-3632 ◽  
Author(s):  
Barbara Plaimauer ◽  
Klaus Zimmermann ◽  
Dirk Völkel ◽  
Gerhard Antoine ◽  
Randolf Kerschbaumer ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Functional Activity ◽  
Functional Characterization ◽  
Eukaryotic Expression ◽  
Gene Encoding ◽  
Hek 293 ◽  
293 Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designatedADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.

scholarly journals Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells

1996 ◽  
Vol 118 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
Anthony G. Hope ◽  
John A. Peters ◽  
Angus M. Brown ◽  
Jeremy J. Lambert ◽  
Thomas P. Blackburn
Keyword(s):  
Type A ◽  
Receptor Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Characterization of bovine interleukin-2 stably expressed in HEK-293 cells

2020 ◽  
Author(s):  
Shuya MITOMA ◽  
Heba M. EL-KHAIAT ◽  
Tomofumi UTO ◽  
Katsuaki SATO ◽  
Satoshi SEKIGUCHI ◽  
...  
Keyword(s):  
Interleukin 2 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Clinical and Functional Characterization of the Recurrent TUBA1A p.(Arg2His) Mutation

2018 ◽  
Vol 8 (8) ◽  
pp. 145 ◽  
Author(s):  
Jennifer Gardner ◽  
Thomas Cushion ◽  
Georgios Niotakis ◽  
Heather Olson ◽  
P. Grant ◽  
...  
Keyword(s):  
De Novo ◽  
Functional Characterization ◽  
Clinical Manifestations ◽  
Fetal Brain ◽  
Detailed Comparison ◽  
Hek 293 ◽  
293 Cells ◽  
Brain Malformations ◽  
Computer Based

The TUBA1A gene encodes tubulin alpha-1A, a protein that is highly expressed in the fetal brain. Alpha- and beta-tubulin subunits form dimers, which then co-assemble into microtubule polymers: dynamic, scaffold-like structures that perform key functions during neurogenesis, neuronal migration, and cortical organisation. Mutations in TUBA1A have been reported to cause a range of brain malformations. We describe four unrelated patients with the same de novo missense mutation in TUBA1A, c.5G>A, p.(Arg2His), as found by next generation sequencing. Detailed comparison revealed similar brain phenotypes with mild variability. Shared features included developmental delay, microcephaly, hypoplasia of the cerebellar vermis, dysplasia or thinning of the corpus callosum, small pons, and dysmorphic basal ganglia. Two of the patients had bilateral perisylvian polymicrogyria. We examined the effects of the p.(Arg2His) mutation by computer-based protein structure modelling and heterologous expression in HEK-293 cells. The results suggest the mutation subtly impairs microtubule function, potentially by affecting inter-dimer interaction. Based on its sequence context, c.5G>A is likely to be a common recurrent mutation. We propose that the subtle functional effects of p.(Arg2His) may allow for other factors (such as genetic background or environmental conditions) to influence phenotypic outcome, thus explaining the mild variability in clinical manifestations.

scholarly journals Characterization of α3 Glycine Receptors with Ginkgolide B and Picrotoxin

2018 ◽  
Author(s):  
Sampurna Chakrabarti ◽  
Anil Neelakantan ◽  
Malcolm M. Slaughter
Keyword(s):  
Beta Subunits ◽  
Ginkgolide B ◽  
Glycine Receptors ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Voltage Dependent ◽  
The Central Nervous System ◽  
Subunit Isoforms

