Laminin Fragments Research Articles

Polypyrrole doped with 2 peptide sequences from laminin

In the field of neural tissue engineering, electrically conducting, biocompatible surfaces are of great interest. Over the past several decades conducting polymers have been studied as candidate surfaces because they fit these criteria. Several attempts have been made to combine the conductivity and biocompatibility of conducting polymers with biomolecules that could promote specific cell attachment and growth. In this report the laminin fragments CDPGYIGSR (p31) and RNIAEIIKDI (p20) are used as dopants in electropolymerization of the conducting polymer polypyrrole (PPy). The electrical properties of the resulting films are analyzed by impedance spectroscopy and cyclic voltammetry and compared to gold. PPy/p20 surfaces consistently demonstrate the lowest impedance and largest charge capacity for a given deposition charge. Next, in vitro studies using primary neurons cultured in a defined media and primary astrocytes in a serum containing media were performed; neuron density and neurite length, as well as astrocyte density, were quantified. Surfaces doped with a combination of the two peptides (PPy/p20-p31) consistently supported the highest neuronal density. It is shown that surfaces doped with the laminin fragment p20 had significantly longer primary neurites than either the p31 doped or poly(styrenesulfonate) doped PPy surfaces. Finally, the astrocyte studies demonstrate that PPy surfaces have significantly less astrocyte adhesion in culture than the common electrode material, gold.

Spectroscopic techniques applied to the study of laminin fragments inserted into model membranes

The influence of four laminin-derived peptides on bilayer organization is studied. Spectroscopic methods applied were based on pyrene fluorescence properties (quenching, I 1/I 3, and monomer/excimer equilibrium), asymmetric membrane fluorescence (NBD-PE/dithionite), and polarization fluorescence (TMA-DPH). Also, the ability of these peptides to release carboxyfluorescein entrapped in vesicles was determined. Results suggest that these peptides do not noticeably modify the packing and motion of lipids (in the gel state), but coat its surface, preventing penetration of quenchers and chemical reactants. Nevertheless, their presence promotes a soft release of entrapped CF after incubation at 37 °C.

Effect of a laminin amphiphatic sequence on DPPC ordered bilayers

A peptide sequence, stearoyl-GESIKVAVS(NH2), related to a laminin fragment, has been synthesized. Formation of aggregates was controlled by titrating a sodium anilinonaphthalene sulphonate (ANS) solution with peptide and recording fluorescence intensity increases. The results show that this system experiences a sudden increase in fluorescence at peptide concentrations around 2.5 x 10(-4) mol/L. The interaction of this hydrophobic peptide with DPPC vesicles has been studied using fluorescence techniques. Its influence on the microviscosity of bilayers was determined by studying polarization/temperature dependence for ANS and diphenyl hexatriene (DPH) fluorescent probes. With both markers the presence of peptide promotes a clear increase in anisotropy values. This indicates a rigidifying effect. Leakage studies carried out with liposomes loaded with carboxyfluorescein (CF) indicate a stabilizing effect of the peptide on bilayers, in agreement with results obtained with fluorescent probes.

Nephrotoxicity of organic solvents: biomarkers for early detection

Evidence for a relationship between chronic kidney diseases or progression of already existing diseases (glomerulonephritides) and occupational solvent exposure has been found in case reports, in case-control studies and also in cross-sectional studies. An analysis of the available literature was performed with respect to markers measured in cross-sectional studies that might be useful for an early detection of solvent-induced effects on the kidney. The relevant cross-sectional studies were evaluated and the following markers were analyzed with respect to their suitability as biomarker for renal damage: total protein, albumin, transferrin, IgG, beta(2)-microglobulin, retinol-binding protein, N-acetyl-beta-D: -glucosaminidase, alanine aminopeptidase, beta-galactosidase, beta-glucuronidase, leucin aminopeptidase, alkaline phosphatase, lysozyme, Tamm-Horsfall protein and laminin fragments in urine as well as E-selectin, laminin and anti-laminin antibodies and anti-glomerular basement membrane antibodies in serum. An increased albumin excretion was observed more frequently in groups of workers exposed to various solvents (like toluene, styrene, aliphatic/aromatic hydrocarbon mixtures, tetrachloroethene, mixtures of chlorinated hydrocarbons) than in controls. No clear pattern emerged for the other markers. The determination of albumin excretion in the urine appears to be a useful parameter for monitoring solvent-exposed workers.

