Laminin-binding Protein Research Articles

Characterization of glycoprotein E C-End of West Nile virus and evaluation of its interaction force with αVβ3 integrin as putative cellular receptor

Recombinant polypeptide containing the 260-466 amino acid sequence of West Nile virus (WNV) strain LEIV-Vlg99-27889-human glycoprotein E (gpE, E(260-466)) was constructed. Immunochemical similarity between the E(260-466) and gpE of WNV was proven by enzyme immunoassay (EIA), immunoblot, competitive EIA, hemagglutination inhibition, and neutralization tests using polyclonal and monoclonal antibodies against the viral gpE and recombinant E(260-466). Polypeptide E(260-466) induced formation of virus neutralizing and cross-reactive antibodies that were interactive with various epitopes of this recombinant protein. It is shown by evaluation of the interaction of E(260-466) with one of the proposed cell receptors of WNV that average E(260-466)-alphaVbeta3 integrin-specific interaction force measured using atomic force spectroscopy was 80 and 140 pN for single and double interactions, correspondingly. Taken together with previously described interaction between laminin-binding protein (LBP) and WNV gpE domain II, it is proposed that WNV gpE can interact specifically with two cellular proteins (LBP and alphaVbeta3 integrin) during virus entry.

English

  Staphylococcus aureus is a causative agent of acute and infectious diarrhoea. In Africa, there is no sufficient information on the virulence and the degree of factors produced by its diarrhoea-isolated strains. Clinical features and virulence factors produced by S. aureus isolated from diarrhoeal-patients admitted at the Hospital Hubert Koutoukou Maga (HKM) in Cotonou was investigated. The virulence factors were screened by radial immunoprecipitation and multiplex polymerase chain reaction (PCR). Fifteen antibiotics were tested. Among independent 115 patients examined for diarrhoea, 32 had faeces positive for S. aureus isolated as pure culture. Most of these patients were hospitalized (21/32) and developed aqueous, bloody and painful diarrhoea, after antimicrobial therapy. About 62% were resistant to oxacillin. Genes encoding for clumping factor B and for laminin binding protein were detected in 62% of S. aureus isolates. About 94% of LukE-LukD producing strains have been isolated from patients developing post-antibiotic associated diarrhoea (PAAD). The Panton-Valentine Leucocidin (PVL) was produced by 19% of isolates, all from PAAD. This study points out new data concerning virulence factors and adhesion factor produced by S. aureus strains isolated from diarrhoea in Benin. The culture of the faeces will not always allow the diagnosis. It is important to update a technique, which enables researchers to carry out the virulence factors produced by these bacteria.   Key words: Adhesion factors, diarrhoea, enterotoxins, Staphylococcus aureus, leucotoxins, Benin, Africa, PCR.

Virulence characteristics of genetically related isolates of group B streptococci from bovines and humans

The present study had the objective of evaluating the pathogenic potential of the genetically related strains of Streptococcus agalactiae no. 80427 (human origin) and no. 87159 (bovine origin), and comparing the results with two other strains isolated from bovine mastitis (no. 87244) and invasive human infection (no. 90356), with no genetic or epidemiologic relationship between them or with the first 2 isolates. Virulence genes hylB (hyaluronidase) and lmb (laminin-binding protein) were detected in the 4 strains, and genes bac (beta protein) and bca (alpha protein) were only detected in human strains. The protein profile obtained using SDS-PAGE did not indicate any differences between the 4 strains. No significant difference was detected between human and bovine strains in the assays of adherence to and invasion of 16HBe cells, as well as in the resistance assay for intracellular bacterial survival in macrophages. However, the strain 87159 exhibited a greater survival in the killing test with whole human blood and was more virulent in newborn mice than the 80427 strain. The strain 87244 was not virulent in mice. These data suggest that isolates of human and bovine origins may express similar virulence attributes, leading to a possible, however limited, dissemination.

C-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis viruses

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alpha- and flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187-210, and MAB 4F6 interacts with the fragment of residues 263-278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187-210 and 263-278) as well as the central beta-folded region turning into the alpha-helical site including residues 200-216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.

Expression of Laminin Isoforms, Receptors, and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin gamma1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin alpha6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors, and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin alpha5 chain, laminin receptors (integrin alpha3, alpha6, beta4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors, and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

The Laminin-Binding Protein Lbp from Streptococcus pyogenes Is a Zinc Receptor

The common pathogen Streptococcus pyogenes colonizes the human skin and tonsils and can invade underlying tissues. This requires the adhesion of S. pyogenes to host surface receptors mediated through adhesins. The laminin-binding protein Lbp has been suggested as an adhesin, specific for the human extracellular matrix protein laminin. Sequence alignments, however, indicate a relationship between Lbp and a family of bacterial metal-binding receptors. To further analyze the role of Lbp in S. pyogenes and its potential role in pathogenicity, Lbp has been crystallized, and its structure has been solved at a resolution of 2.45 A (R = 0.186; R(free) = 0.251). Lbp has the typical metal-binding receptor fold, comprising two globular (beta/alpha)(4) domains connected by a helical backbone. The two domains enclose the metal-binding site, which contains a zinc ion. The interaction of Lbp with laminin was further investigated and shown to be specific in vitro. Localization studies with antibodies specific for Lbp show that the protein is attached to the membrane. The data suggest that Lbp is primarily a zinc-binding protein, and we suggest that its interaction with laminin in vivo may be mediated via zinc bound to laminin.

