Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-β-ACPC) Scaffolds

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvβ3 and α5β1 receptors using biotinylated vitronectin (αvβ3) and fibronectin (α5β1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvβ3 over α5β1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvβ3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVβ3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

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Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

Biochemical Journal ◽

10.1042/bj20080234 ◽

2008 ◽

Vol 415 (1) ◽

pp. 27-33 ◽

Cited By ~ 45

Author(s):

Meghna Thakur ◽

Pradip K. Chakraborti

Keyword(s):

Protein Kinases ◽

Solid Phase ◽

Morphological Changes ◽

Binding Assays ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Peptidoglycan Biosynthesis ◽

Vitro Binding ◽

Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽

10.3390/cells10082005 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2005

Author(s):

Irene Vorontsova ◽

James E. Hall ◽

Thomas F. Schilling ◽

Noriaki Nagai ◽

Yosuke Nakazawa

Keyword(s):

Cell Adhesion ◽

Single Amino Acid ◽

Amino Acid Difference ◽

Adhesion Assay ◽

Loss Of Function ◽

Adhesive Properties ◽

The Difference ◽

In Vitro Adhesion ◽

Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

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Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

Journal of Cell Science ◽

10.1242/jcs.113.13.2471 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2471-2483 ◽

Cited By ~ 3

Author(s):

I. Hofmann ◽

C. Mertens ◽

M. Brettel ◽

V. Nimmrich ◽

M. Schnolzer ◽

...

Keyword(s):

Epithelial Cells ◽

Intermediate Filament ◽

Solid Phase ◽

Specific Interaction ◽

Binding Assays ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Vitro Analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

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Thranbospondin Promotes Cell-and Platelet-Substratum Adhesion

10.1055/s-0038-1643820 ◽

1987 ◽

Author(s):

George P Tuszynski ◽

Vicki L Rothman ◽

Andrew Murphy ◽

Katherine Siegler ◽

Linda Smith ◽

...

Keyword(s):

Cell Adhesion ◽

Cell Surface ◽

Polyacrylamide Gel Electrophoresis ◽

Cell Types ◽

Preliminary Evidence ◽

Human Platelets ◽

Cell Surface Receptor ◽

Binding Assays ◽

Surface Receptor

Thrombospondin (TSP), isolated from human platelets, promotes the in vitro, calcium-specific adhesion of a variety of cells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. The cell adhesion-promoting activity of TSP is species independent since human, bovine, pig, rat and mouse cells all adhered to TSP. Furthermore, the cell adhesion-promoting activity of TSP is specific and not due to a nonspecific protein effect or to contamination by fibronectin, vitronectin, or laminin. That is, neither bovine serum albumin nor TSP preparations treated with a monospecific anti-TSP antibody support cell adhesion. As analyzed by polyacrylamide-gel electrophoresis and specific antibody binding assays, the TSP preparations used in these studies contained no detectable fibronectin or laminin and less than 0.04% vitronectin. The cell surface receptor for TSP appears distinct frcm that of fibronectin since an antiserum that blocks cell adhesion to fibronectin has no effect on adhesion to TSP. In addition, The platelet cell surface receptor for TSP appears distinct, frcm that of fibrinogen since thrcmbasthenic platelets adhere to TSP as well as control platelets. Antibodies to the GPIIb-GPIIIa complex block platelet adhesion to fibrinogen but have no effect on adhesion to TSP. Initial characterization of the cell surface receptor for TSP shows it to be protein in nature since cells treated with trypsin fail to adhere to TSP. In summary, our results provide the first clear evidence that TSP specifically promotes cell-substratum adhesion of a variety of cell types independent of the animal species. Our preliminary evidence suggests that the cell-surface receptor(s) for TSP is protein and that it is distinct for the receptor for fibronectin and fibrinogen. Our data suggest that TSP may play a central role in normal adhesive events mediated by platelets and other cells, such as those involved in hemostasis and wound healing. In addition, TSP may be involved in pathological adhesive events mediated by platelets and tumor cells, such as those involved in cardiovascular disease and tumor cell metastasis.