Dystroglycan in skin and cutaneous cells: beta-subunit is shed from the cell surface.

In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. In this study, we characterized the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. We show that DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the alpha- and beta-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous beta-subunit is shed from the cell surface by metalloproteinases. Shedding of the beta-subunit releases the alpha-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1beta and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of alpha-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing.

Patterned networks of mouse hippocampal neurons on peptide-coated gold surfaces

Patterned networks of hippocampal neurons were generated on peptide-coated gold substrates prepared by microscope projection photolithography and microcontact printing. A 19 amino acid peptide fragment of laminin A (PA22-2) that includes the IKVAV cell adhesion domain was used to direct patterns of cell adhesion in primary culture. Microscale grid patterns of peptide were deposited on gold-coated glass cover slips by soft lithography using “stamps” fashioned from polydimethylsiloxane. Strong coordination bonding between gold atoms on the surface and the sulfur atoms of the N-terminal cysteine residues supported stable adhesion of the peptide, which was confirmed by immunofluorescence using anti-IKVAV antiserum. Dispersed hippocampal cells isolated from neonatal mouse pups were grown on peptide-patterned gold substrates for 7 days. Neurons preferentially adhered to peptide-coated regions of the gold surface and restricted their processes to the peptide patterns. Whole cell recordings of neurons grown in patterned arrays revealed an average membrane potential of −50 mV, as well as the presence of voltage-gated ion conductances. Peptide-modified gold surfaces serve as convenient and effective substrates for growing ordered neural networks that are compatible with existing multi-electrode array recording technology.

Patterned networks of mouse hippocampal neurons on peptide-coated gold surfaces

Patterned networks of hippocampal neurons were generated on peptide-coated gold substrates prepared by microscope projection photolithography and microcontact printing . A 19 amino acid peptide fragment of laminin A (PA22-2) that includes the IKVAV cell adhesion domain was used to direct patterns of cell adhesion in primary culture. Microscale grid patterns of peptide were deposited on gold-coated glass cover slips by soft lithography using “stamps” fashioned from polydimethylsiloxane . Strong coordination bonding between gold atoms on the surface and the sulfur atoms of the N-terminal cysteine residues supported stable adhesion of the peptide, which was confirmed by immunofluorescence using anti-IKVAV antiserum. Dispersed hippocampal cells isolated from neonatal mouse pups were grown on peptide-patterned gold substrates for 7 days. Neurons preferentially adhered to peptide-coated regions of the gold surface and restricted their processes to the peptide patterns. Whole cell recordings of neurons grown in patterned arrays revealed an average membrane potential of −50 mV, as well as the presence of voltage-gated ion conductances . Peptide-modified gold surfaces serve as convenient and effective substrates for growing ordered neural networks that are compatible with existing multi-electrode array recording technology.

New analogues of laminin active fragment YIGSR: synthesis and biological activity in vitro and in vivo.

Eleven analogues of the laminin pentapeptide amide fragment Tyr-Ile-Gly-Ser-Arg-NH2 (YIGSR-NH2) corresponding to a B1 chain fragment of the glycoprotein laminin have been synthesized by the solid phase method, and their biological activity has been studied in vitro by a cell adhesion assay: all of them inhibited the adhesion of LLC tumor cells to laminin. The analogues were found to be more resistant to enzymatic degradation in human serum than YIGSR-NH2 itself. Analogue DatIGSHar-NH2 was selected for an experimental pulmonary metastasis assay in vivo: it had higher antimetastatic activity than YIGSR-NH2.

Complex between nidogen and laminin fragments reveals a paradigmatic β-propeller interface

Basement membranes are fundamental to tissue organization and physiology in all metazoans. The interaction between laminin and nidogen is crucial to the assembly of basement membranes. The structure of the interacting domains reveals a six-bladed Tyr-Trp-Thr-Asp (YWTD) beta-propeller domain in nidogen bound to laminin epidermal-growth-factor-like (LE) modules III3-5 in laminin (LE3-5). Laminin LE module 4 binds to an amphitheatre-shaped surface on the pseudo-6-fold axis of the beta-propeller, and LE module 3 binds over its rim. A Phe residue that shutters the water-filled central aperture of the beta-propeller, the rigidity of the amphitheatre, and high shape complementarity enable the construction of an evolutionarily conserved binding surface for LE4 of unprecedentedly high affinity for its small size. Hypermorphic mutations in the Wnt co-receptor LRP5 (refs 6-9) suggest that a similar YWTD beta-propeller interface is used to bind ligands that function in developmental pathways. A related interface, but shifted off-centre from the pseudo-6-fold axis and lacking the shutter over the central aperture, is used in the low-density lipoprotein receptor for an intramolecular interaction that is regulated by pH in receptor recycling.