Identification and characterization of a laminin-binding protein of Aspergillus fumigatus: extracellular thaumatin domain protein (AfCalAp)

Aspergillus fumigatus, an opportunistic fungal pathogen, infects the human host via inhalation of airborne conidia. Adhesion of fungal conidia, to host cells and extracellular matrix (ECM) components associated with host tissue surfaces, is thought to be the primary step in the pathogenesis and dissemination of infection. To identify novel adhesion proteins (adhesins) of A. fumigatus, we screened its proteome in silico using SPAAN (software program for prediction of adhesins and adhesin-like proteins using neural networks). One of the predicted adhesin-encoding genes with a P(ad) (probability of being adhesin) value >0.9, the gene encoding extracellular thaumatin domain protein (AfCalA), was cloned and expressed in Escherichia coli. Recombinant AfCalAp showed significant binding with laminin and murine lung cells. Anti-AfCalAp antibodies inhibited the binding of AfCalAp to laminin in a dose-dependent manner. Significant binding of anti-AfCalAp antibodies to 2 h swollen conidia suggests the presence of AfCalAp on the conidial surface. AfCalA transcript was not detectable in resting conidia but was detected in conidia incubated with RPMI 1640 medium in the presence and absence of lung epithelial cell line (A539)-derived ECM. Elevated levels of IgE antibodies specific to AfCalAp were observed in the sera of two out of seven patients with allergic bronchopulmonary aspergillosis. The study confirms the relevance of the bioinformatic approach for predicting fungal adhesins and establishes AfCalAp as a novel laminin-binding protein of A. fumigatus.

Down-regulation of 67LR reduces the migratory activity of human glioma cells in vitro

Objectives Glioma is the most common brain tumor in central nervous system. Traditional therapies are not effective to cure this disease. Experimental evidence indicates that the 67 kDa elastin-laminin receptor (67LR) subunit is a high-affinity non-integrin laminin-binding protein that is over-expressed on the tumor cell surface in a variety of human carcinomas, and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasise. However, little is known of the expression and function of 67LR in glioma cells. Methods In this study, we estimated whether 67LR was constitutively over-expressed in high-grade astrocytomas by immunohistochemical staining and Western blotting, and investigated the role of a low level of 67LR expression in glioma cell line-U251 by constructing an interfering RNA expression plasmid. Results The results showed that the 67LR had an enhanced over-expression in high-grade astrocytomas against normal brain tissues samples, and that the migratory activity of glioma cells was reduced after the down-regulation of the 67LR gene by RNAi. Discussion It was hypothesized that a low level of 67LR expression could reduce migratory activity of glioma cells, which further proved that 67LR played an important role in glioma invasion by mediating tumor cell functions leading to sarcomata. This study provided a new alternative to gene therapy for glioma treatment.

Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The alpha(7)beta(1)-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the alpha(7)-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate alpha(7)-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for alpha(7)-integrin expression. We show that laminin-111 (alpha(1), beta(1), gamma(1)), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased alpha(7)-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of alpha(7)-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.

Acanthamoeba Interaction with Extracellular Matrix Glycoproteins: Biological and Biochemical Characterization and Role in Cytotoxicity and Invasiveness

Acanthamoeba are free-living amoebae that are dispersed in most environments. Occasionally, Acanthamoeba cause serious human infections, such as keratitis and encephalitis. During the infection process, amoebic adhesion to, and degradation of, host cells and their extracellular matrix (ECM) appear to be important requirements. We examined the interaction of Acanthamoeba with the ECM, and related this event to host cell destruction and tissue invasion. Pathogenic Acanthamoeba culbertsoni differentially attached on the ECM glycoproteins laminin-1, collagen-I, and fibronectin, as compared with non-pathogenic Acanthamoeba astronyxis. Binding to collagen-I and laminin-1 induced A. culbertsoni to become flattened and elongated. Because attachment on laminin-1 was higher in A. culbertsoni, laminin-1 was chosen for further analysis. A 55-kDa laminin-binding protein was identified in pathogenic amoebae, but it was not found in non-pathogenic amoebae. No differential cytotoxicity against distinct cell types was observed between A. culbertsoni incubated with or without ECM. On the other hand, binding on collagen-I or matrigel scaffolds induced a differential effect where A. culbertsoni invaded collagen-I matrices more rapidly. These results indicate that ECM recognition, as an antecedent to tissue invasion, may be a trait characteristic of pathogenic Acanthamoeba.

Expression, purification, crystallization and preliminary crystallographic analysis of laminin-binding protein (Lmb) from Streptococcus agalactiae.

