Tumor cell haptotaxis on immobilized N-acetylglucosamine gradients

Polyacrylamide surfaces covalently derivatized with quantifiable gradients of glycosides superimposed on a uniform adhesive background of coimmobilized Arg-Gly-Asp-containing adhesion peptide were synthesized. Substrate-directed cell redistribution (haptotaxis) was measured by seeding derivatized surfaces uniformly with B16F10 murine melanoma cells. After 4-32 hr, cells on gradients of N-acetylglucosamine (GlcNAc) redistributed markedly; higher cell densities were found at gel positions having a higher immobilized GlcNAc density. In contrast, cells seeded on otherwise identical gels having a uniform concentration of immobilized GlcNAc, or on gels having gradients of glucose or galactose, did not redistribute. Soluble inhibitors containing nonreducing terminal GlcNAc (but not those with terminal GalNAc or Gal) blocked redistribution on immobilized GlcNAc gradients. Redistribution was not affected by the presence or absence of serum in the medium. An affinity-purified antibody against beta-1,4-galactosyltransferase, a GlcNAc-binding protein reported to be expressed on B16F10 cell surfaces, attenuated GlcNAc-directed redistribution. When cells were seeded on surfaces derivatized with various uniform densities of immobilized GlcNAc coimmobilized with an invariant density of immobilized Arg-Gly-Asp-peptide, neither cell attachment nor proliferation rate were enhanced on the gels having a higher GlcNAc density. These data indicate that the redistribution on immobilized GlcNAc gradients was due to cell motility. Although gels derivatized with Arg-Gly-Asp-peptide alone supported strong B16F10 cell adhesion, surfaces derivatized with uniform high concentrations of GlcNAc did not. We conclude that cell recognition of substratum gradients that support, at best, weak adhesion (GlcNAc) on an otherwise uniform strongly adhesive background (Arg-Gly-Asp-peptide) may be sufficient to direct cell migration.     

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [¹²⁵I]-TSP1 and [¹²⁵I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,^18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd.     

Carbohydrate-mediated cell adhesion to glycoproteins immobilized on nitrocellulose

A method that allows the estimation of carbohydrate-mediated cell adhesion to glycoproteins and polysaccharides immobilized to a nitrocellulose matrix is described. Specificity of adhesion by indicator cells (Chang liver) has been verified using glycoconjugates with defined carbohydrate structure. Two independent receptor systems with beta-galactose or alpha-fucose specificity, respectively, have been demonstrated by this method to occur on Chang liver cells. The method is also applicable for other indicator cells like murine fibrosarcoma cells and has been used for the analysis of dot-blots and Western blots of glycoproteins.     

Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.     

The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.     

Lysis ofMicrococcus lysodeikticus by lysozyme covalently immobilized on cellulose and polyacrylamide

Lysozyme immobilized on polyacrylamide beads or cellulose fibers is found to retain activity for hydrolysis of the cell walls of Micrococcus lysodeikticus. The immobilization on cellulose is somewhat reversible; the polyacrylamide immobilized lysozyme does not release any enzyme upon washing as evidenced by UV and lytic activity tests. The specific catalytic activity of the lysozyme-polyacrylamide system is found to decline as the density of derivatized surface groups is increased; a model of protein deactivation due to excess surface coupling is presented as a possible rationale for such specific activity variations.     

Electrochemical monitoring of chlorhexidine digluconate effect on polyelectrolyte immobilized bacteria and kinetic cell adhesion

The electrochemical impedance spectroscopy (EIS) technique has been used as a sensitive method to explore the effect of antibacterial molecules on immobilized bacteria and biofilm formation. In this work, we describe the electrochemical spectroscopy as a powerful method to monitor the effect of Chlorhexidine Digluconate (CHX-Dg) on polyelectrolyte immobilized Escherichia coli K12 MG1655 and the kinetics of cell adhesion on gold electrodes. The experimental impedance data were modelised with a Zview program to find the best equivalent electrical circuit and analyse its parameter's properties. Polyelectrolyte multilayer formation on the electrode surface and bacteria immobilization greatly increased the electron-transfer resistance (R_et) and reduced the constant phase element (CPE_dl). The effect of CHX-Dg was studied in a 0.5 × 10⁻⁴ mmol l⁻¹ to 0.5 mmol l⁻¹ range. The relation between the evolution of R_et and CHX-Dg concentration was found to be negatively correlated. When CHX-Dg was added, the electrochemical monitoring of the bacterial kinetic adhesion showed that the electrode's capacity (C_P) variation remained stable, demonstrating that the addition of CHX-Dg in the broth inhibited bacterial adhesion.     