Expression of the nidogen-binding site of the laminin gamma1 chain disturbs basement membrane formation and maintenance in F9 embryoid bodies.

Basement membranes contain two major molecular networks consisting of laminin and collagen IV. Previous antibody perturbation experiments suggest that the interaction between laminin and nidogen-1 is necessary for proper basement membrane formation and epithelial development, whereas results from gene ablation experiments in mice show that both basement membranes and general development are grossly normal in the absence of nidogen-1. To refine the perturbation approach, we produced F9-teratocarcinoma-cell-derived embryoid bodies in the presence of recombinantly expressed nidogen-binding sites localized within the gamma1III3-5 laminin fragment. We found basement membranes were disrupted in gamma1III3-5-expressing embryoid bodies. As a measurement of basement membrane function, we tested permeability and detected drastically increased diffusion rates in correlation with basement membrane disruption. Furthermore, TROMA-1 localization in embryoid bodies expressing the nidogen-binding site was altered, suggesting separation of epithelium-specific gene expression from the formation of the actual epithelium when occurring in the absence of an organized basement membrane.

Identification of Biologically Active Sequences in the Laminin α4 Chain G Domain

Laminins are a family of trimeric extracellular matrix proteins consisting of alpha, beta, and gamma chains. So far five different laminin alpha chains have been identified. The laminin alpha 4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin alpha 4 chain. Here we have focused on the biological functions of the laminin alpha 4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-alpha 4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-alpha 4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-alpha 4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-alpha 4G. These results suggest that the four active sites are involved in the biological functions of the laminin alpha 4 chain G domain. Furthermore, rec-alpha 4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.

Plasma from Cancer Patients Featuring a Characteristic Protein Composition Mediates Protection against Apoptosis

By comparative proteome analysis we searched for characteristic alterations of human plasma accompanying neoplastic disease. We identified protein alterations in plasma of prostate-, lung-, and breast-cancer patients in comparison to controls, comprising elevated levels of fibrinogen gamma-chain dimer, degradation products of antiplasmin and laminin gamma-chain, and elevated levels of acute phase proteins. The latter proteins and laminin fragments have been described as anti-apoptotic factors. We raised the question whether these alterations may have any relevance for the regulation of apoptosis. In contrast to plasma derived from healthy donors, samples from prostate-, lung-, and breast-cancer patients selectively inhibited Fas- and staurosporine-induced apoptosis in Jurkat cells but remained ineffective upon UV light-induced apoptosis. These data suggested that inhibition occurred by extracellular interference with apoptosis induction. Supporting this hypothesis, we found that formation of the CD95 death-inducing signal complex was strongly inhibited in the presence of plasma from cancer patients.

Cell Adhesion Proteins As Tumor Suppressors

We summarize recent progress on the role of cell adhesion molecules in biology and discuss the potential application of cell adhesion molecules for managing urological cancer. We comprehensively reviewed the literature from 1982 to 2001, including peer reviewed publications and recent abstracts from national meetings, relevant to cell adhesion molecules in urological cancer. A growing body of evidence suggests that alterations in the adhesion properties of neoplastic cells have a pivotal role in the development and progression of cancer. Loss of intercellular adhesion and desquamation of cells from the underlying lamina propria allows malignant cells to escape from their site of origin, degrade the extracellular matrix, acquire a more motile and invasion phenotype, and invade and metastasize. In addition to participating in tumor invasiveness and metastasis, adhesion molecules regulate or significantly contribute to various functions, including signal transduction, cell growth, differentiation, site specific gene expression, morphogenesis, immunological function, cell motility, wound healing and inflammation. To date a diverse system of transmembrane glycoproteins has been identified that mediates cell-cell and cell-extracellular matrix adhesion. The main families of adhesion molecules include members of the Ig superfamily, cadherins, integrins and selectins. Multiple and diverse cell adhesion molecules participate in intercellular and cell-extracellular matrix interactions of cancer. Cancer progression is a multistep process, in which some adhesion molecules have a pivotal role in the development of recurrent, invasive and distant metastasis. Recent data implicate some of these molecules in cell signaling and tumor suppression, which has important consequences for tumor growth.

