scholarly journals Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

2020 ◽  
Vol 295 (11) ◽  
pp. 3719-3733 ◽  
Author(s):  
Fajr A. Aleisa ◽  
Kosuke Sakashita ◽  
Jae Man Lee ◽  
Dina B. AbuSamra ◽  
Bader Al Alwan ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Expression System ◽  
Critical Role ◽  
Structural Features ◽  
Cellular Adhesion ◽  
Dissociation Rate ◽  
Association Rate ◽  
Lectin Domain ◽  
Selectin Ligand ◽  
And Migration

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

scholarly journals Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

1999 ◽  
Vol 10 (9) ◽  
pp. 2861-2878 ◽  
Author(s):  
Margaret A. Adelsman ◽  
James B. McCarthy ◽  
Yoji Shimizu
Keyword(s):  
Cell Adhesion ◽  
Growth Factor ◽  
Breast Carcinoma ◽  
Carcinoma Cell ◽  
Critical Role ◽  
Metastatic Breast ◽  
Cellular Adhesion ◽  
Breast Carcinoma Cell Line ◽  
Β1 Integrin ◽  
And Migration

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.

scholarly journals Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus

2004 ◽  
Vol 382 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Teresa FREIRE ◽  
Cecilia FERNÁNDEZ ◽  
Cora CHALAR ◽  
Rick M. MAIZELS ◽  
Pedro ALZARI ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Maximal Activity ◽  
Structural Features ◽  
Helminth Parasites ◽  
Lectin Domain ◽  
C Terminus ◽  
The One

As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 °C, in the range 6.5–7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.

scholarly journals Identification of Podocalyxin-like Protein as a High Endothelial Venule Ligand for L-selectin: Parallels to CD34

1998 ◽  
Vol 187 (12) ◽  
pp. 1965-1975 ◽  
Author(s):  
Christopher Sassetti ◽  
Kirsten Tangemann ◽  
Mark S. Singer ◽  
David B. Kershaw ◽  
Steven D. Rosen
Keyword(s):  
Adhesion Molecule ◽  
Leukocyte Adhesion ◽  
Structural Features ◽  
High Endothelial Venules ◽  
Lectin Domain ◽  
Kidney Glomerulus ◽  
Selectin Ligand ◽  
Definition Of ◽  
Secondary Lymphoid Organs

The leukocyte adhesion molecule, L-selectin, mediates the recruitment of lymphocytes to secondary lymphoid organs via interactions with specific ligands presented on high endothelial venules (HEV). Although the HEV-derived ligands for L-selectin are still incompletely defined, they share a common sialomucin-like structure which is thought to present clustered oligosaccharides to the lectin domain of L-selectin. Podocalyxin-like protein (PCLP) is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34. PCLP has been shown previously to be expressed on the foot processes of podocytes in the kidney glomerulus as well as on vascular endothelium at some sites. We have determined that PCLP is present on HEV, where it binds to both recombinant L-selectin and the HEV-specific monoclonal antibody MECA-79. Furthermore, purified HEV-derived PCLP is able to support the tethering and rolling of lymphocytes under physiological flow conditions in vitro. These results suggest a novel function for PCLP as an adhesion molecule and allow the definition of conserved structural features in PCLP and CD34, which may be important for L-selectin ligand function.

scholarly journals An Autotransporter Protein fromOrientia tsutsugamushiMediates Adherence to Nonphagocytic Host Cells

2011 ◽  
Vol 79 (4) ◽  
pp. 1718-1727 ◽  
Author(s):  
Na-Young Ha ◽  
Nam-Hyuk Cho ◽  
Yeon-Sook Kim ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim
Keyword(s):  
Mammalian Cells ◽  
Scrub Typhus ◽  
Expression System ◽  
Critical Role ◽  
Cellular Adhesion ◽  
Host Cells ◽  
Mammalian Host ◽  
Functional Conservation ◽  
Pulldown Assay ◽  
Obligate Intracellular Pathogen

ABSTRACTOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen whose mechanism of cellular adhesion and invasion is poorly characterized. Bioinformatic analyses of twoO. tsutsugamushigenomes revealed the presence of a group of genes that encode autotransporter proteins. In this study, we identified 10 autotransporter gene products and categorized them into five groups of orthologs (ScaA to ScaE) based on their sequence similarities. Sequence homology was highest between members of ScaC group, suggesting the functional conservation of bacterium-host interactions. ScaC was actively expressed on the surface ofO. tsutsugamushiand induced antibody responses in scrub typhus patients. Experiments using microbeads conjugated to recombinant ScaC or a surrogateEscherichia coliexpression system showed that ScaC was sufficient to mediate attachment to, but not invasion of, nonphagocytic mammalian cells. In addition, preincubation of host cells with recombinant ScaC significantly inhibited their interaction withO. tsutsugamushi. Finally, fibronectin was identified as a potential receptor for ScaC by using yeast two-hybrid screening, and this was confirmed using a glutathioneS-transferase (GST) pulldown assay. Taken together, these results demonstrate that ScaC is involved in the interaction ofO. tsutsugamushiwith mammalian host cells and suggest that ScaC may play a critical role in bacterial pathogenesis.

Exosomal miR-214-5p Released from Glioblastoma Cells Modulates Inflammatory Response of Microglia after Lipopolysaccharide Stimulation through Targeting CXCR5

2019 ◽  
Vol 18 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Jian-kai Yang ◽  
Hong-jiang Liu ◽  
Yuanyu Wang ◽  
Chen Li ◽  
Ji-peng Yang ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Clinical Prognosis ◽  
Direct Target ◽  
And Migration ◽  
High Level ◽  
Cxcr5 Expression ◽  
Proliferation And Migration

Background and Objective: Exosomes communicate inter-cellularly and miRNAs play critical roles in this scenario. MiR-214-5p was implicated in multiple tumors with diverse functions uncovered. However, whether miR-214-5p is mechanistically involved in glioblastoma, especially via exosomal pathway, is still elusive. Here we sought to comprehensively address the critical role of exosomal miR-214-5p in glioblastoma (GBM) microenvironment.Methods:The relative expression of miR-214-5p was determined by real-time PCR. Cell viability and migration were measured by MTT and transwell chamber assays, respectively. The secretory cytokines were measured with ELISA kits. The regulatory effect of miR-214-5p on CXCR5 expression was interrogated by luciferase reporter assay. Protein level was analyzed by Western blot.Results:We demonstrated that miR-214-5p was aberrantly overexpressed in GBM and associated with poorer clinical prognosis. High level of miR-214-5p significantly contributed to cell proliferation and migration. GBM-derived exosomal miR-214-5p promoted inflammatory response in primary microglia upon lipopolysaccharide challenge. We further identified CXCR5 as the direct target of miR-214- 5p in this setting.Conclusion:Overexpression of miR-214-5p in GBM modulated the inflammatory response in microglia via exosomal transfer.

scholarly journals MIR21-induced loss of junctional adhesion molecule A promotes activation of oncogenic pathways, progression and metastasis in colorectal cancer

2021 ◽  
Author(s):  
Andrea Lampis ◽  
Jens C. Hahne ◽  
Pierluigi Gasparini ◽  
Luciano Cascione ◽  
Somaieh Hedayat ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Critical Role ◽  
Tumour Volume ◽  
Cell Permeability ◽  
Oncogenic Pathways ◽  
Morphogenetic Protein ◽  
Cancer Phenotype ◽  
And Migration ◽  
2D And 3D

AbstractJunctional adhesion molecules (JAMs) play a critical role in cell permeability, polarity and migration. JAM-A, a key protein of the JAM family, is altered in a number of conditions including cancer; however, consequences of JAM-A dysregulation on carcinogenesis appear to be tissue dependent and organ dependent with significant implications for the use of JAM-A as a biomarker or therapeutic target. Here, we test the expression and prognostic role of JAM-A downregulation in primary and metastatic colorectal cancer (CRC) (n = 947). We show that JAM-A downregulation is observed in ~60% of CRC and correlates with poor outcome in four cohorts of stages II and III CRC (n = 1098). Using JAM-A knockdown, re-expression and rescue experiments in cell line monolayers, 3D spheroids, patient-derived organoids and xenotransplants, we demonstrate that JAM-A silencing promotes proliferation and migration in 2D and 3D cell models and increases tumour volume and metastases in vivo. Using gene-expression and proteomic analyses, we show that JAM-A downregulation results in the activation of ERK, AKT and ROCK pathways and leads to decreased bone morphogenetic protein 7 expression. We identify MIR21 upregulation as the cause of JAM-A downregulation and show that JAM-A rescue mitigates the effects of MIR21 overexpression on cancer phenotype. Our results identify a novel molecular loop involving MIR21 dysregulation, JAM-A silencing and activation of multiple oncogenic pathways in promoting invasiveness and metastasis in CRC.