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Neutrophils Rolling on Immobilised Platelets Migrate into Homotypic Aggregates after Activation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615037 ◽

1998 ◽

Vol 79 (06) ◽

pp. 1177-1183 ◽

Cited By ~ 14

Author(s):

Christopher Buckley ◽

David Simmons ◽

Gerard Nash ◽

G. E. Rainger

Keyword(s):

Cell Adhesion ◽

Vascular Disease ◽

Adhesion Molecules ◽

Platelet Activating Factor ◽

Vascular Occlusion ◽

Adhesion Assay ◽

Αvβ3 Integrin ◽

Projected Area ◽

Activated Platelets ◽

Cell Cell

SummaryInteractions between platelets and leucocytes are implicated in the pathology of thrombotic vascular disease. Using a flow-based adhesion assay we have investigated a novel route for the formation of neutrophil aggregates on the surface of immobilised activated platelets. Neutrophils perfused over a platelet monolayer formed numerous rolling attachments but rapidly stopped and spread after the superfusion of N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor (both at 10–7 M). Subsequent integrin-mediated migration across the platelet monolayer enabled formation of homotypic neutrophil aggregates, which was significant within 2.5 min of receipt of either stimulus. Aggregates increased in size with time and had an average projected area of ~500 μm2 after 10 min. Increasing size was correlated with an increasing tendency for movement downstream and large aggregates sometimes tumbled in that direction. The formation and stability of homotypic aggregates was dependent on several adhesive mechanisms. Antibody blockade demonstrated that interactions involving CD11a/ CD18 and ICAM-3, between αvβ3-integrin and CD31 and between L-selectin and an unidentified counter-ligand were all required for the complete aggregatory response. Furthermore, blockade of L-selectin allowed initial aggregation which then reversed, suggesting that this receptor might regulate the interactions between other adhesion molecules that directly supported cell-cell adhesion. We propose that this novel route for leucocyte aggregation could promote vascular occlusion in thrombotic vessels or at distal sites in the event of embolisation.

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New insights into the structural elements involved in the skin haemorrhage induced by snake venom metalloproteinases

Thrombosis and Haemostasis ◽

10.1160/th09-12-0855 ◽

2010 ◽

Vol 104 (09) ◽

pp. 485-497 ◽

Cited By ~ 42

Author(s):

Ana Oliveira ◽

Adriana Paes Leme ◽

Amanda Asega ◽

Antonio Camargo ◽

Jay Fox ◽

...

Keyword(s):

Extracellular Matrix ◽

Snake Venom ◽

Solid Phase ◽

Structural Elements ◽

Binding Assays ◽

Domain Organisation ◽

Solid Phase Binding ◽

A Minor ◽

Von Willebrand

SummaryHaemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extravasation. This study analysed structural elements important for the interaction of four Bothrops jararaca SVMPs of different domain organisation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ~80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbohydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel™; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin interacted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI to these proteins was detected. N-deglycosylation caused loss of structural stability of bothropasin and BJ-PI but HF3 remained intact, although its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affinity to collagens I and VI, although its proteolytic activity upon these collagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage generation by SVMPs.

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Adhesive properties of carcinoembryonic antigen glycoforms expressed in glycosylation-deficient Chinese hamster ovary cell lines.

Acta Biochimica Polonica ◽

10.18388/abp.2002_3846 ◽

2002 ◽

Vol 49 (1) ◽

pp. 273-283 ◽

Cited By ~ 1

Author(s):

Anna Krop-Watorek ◽

Arkadiusz G Klopocki ◽

Marcin Czerwinski ◽

Elwira Lisowska

Keyword(s):

Cell Adhesion ◽

Carcinoembryonic Antigen ◽

Chinese Hamster Ovary ◽

Solid Phase ◽

Chinese Hamster ◽

Ovary Cell ◽

Phase Cell ◽

Adhesion Assay ◽

Cell Surface Glycoprotein ◽

Polypeptide Structure

Carcinoembryonic antigen (CEA) is an oncofoetal cell surface glycoprotein that serves as an important tumour marker for colorectal and some other carcinomas. Its immunoglobulin-like structure places CEA within the immunoglobulin superfamily. CEA functions in several biological roles including homotypic and heterotypic (with other CEA family members) cell adhesion. Cell-cell interaction can be modulated by different factors, e.g., post-translational modifications such as glycosylation. The purpose of this study was to examine whether changes in carbohydrate composition of CEA oligosaccharides can influence homotypic (CEA-CEA) interactions. In order to modulate glycosylation of CEA we used two different glycosylation mutants of Chinese hamster ovary (CHO) cells, Lec2 and Lec8. Lec2 cells should produce CEA with nonsialylated N-glycans, while Lec8 cells should yield more truncated sugar structures than Lec2. Parental CHO (Pro5) cells and the glycosylation deficient mutants were stably transfected with CEA cDNA. All three CEA glycoforms, tested in a solid-phase cell adhesion assay, showed an ability to mediate CEA-dependent cell adhesion, and no qualitative differences in the adhesion between the glycoforms were observed. Thus, it may be assumed that carbohydrates do not play a role in homotypic adhesion, and the interactions between CEA molecules depend solely on the polypeptide structure.