Interaction of the leucine-rich repeats of polycystin-1 with extracellular matrix proteins: possible role in cell proliferation.

Polycystin-1, the product of the PKD1 gene, is a membrane-bound multidomain protein with a unique structure and a molecular weight of approximately 460 kD. The purpose of this study is to investigate the binding of the cystein-flanked leucine-rich repeats (LRR) of polycystin-1 to extracellular matrix (ECM) components. These interactions may play a role in normal renal development as well as the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). In vitro assays were used to assess the binding of a fusion protein containing the LRR of polycystin-1 and that of affinity purified polycystin-1 to a number of ECM components. The results showed that the LRR modulate the binding of polycystin-1 to collagen I, fibronectin, laminin, and cyst fluid-derived laminin fragments. The addition of the LRR fusion protein to cells in culture resulted in a significant dose-dependent reduction in the rate of proliferation. Cyst fluid-derived laminin fragments had a stimulatory effect on cell proliferation, which was reversed by the LRR fusion protein. These results suggest that the LRR of polycystin-1 act as mediators of the polycystin-1 interaction with the ECM. The observed suppression effect of the LRR on cell proliferation suggests a functional role of the LRR-mediated polycystin-1 involvement in cell-matrix and cell-cell interactions. These interactions may result in the enhanced cell proliferation that is a characteristic feature of ADPKD.

Acquisition and alteration of adhesion molecules during cultured human mast cell differentiation

Background: Mature human mast cells express several types of adhesion molecules on their surface. Interactions between extracellular matrix (ECM) and adhesion molecules may be important for the migration and localization of mast cells and their precursors in tissues. Little is known about the regulation of adhesion molecules on mast cells during their differentiation. Objectives: To clarify the evolution of adhesion phenotype and function, we examined the expression of adhesion molecules during cultured human mast cell (CHMC) differentiation and tested adhesion of mature CHMCs to various ECM proteins. Methods: CHMCs were obtained by culturing human cord blood–derived CD34+ cells in the presence of stem cell factor and IL-6. Indirect immunofluorescence and flow cytometry was used to study cell surface expression of adhesion molecules and other markers. Mature CHMCs were tested for adhesion molecule function with immobilized matrix proteins. Results: At 1 week of culture, cells expressed CD11a, CD18, CD29, CD49d, and CD49e. At 14 weeks of culture, more mature CHMCs expressed CD11b, CD11c, CD29, CD49b, CD49c, CD49d, CD49e, CD51, CD61, and CD54 and weakly expressed CD18 and CD11a. CD11c, CD51, and CD61 appeared de novo by 4 weeks of culture, whereas CD49b and CD49c appeared by 8 weeks. CD29 decreased at 4 weeks but returned to the identical levels of 1-week-old cells by 8 weeks. Compared with levels at week 1, the levels of CD11a, CD18, CD49d, and CD49e at 4 weeks and beyond decreased during culture. Expression of CD49a, CD49f, and αd integrin was never detectable during CHMC differentiation. Fourteen-week-old CHMCs significantly adhered to the leucine-aspartic acid-valine—containing connecting segment 1 fragment of fibronectin, the 120-kd argine-glycine-aspartic acid—containing fragment of fibronectin, vitronectin, and laminin through specific integrins. Conclusion: Expression of integrins and CD54 is differentially regulated during CHMC differentiation, and mature CHMCs can adhere to many ECM proteins. These changes may facilitate emigration from the bone marrow into the circulation and ultimately contribute to the tissue homing and localization pattern seen with mature mast cells. (J Allergy Clin Immunol 2001;107:302-9.)

A conformational analysis of biologically active RGD-containing cyclopentapeptides

The theoretical conformational analysis of the biologically active RGD-containing pentapeptide cyclo(-Arg-Gly-Asp-Phe-DVal-), an inhibitor of laminin P1 interaction with its receptor, was performed. The space of permissible torsional angles of the backbone of the molecule was studied by the Monte Carlo method. From the large number of predicted low-energy conformers with various packings of the cyclic moiety of this pentapeptide, only those were selected that corresponded to stable structures of the model linear tripeptide Ac-Ala-Gly-Asp-NHMe. This peptide simulated the spatial possibilities of the backbones of RGD-containing fragments of laminin, vitronectin, and fibronectin. We selected several dozen structures that may be potential biologically active conformers, but only a few of them were capable of forming stable intramolecular hydrogen bonds. We assumed that a biologically active conformer of cyclo(-Arg-Gly-Asp-Phe-DVal-) can be present in significant amounts in an equilibrium mixture in solution along with other conformers without necessarily dominating among them.