scholarly journals Sp1 mediates cell proliferation-dependent regulation of rat DNA topoisomerase IIα gene promoter

1999 ◽  
Vol 344 (2) ◽  
pp. 367-374 ◽  
Author(s):  
Jeong Ho YOON ◽  
Jeong Kee KIM ◽  
Geun Bae RHA ◽  
Misook OH ◽  
Se-Ho PARK ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Promoter Activity ◽  
Expression System ◽  
Critical Role ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Specific Regulation ◽  
Dna Topoisomerase Iiα

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIα is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5′ and/or 3′ serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIα. The luciferase activities of the mutated reporter plasmids in G0-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIα in growing cells.

scholarly journals Effects of a New Bioceramic Material on Human Apical Papilla Cells

2018 ◽  
Vol 9 (4) ◽  
pp. 74 ◽  
Author(s):  
Diana Sequeira ◽  
Catarina Seabra ◽  
Paulo Palma ◽  
Ana Cardoso ◽  
João Peça ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mineral Trioxide Aggregate ◽  
Cellular Adhesion ◽  
Cellular Migration ◽  
Cellular Viability ◽  
Migration Rates ◽  
Apical Papilla ◽  
And Migration ◽  
Proliferation And Migration

Background: The development of materials with bioregenerative properties is critically important for vital pulp therapies and regenerative endodontic procedures. The aim of this study was to evaluate the cytocompatibility and cytotoxicity of a new endodontic biomaterial, PulpGuard, in comparison with two other biomaterials widely used in endodontic procedures, ProRoot Mineral Trioxide Aggregate (MTA) and Biodentine. Methods: Apical papilla cells (APCs) were isolated from third molars with incomplete rhizogenesis from patients with orthodontic indication for dental extraction. Cultured APCs were incubated for 24, 48, or 72 h with different dilutions of eluates prepared from the three materials. Cellular viability, mobility, and proliferation were assessed in vitro using the Alamar Blue assay and a wound-healing test. The cells were also cultured in direct contact with the surface of each material. These were then analyzed via Scanning Electron Microscopy (SEM), and the surface chemical composition was determined by Energy-Dispersive Spectroscopy (EDS). Results: Cells incubated in the presence of eluates extracted from ProRoot MTA and PulpGuard presented rates of viability comparable to those of control cells; in contrast, undiluted Biodentine eluates induced a significant reduction of cellular viability. The wound-healing assay revealed that eluates from ProRoot MTA and PulpGuard allowed for unhindered cellular migration and proliferation. Cellular adhesion was observed on the surface of all materials tested. Consistent with their disclosed composition, EDS analysis found high relative abundance of calcium in Biodentine and ProRoot MTA and high abundance of silicon in PulpGuard. Significant amounts of zinc and calcium were also present in PulpGuard discs. Concerning solubility, Biodentine and ProRoot MTA presented mild weight loss after eluate extraction, while PulpGuard discs showed significant water uptake. Conclusions: PulpGuard displayed a good in vitro cytocompatibility profile and did not significantly affect the proliferation and migration rates of APCs. Cells cultured in the presence of PulpGuard eluates displayed a similar profile to those cultured with eluates from the widely used endodontic cement ProRoot MTA.

scholarly journals Molecular Basis for CCRL2 Regulation of Leukocyte Migration

2020 ◽  
Vol 8 ◽  
Author(s):  
Tiziana Schioppa ◽  
Francesca Sozio ◽  
Ilaria Barbazza ◽  
Sara Scutera ◽  
Daniela Bosisio ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Chemokine Receptors ◽  
Transmembrane Domain ◽  
Nk Cell ◽  
Structural Features ◽  
Negative Regulator ◽  
Leukocyte Migration ◽  
Genetic Ablation