AbstractGinkgolide B (GB) and picrotoxin (PTX) are antagonists of the major inhibitory receptors of the central nervous system: GABA and glycine receptors (GlyRs). GlyRs contain one or more of the four alpha subunit isoforms of which α1 and α2 have been extensively studied. This report compares GB and PTX block of α3 GlyRs expressed in HEK 293 cells, using whole-cell patch clamp techniques. In CNS, α3 exists as a heteropentamer in conjunction with beta subunits in a 2α:3β ratio. Thus, the nature of block was also tested in α3β heteromeric glycine receptors. GB and PTX blocked α3 GlyRs both in the presence (liganded state) and absence of glycine (unliganded state). This property is unique to α3 subunits; α1 and α2 subunits are only blocked in the liganded state. The GB block of α3 GlyRs is voltage-dependent (more effective when the cell is depolarized) and non-competitive, while the PTX block is competitive and not voltage-dependent. The heteromeric and homomeric α3 GlyRs recovered significantly faster from unliganded GB block compared to liganded GB block, but no such distinction was found for PTX block suggesting more than one binding site for GB. This study sheds light on features of the α3 GlyR that distinguish it from the more widely studied α1 and α2 subunits. Understanding these properties can help decipher the physiological functioning of GlyRs in the CNS and may permit development of subunit specific drugs.

scholarly journals Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter

1999 ◽  
Vol 277 (6) ◽  
pp. C1210-C1219 ◽  
Author(s):  
Joanne E. Race ◽  
Fadi N. Makhlouf ◽  
Paul J. Logue ◽  
Frederick H. Wilson ◽  
Philip B. Dunham ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Amino Acid Level ◽  
Transmembrane Domains ◽  
Chromosome 15 ◽  
Hek 293 ◽  
293 Cells ◽  
Liver And Pancreas ◽  
Large Extracellular Loop ◽  
Hydropathy Analysis

We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75–76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and ∼20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH2 and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.

scholarly journals Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion

2003 ◽  
Vol 370 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Slavoljub VUJCIC ◽  
Ping LIANG ◽  
Paula DIEGELMAN ◽  
Debora L. KRAMER ◽  
Carl W. PORTER
Keyword(s):  
Northern Blot Analysis ◽  
Biochemical Characterization ◽  
Polyamine Oxidase ◽  
Preference Ranking ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Polyamine Analogues ◽  
293 Cells ◽  
Back Conversion

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1-acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2—4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine = N1-acetylspermidine>N1,N12-diacetylspermine>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogues and inhibitors.

Characterization of an epilepsy-associated variant of the human Cl−/HCO3−exchanger AE3

2009 ◽  
Vol 297 (3) ◽  
pp. C526-C536 ◽  
Author(s):  
Gonzalo L. Vilas ◽  
Danielle E. Johnson ◽  
Paul Freund ◽  
Joseph R. Casey
Keyword(s):  
Protein Trafficking ◽  
Specific Inhibitor ◽  
Similar Degree ◽  
Transport Activity ◽  
Wild Type ◽  
Hek 293 ◽  
293 Cells ◽  
The Brain ◽  
Exchange Activity

Anion exchanger 3 (AE3), expressed in the brain, heart, and retina, extrudes intracellular HCO3−in exchange for extracellular Cl−. The SLC4A3 gene encodes two variants of AE3, brain or full-length AE3 (AE3fl) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. The AE3-A867D allele in humans has been associated with the development of IGE (IGE), which accounts for ∼30% of all epilepsies. To examine the molecular basis for the association of the A867D allele with IGE, we characterized wild-type (WT) and AE3fl-A867D in transfected human embryonic kidney (HEK)-293 cells. AE3fl-A867D had significantly reduced transport activity relative to WT (54 ± 4%, P < 0.01). Differences in expression levels or the degree of protein trafficking to the plasma membrane did not account for the defect of AE3fl-A867D. Treatment with 8-bromo-cAMP (8-Br-cAMP) increased Cl−/HCO3−exchange activity of WT and AE3fl-A867D to a similar degree, which was abolished by preincubation with the protein kinase A (PKA)-specific inhibitor H89. This indicates that PKA regulates WT and AE3fl-A867D Cl−/HCO3−exchange activity. No difference in Cl−/HCO3−exchange activity was found between cultures of mixed populations of neonatal hippocampal cells from WT and slc4a3−/−mice. We conclude that the A867D allele is a functional (catalytic) mutant of AE3 and that the decreased activity of AE3fl-A867D may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexcitability and the generation of seizures.

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