Bioactive microarrays immobilized on low-fouling surfaces to study specific endothelial cell adhesion

With the aim to study how to modulate the specific endothelial cell patterning and responses on biomaterials surfaces, bioactive microarrays were developed and validated for specific cell patterning. These microarrays were made of low-fouling surfaces, that prevent nonspecific cell adhesion, bearing bioactive molecules at given known locations by presenting specific ligands to cell receptors. Arrays of bioactive molecules (RGD, REDV, and SVVYGLR sequences and vascular endothelial growth factor (VEGF)) were immobilized on a carboxy-methyl-dextran low-fouling surface and were exposed to human endothelial cells and fibroblasts to screen for the effect of bioactive spot molecular composition on cell adhesion. Endothelial cells only were sensitive to RGD peptide co-immobilized with REDV or SVVYGLR sequences: they induced a reduction in cell spreading and a loss of actin stress fibers. RGD co-immobilized with VEGF also resulted in the reorganization of actin filaments and focal points in endothelial cells. Combination of RGD with these endothelial cell-selective biomolecules did not elicit a strong adhesion phenotype but rather one characteristic of migrating cells.     

Effect of cyclic RGD peptide on cell adhesion and tumor metastasis.

Several kinds of cyclic peptides containing an L-arginine-glycine-L-aspartic acid RGD sequence were synthesized by the liquid phase method, and we investigated their effects on the attachment of mouse B16 melanoma cells onto fibronectin-coated well. Cyclo (GRGDSPA) inhibited the cell attachment at a 20-fold lower concentration than the linear form. The cell adhesion was inhibited by the synthetic peptides with the following relative order of activity: cyclo (GRGDSPA) much greater than cyclo (GRGD) greater than cyclo (RGDS), cyclo (GRGDSP) greater than cyclo (GRGDS) greater than cyclo (RGDSP), cyclo (RGDSPA). Cyclo (GRGDSPA) was more effective at inhibiting cell attachment to vitronectin than it was at competing with fibronectin attachment, as reported in the case of GRGDSP. Moreover, cyclo (GRGDSPA) significantly reduced the formation of colonies in mice injected with B16-FE7 melanoma cells.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification with HA on physicochemical surface properties of these substrata and estimates of the quantities of immobilized HA were obtained by different physical methods such as contact angle measurements, ellipsometry, and atomic force microscopy. The bioactivity of aHA and tHA toward their natural binding partner aggrecan was studied by comparing surface plasmon resonance to native HA; this shows that binding of aggrecan was achieved in a similar way. Dermal human fibroblasts were used as a model cell to study how chemical modification and immobilization of HA impact adhesion and spreading of cells, which also affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated primarily by HA receptor CD44, indicating that bioactivity of HA was not significantly reduced by chemical modification.     

Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate.

A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules.     

Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support

Glucose oxidase (GOD) was covalently immobilized on amorphous AlPO₄ as well as on an AlPO₄/clay mineral Sepiolite system. Immobilization of the enzyme was carried out through the -amino group of lysine residues through an aromatic Schiff's-base. Activation of the support was obtained after reaction of appropriate molecules with support surface –OH groups. The enzymatic activities of native, and different immobilized GOD systems and filtrates, were followed by the amount of liberated -gluconic acid obtained in the enzymatic β- -glucose oxidation with the aid of an automatic titrator. The kinetic properties of native and immobilized GOD were obtained for glucose concentrations in the range of physiological conditions and at different working conditions such as reaction temperature, reaction pH, and enzyme concentration.