Serum laminin as a marker of diabetic retinopathy development: a 4-year follow-up study

PURPOSE: The usefulness of laminin as a serum marker of diabetic retinopathy is a topic that generates conflicting views. The aim of the present study was to investigate the effect of diabetic retinopathy on serum laminin-P1, the larger pepsin resistant fragment of laminin, and to elucidate whether serum laminin-P1 could be an indicator of the risk for development of diabetic retinopathy. METHODS: In a prospective study, 97 consecutive diabetic patients (35 type 1 and 62 type 2) without diabetic retinopathy and a urinary albumin excretion rate lower than 20 μg per minute were enrolled in a 4-year follow-up study. Patients who developed microalbuminuria during the study were excluded in order to avoid the influence of diabetic nephropathy on serum laminin-P1. At the end of follow-up, data from ophthalmologic studies and serum laminin-P1 were evaluated in the 66 normoalbuminuric diabetic patients who completed the study. RESULTS: No statistical differences were observed in baseline laminin-P1 serum concentrations between patients who developed diabetic retinopathy (n = 15) and patients who remained without it during follow-up (n = 51). However, serum laminin-P1 levels obtained at the end of the study were significantly higher in patients who developed diabetic retinopathy (1.75 ± 0.33 U/ml versus 1.47 ± 0.27 U/ml; P = .002). Furthermore, statistical difference was observed when initial and final values of serum laminin-P1 were compared in patients who developed diabetic retinopathy (1.56 ± 0.27 U/ml versus 1.75 ± 0.33 U/ml; P = .001). Remarkably, an increase in serum laminin-P1 concentration was detected in all but two of the patients who developed diabetic retinopathy. The relative risk of development of diabetic retinopathy in patients who showed an increase in serum laminin-P1 during follow-up was 5.4 (95% confidence interval, 1.32 to 22.13). CONCLUSIONS: Serum laminin-P1 is a marker and a risk indicator of diabetic retinopathy but is not an early predictor of its development.

Effect of panretinal photocoagulation on serum levels of laminin in patients with diabetes: a prospective study

BACKGROUND/AIMLaminin, a major specific non-collagenous glycoprotein of basement membrane, has been proposed as an index of diabetic retinopathy and high serum concentrations have been reported in patients with proliferative diabetic...

Division of labor among the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells.

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein beta-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor. Signals from laminin for beta-casein expression were inhibited in the presence of function-blocking antibodies against both the alpha6 and beta1 integrin subunits and by the laminin E3 fragment. The alpha6-blocking antibody perturbed signals mediated by the alpha6beta4 integrin, and the beta1-blocking antibody perturbed signals mediated by another integrin, the alpha subunit(s) of which remains to be determined. Neither alpha6- nor beta1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

Soluble factors from the target organ enhance tumor cell angiogenesis: role of laminin SIKVAV sequence.

The ability of tumor cells to respond to microenvironmental factors present in the target organ determines in part the successful development of a metastasis. In a previous work it was demonstrated that the conditioned medium (CM) from lungs of normal mice stimulates in vitro migration, proliferation and uPA activity of cells from a murine mammary adenocarcinoma moderately metastatic to lung. This CM also enhanced local and metastatic tumor growth. Here, we show that lung CM enhanced neovascularization when inoculated together with LM3 tumor cells into the skin of syngeneic mice. A similar tumor-induced angiogenesis response was obtained when lung CM was injected systemically. Western blot analysis of lung CM revealed the presence of some laminin fragments containing the sequence SIKVAV. To determine whether those molecules were responsible for the observed angiogenic effects, the CM was depleted of the peptides containing the SIKVAV sequence. We observed that the SIKVAV-depleted lung CM lost its ability to induce an enhancement of the tumor neovascular response. Our results suggest a role for the target organ in facilitating the neovascularization of tumor cells, probably through the participation of active peptides derived from the proteolytic degradation of the basement membrane component laminin.