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Fibrinogen in equine pregnancy as a mediator of cell adhesion, an epigenetic and functional investigation

Biology of Reproduction ◽

10.1093/biolre/ioz157 ◽

2019 ◽

Author(s):

Danielle M Grant ◽

Alysson Macedo ◽

Derek Toms ◽

Claudia Klein

Keyword(s):

Dna Methylation ◽

Cell Adhesion ◽

Tumor Stroma ◽

Transcript Abundance ◽

Metastatic Potential ◽

Adhesion Assay ◽

Dependent Manner ◽

Clotting Cascade ◽

Functional Investigation

Abstract Preimplantation equine embryos synthesize and secrete fibrinogen, which is a peculiar finding as fibrinogen synthesis almost exclusively occurs in the liver. This study investigated the hypothesis that conceptus-derived fibrinogen mediates cell adhesion during fixation. On day 21 of pregnancy, five integrin subunits, including ITGA5, ITGB1, ITGAV, and ITGB1, displayed significantly higher transcript abundance than on day 16 of pregnancy. Endometrial epithelial cells adhered to fibrinogen in an integrin-dependent manner in an in vitro cell adhesion assay. Bilaminar trophoblast and allantochorion expressed fibrinogen transcript, indicating that fibrinogen expression persists past fixation. Preimplantation-phase endometrium, conceptuses, and microcotyledonary tissue expressed components of the clotting cascade regulating fibrin homeostasis, leaving open the possibility that fibrinogen is converted to fibrin. Fibrinogen is likely to have functions beyond mediating cell adhesion, such trapping growth factors and triggering signaling cascades, and has remarkable parallels to the expression of fibrinogen by some tumors. The deposition of fibrinogen within tumor stroma is characteristic of breast carcinoma, and tumor-derived fibrinogen has been implicated in the metastatic potential of circulating tumor cells. DNA methylation of the fibrinogen locus in equine conceptuses was examined in comparison to liver and endometrium, and across the full gene cluster, was significantly higher for endometrium than liver and conceptus. DNA methylation of regulatory regions did not differ between liver and conceptus, and was significantly lower than in endometrium. These results, therefore, support the hypothesis of DNA methylation being a regulator of fibrinogen expression in the conceptus.

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Bisphenol A impaired cell adhesion by altering the expression of adhesion and cytoskeleton proteins on human podocytes

Scientific Reports ◽

10.1038/s41598-020-73636-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Rafael Moreno-Gómez-Toledano ◽

María I. Arenas ◽

Clara González-Martínez ◽

Nuria Olea-Herrero ◽

Paula Reventún ◽

...

Keyword(s):

Nitric Oxide ◽

Estrogen Receptor ◽

Cell Adhesion ◽

Bisphenol A ◽

Population Studies ◽

Renal Diseases ◽

Adhesion Assay ◽

Nitric Oxide Production ◽

In Vitro Adhesion

Abstract Bisphenol A (BPA), a chemical -xenoestrogen- used in food containers is present in the urine of almost the entire population. Recently, several extensive population studies have proven a significant association between urinary excretion of BPA and albuminuria. The alteration of glomerular podocytes or "podocytopathy" is a common event in chronic albuminuric conditions. Since many podocytes recovered from patients' urine are viable, we hypothesized that BPA could impair podocyte adhesion capabilities. Using an in vitro adhesion assay, we observed that BPA impaired podocyte adhesion, an effect that was abrogated by Tamoxifen (an estrogen receptor blocker). Genomic and proteomic analyses revealed that BPA affected the expression of several podocyte cytoskeleton and adhesion proteins. Western blot and immunocytochemistry confirmed the alteration in the protein expression of tubulin, vimentin, podocin, cofilin-1, vinculin, E-cadherin, nephrin, VCAM-1, tenascin-C, and β-catenin. Moreover, we also found that BPA, while decreased podocyte nitric oxide production, it lead to overproduction of ion superoxide. In conclusion, our data show that BPA induced a novel type of podocytopathy characterizes by an impairment of podocyte adhesion, by altering the expression of adhesion and cytoskeleton proteins. Moreover, BPA diminished production of podocyte nitric oxide and induced the overproduction of oxygen-free metabolites. These data provide a mechanism by which BPA could participate in the pathogenesis and progression of renal diseases.

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Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization

Journal of Cell Science ◽

10.1242/jcs.114.22.4127 ◽

2001 ◽

Vol 114 (22) ◽

pp. 4127-4136

Author(s):

Elizabeth Howes ◽

John C. Pascall ◽

Wolfgang Engel ◽

Roy Jones

Keyword(s):

Zona Pellucida ◽

Solid Phase ◽

Binding Assays ◽

Vitro Fertilization ◽

Sperm Binding ◽

Secondary Receptor ◽

Specificity Of Binding ◽

Solid Phase Binding ◽

Complementary Binding

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

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