The binding percentage of enzymes was in the 50–80% range, with residual and specific activities in the 65–80% and 90–150% ranges, respectively. No change in the pH optimum and only slight changes in the V_max and K_M kinetic parameters with respect to native GOD were observed, so that not only was little deactivation of enzyme obtained throughout the immobilization process but also that the stability of the covalently bound enzyme in the two supports appeared to have increased with respect to the soluble enzyme. GOD immobilization also increased its efficiency and operational stability in repeated uses on increasing the amount of immobilized enzyme.     

A quantitative microassay of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on polystyrene plates.

A microadhesion assay that allows the quantitative determination of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on 96-well polystyrene plates has been developed. After dislodging nonadherent cells by centrifugation, specifically bound cells are quantified by colorimetric analysis of a blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide by enzymatic reduction. Carbohydrate specificity of the cell adhesion was demonstrated by inhibition analyses and the general applicability of the assay was proved with indicator cells of three different origins: mouse fibrosarcoma cells, Chang liver cells, and human breast carcinoma cells (MDA-MB 231).     

Structural investigations of a GYF domain covalently linked to a proline-rich peptide

Protein structure determination of low affinity complexes of interacting macromolecules is often hampered by a lack of observable NOEs between the binding partners. Covalent linkage offers a way to shift the equilibrium of the interaction partners to the bound state. Here we show that a single-chain protein containing the GYF domain of CD2BP2 and the target peptide SHRPPPPGHRV from CD2 allows for the intramolecular association of the binding partners. We obtained NOEs between the GYF domain and the peptide that could define the principal orientation of the peptide in the complex. In conjunction with general recognition rules for proline-rich sequence recognition these NOEs allowed the accurate modeling of the protein-peptide complex.     

Identification of covalently bound fatty acids on acylated proteins immobilized on nitrocellulose paper

A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described. As demonstrated for [3H]palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices. Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography. The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system.     

Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.     

A peptide containing the cell adhesion sequence RGD can mediate degranulation and cell adhesion of crayfish granular haemocytes in vitro

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (which corresponds to a fragment of fibronectin and contains its cell adhesion sequence RGD) caused degranulation and spreading of monolayers of isolated granular haemocytes of the crayfish Pacifastacus leniusculus in vitro. When coated on glass coverslips, this RGD-containing peptide could mediate cell attachment of granular cells in vitro. A control peptide, Gly-Arg-Gly-Glu-Ser (GRGES), did not have these activities.Thus, GRGDS imitates the biological activities in vitro of the cell adhesion factor recently purified from crayfish haemocytes. This suggests that the sequence Arg-Gly-Asp (RGD), which is responsible for the cell adhesion activities of a number of vertebrate proteins, may also be involved in degranulation and cell adhesion of arthropod haemocytes.This is the first report describing direct activities by an RGD-containing peptide towards invertebrate cells in vitro, and the first indication of the presence of an RGD-recognizing receptor on invertebrate haemocytes.     

Preparation of immobilized papain covalently bound on natural cellulose for treatment of beer

Summary Papain was covalently immobilized on both natural wood chip and cotton without substantial loss of enzymic activity, and its optimum pH was 6–10; optimum temperature, 67°C. The immobilized papain showed high activity for hydrolysis of beer peptide and was used in treatment of beer for 28 days without loss of activity.     

A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme

The G-H loop of foot-and-mouth disease virus is a disordered protrusion of the VP1 protein exposed on the virion surface. This short stretch includes an arginine-glycine-aspartic acid tripeptide, a recognized integrin-binding motif, which is responsible for cell attachment and infection. Eight copies of a peptide reproducing the amino acid sequence of this FMDV ligand have been displayed in solvent-exposed regions on an enzymatically active recombinant beta-galactosidase. This viral peptide segment enables the chimeric enzyme to bind mammalian cell lines with different efficiencies, probably depending on the number of suitable cell receptors present on each of them. Moreover, it also promotes the internalization of the attached enzyme, which is transiently active inside the cells. These results suggest further exploration of the potential use of short adhesion peptides of viral origin as cell attachment tags to direct the targeted delivery of both genes and enzymes, instead of whole, infectious viruses.