Laminin-binding protein (Lmb), a surface-exposed lipoprotein from Streptococcus agalactiae (group B streptococcus), mediates attachment to human laminin and plays a crucial role in the adhesion/invasion of eukaryotic host cells. However, the structural basis of laminin binding still remains unclear. In the context of detailed structural analysis, the lmb gene has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals diffracted to a resolution of 2.5 A and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.63, b = 70.60, c = 75.37 A, beta = 96.77 degrees .

Regulation of Prostate Cancer Progression by Galectin-3

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.

The Borrelia burgdorferi outer-surface protein ErpX binds mammalian laminin.

The Lyme disease spirochaete, Borrelia burgdorferi, can invade and persistently infect its hosts' connective tissues. We now demonstrate that B. burgdorferi adheres to the extracellular matrix component laminin. The surface-exposed outer-membrane protein ErpX was identified as having affinity for laminin, and is the first laminin-binding protein to be identified in a Lyme disease spirochaete. The adhesive domain of ErpX was shown to be contained within a small, unstructured hydrophilic segment at the protein's centre. The sequence of that domain is distinct from any previously identified bacterial laminin adhesin, suggesting a unique mode of laminin binding.

Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly

Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.

Upregulation in Rat Spinal Cord Microglia of the Nonintegrin Laminin Receptor 37 kDa-LRP following Activation by a Traumatic Lesion or Peripheral Injury

The molecular mechanisms triggering microglial activation after injury to the central nervous system, involving cell-extracellular matrix interactions and cytokine signaling, are not yet fully understood. Here, we report that resident microglia in spinal cord express low levels of the non-integrin laminin receptor precursor (LRP), also found on certain neurons and glial cells in the peripheral nervous system. 37LRP/p40 and its 67-kDa isoform laminin receptor (LR) were the first high-affinity laminin binding proteins identified. While the role of laminin receptor was later attributed to integrins, LRP/LR gained new interest as receptors for prions, and their interaction with laminin seems important for migration of metastatic cancer cells. Using immunohistochemistry and Western blotting, we demonstrate that traumatic spinal cord injury leads to a strong and rapid increase in LRP levels in relation to activated microglia/macrophages. Associated with laminin re-expression in the lesion epicenter, LRP-positive microglia/macrophages exhibit a rounded, ameboid-like shape characteristic of phagocytic cells, whereas in more distant loci they reveal a hypertrophied cell body and short ramifications. The same morphological difference is observed in vitro for purified microglia cultured with or without laminin. Strong, transient upregulation of LRP by activated spinal cord microglia is also induced by transection of the sciatic nerve that affects the spinal cord circuitry without blood-brain barrier dysruption. LRP expression is maximal by 1 week post-lesion, before becoming restricted to dorsal and ventral horns, sites of major structural reorganization. Our findings strongly suggest the involvement of LRP in lesion-induced activation and migration of microglia.

The adaptor protein LAD/TSAd mediates laminin-dependent T cell migration via association with the 67 kDa laminin binding protein

The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin alpha6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration.

Expression of Laminin Isoforms, Receptors, and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin γ1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin α6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors, and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin α5 chain, laminin receptors (integrin α3, α6, β4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors, and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

Mycobacterium smegmatishistone-like protein Hlp is nucleoid associated

Eubacteria encode proteins that are required for nucleoid organization and for regulation of DNA-dependent processes. Of these histone-like proteins (Hlps), Escherichia coli HU has been shown to associate with the nucleoid and to regulate processes such as DNA repair and recombination. In contrast, the divergent HU homologs encoded by mycobacteria have been variously identified as involved in the physiology of dormancy, in the response to cold shock, or as laminin-binding proteins associated with the cell envelope. We show here, contrary to previous reports that the HU-related Hlp from Mycobacterium smegmatis associates with the nucleoid in vivo. Using indirect fluorescent antibody microscopy we show that cold shock causes Hlp to accumulate in the cytoplasm of M. smegmatis. No evidence of surface-associated Hlp was found in M. smegmatis cells treated for cell wall permeabilization. Quantitative Western blots indicate that exponentially growing cells contain c. 120 molecules per cell, with upregulation of Hlp after cold shock estimated to be c. 10-fold. That Hlp associates with the nucleoid in vivo suggests functions in DNA metabolism, perhaps in adaptation to environmental stress.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

Crystal structure of the laminin-binding protein Lpb ofStreptococcus pyogenes

Crystal structure of the laminin-binding protein Lpb of Streptococcus pyogenes Christian Linke, Tom T Caradoc-Davies, Thomas Proft, Edward N Baker University of Auckland, School of Biological Sciences, Thomas Building 110, 3A Symonds Street, Private Bag 92019, Auckland, Auckland, 1142, New Zealand, Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia, University of Auckland, 85 Park Road, Private Bag 92019, Auckland, Auckland, 1142, New Zealand, E-mail:clin180@ ec.auckland.ac.nz