CCRL2 is a seven-transmembrane domain receptor that belongs to the chemokine receptor family. At difference from other members of this family, CCRL2 does not promote chemotaxis and shares structural features with atypical chemokine receptors (ACKRs). However, CCRL2 also differs from ACKRs since it does not bind chemokines and is devoid of scavenging functions. The only commonly recognized CCRL2 ligand is chemerin, a non-chemokine chemotactic protein. CCRL2 is expressed both by leukocytes and non-hematopoietic cells. The genetic ablation of CCRL2 has been instrumental to elucidate the role of this receptor as positive or negative regulator of inflammation. CCRL2 modulates leukocyte migration by two main mechanisms. First, when CCRL2 is expressed by barrier cells, such endothelial, and epithelial cells, it acts as a presenting molecule, contributing to the formation of a non-soluble chemotactic gradient for leukocytes expressing CMKLR1, the functional chemerin receptor. This mechanism was shown to be crucial in the induction of NK cell-dependent immune surveillance in lung cancer progression and metastasis. Second, by forming heterocomplexes with other chemokine receptors. For instance, CCRL2/CXCR2 heterodimers were shown to regulate the activation of β2-integrins in mouse neutrophils. This mini-review summarizes the current understanding of CCRL2 biology, based on experimental evidence obtained by the genetic deletion of this receptor in in vivo experimental models. Further studies are required to highlight the complex functional role of CCRL2 in different organs and pathological conditions.

scholarly journals The Hinge-Helix 1 Region of Peroxisome Proliferator-Activated Receptor γ1 (PPARγ1) Mediates Interaction with Extracellular Signal-Regulated Kinase 5 and PPARγ1 Transcriptional Activation: Involvement in Flow-Induced PPARγ Activation in Endothelial Cells

2004 ◽  
Vol 24 (19) ◽  
pp. 8691-8704 ◽  
Author(s):  
Masashi Akaike ◽  
Wenyi Che ◽  
Nicole-Lerner Marmarosh ◽  
Shinsuke Ohta ◽  
Masaki Osawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Critical Role ◽  
Physiological Role ◽  
Cellular Adhesion ◽  
Dominant Negative ◽  
Peroxisome Proliferator ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Cellular Adhesion Molecule

ABSTRACT Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPARγ have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPARγ activity. We found that activation of ERK5 induced PPARγ1 activation in endothelial cells (ECs). However, we could not detect PPARγ phosphorylation by incubation with activated ERK5 in vitro, in contrast to ERK1/2 and JNK, suggesting a role for ERK5 as a scaffold. Endogenous PPARγ1 was coimmunoprecipitated with endogenous ERK5 in ECs. By mammalian two-hybrid analysis, we found that PPARγ1 associated with ERK5a at the hinge-helix 1 region of PPARγ1. Expressing a hinge-helix 1 region PPARγ1 fragment disrupted the ERK5a-PPARγ1 interaction, suggesting a critical role for hinge-helix 1 region of PPARγ in the ERK5-PPARγ interaction. Flow increased ERK5 and PPARγ1 activation, and the hinge-helix 1 region of the PPARγ1 fragment and dominant negative MEK5β significantly reduced flow-induced PPARγ activation. The dominant negative MEK5β also prevented flow-mediated inhibition of tumor necrosis factor alpha-mediated NF-κB activation and adhesion molecule expression, including vascular cellular adhesion molecule 1 and E-selectin, indicating a physiological role for ERK5 and PPARγ activation in flow-mediated antiinflammatory effects. We also found that ERK5 kinase activation was required, likely by inducing a conformational change in the NH2-terminal region of ERK5 that prevented association of ERK5 and PPARγ1. Furthermore, association of ERK5a and PPARγ1 disrupted the interaction of SMRT and PPARγ1, thereby inducing PPARγ activation. These data suggest that ERK5 mediates flow- and ligand-induced PPARγ activation via the interaction of ERK5 with the hinge-helix 1 region of PPARγ.

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