Laminin Binding Research Articles

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans.

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.

Binding of extracellular matrix laminin to Escherichia coli expressing the Salmonella outer membrane proteins Rck and PagC

Salmonella Rck and PagC are closely related virulence-associated proteins. When expressed in non-adherent, non-invasive laboratory Escherichia coli, both proteins localised to the outer membrane. Only Rck conferred adhesion to culture cells, but both proteins induced bacterial binding to the cell monolayer background, to extracellular matrix (ECM) preparations, and to the ECM component laminin. Laminin binding was saturable and competitive, and was reduced by removal of carbohydrate side chains. Pre-incubation with laminin targeted recombinant Rck+ and PagC+ bacteria directly to the eukaryotic cell surface, and eliminated background binding.

Binding of extracellular matrix laminin to Escherichia coli expressing the Salmonella outer membrane proteins Rck and PagC.

Salmonella Rck and PagC are closely related virulence-associated proteins. When expressed in non-adherent, non-invasive laboratory Escherichia coli, both proteins localised to the outer membrane. Only Rck conferred adhesion to culture cells, but both proteins induced bacterial binding to the cell monolayer background, to extracellular matrix (ECM) preparations, and to the ECM component laminin. Laminin binding was saturable and competitive, and was reduced by removal of carbohydrate side chains. Pre-incubation with laminin targeted recombinant Rck and PagC bacteria directly to the eukaryotic cell surface, and eliminated background binding.

Inhibition of experimental metastasis of human fibrosarcoma cells by anti-recombinant 37-kDa laminin binding protein antibody.

The laminin binding protein of 37 kDa (37LBP) is regarded as a precursor protein of the high‐affinity 67‐kDa laminin receptor (67LR). Expression of 67LR/37LBP is well correlated with biological aggressiveness of cancer, particularly with invasive and metastatic potential. To investigate in detail the role of 37LBP in cancer cells, we synthesized recombinant 37LBP (r37LBP) as a fusion protein and generated an IgG‐type polyclonal antibody P4G against r37LBP. Western blot analysis with P4G showed a single band of 67LR under both nonreducing and reducing conditions using cell extract of human fibrosarcoma cells HT1080. It was shown that P4G inhibited cell attachment to immobilized laminin in a dose‐dependent manner. Further, the intravenous injection of HT1080 cells pretreated with P4G, compared with that of cells pretreated with normal rabbit serum, resulted in a reduced number of experimental metastases (3.3 ± 5.1 vs. 58.0 ± 38.0 nodules per mouse, respectively) (P < 0.005). These results suggest that P4G inhibits the colonization and growth of HT1080 cells in the lungs of mice, and that the blocking of r37LBP with the specific antibody P4G may offer a potential strategy for preventing cancer metastasis.

Hepatocyte-matrix interaction

Various components of extracellular matrices have unique patterns of distribution in the liver, which change under hepatic regeneration and in pathological conditions.In vitro studies using isolated hepatocytes maintained in culture on different matrix proteins or tissue biomatrices revealed that the matrix influences the biochemical activity of hepatocytes in culture on a selective basis. Hepatocyte-matrix interactions are mediated by specific receptors for matrix proteins belonging to both integrin and non-integrin group of cell surface molecules. The level ofα 1 β 1 integrin, which is a common receptor for Col IV and Ln in liver appears to change under hepatic regeneration; cat ions appear to modulate its interaction with Col IV and Ln in a differential manner. A number of other integrin receptors including that for fibronectin (α 5 β 1) and vitronectin (α v β 1) are also present in liver. Apart from integrin receptors, non-integrin receptors such as 67 kDa laminin binding protein, 68 kDa collagen IV binding protein, 110 kDa Fn receptor are also present in liver cells. HSPG present on the hepatocyte surface also appears to have an augmenting effect in mediating cell adhesion. Although matrix components appear to exert their effect through interaction with matrix receptors on cell surface, details about the intracellular signalling process in liver cells are not clear.

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection, probably contributing to the attachment of fungal cells to host tissues.

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Ribosome-Associated Protein LBP/p40 Binds to S21 Protein of 40S Ribosome: Analysis Using a Yeast Two-Hybrid System

The ribosome-associated protein LBP/p40, which was originally named after “laminin binding protein precursor p40,” is distributed on the cell surface as laminin binding protein p67 (LBP/p67), in the nucleus, and on 40S ribosomes. In a broad range of eukaryotes, the localization of LBP/p40 on the 40S ribosome is well conserved. Two yeast homologs of LBP/p40 are believed to be essential for cell viability and each gene product probably corresponds to the assembly and/or stability of the 40S ribosomal subunit. The precise role of LBP/p40 in translation, however, remains to be elucidated, especially in higher eukaryotes. In this report, we used a yeast two-hybrid screening method to isolate molecules associated with human LBP/p40 protein on ribosomes. We found that the 40S ribosomal protein S21 was tightly bound with LBP/p40 in this yeast two-hybrid system and inin vitroanalysis. Further, we discovered that the association required a broad region of the LBP/p40 amino acid sequence, which corresponds to the highly conserved region of LBP/p40 homologs among eukaryotes.

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues.

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.

Role of the 37 kDa laminin receptor precursor in the life cycle of prions

Prions are thought to consist of infectious proteins that cause, in the absence of detectable nucleic acid, a group of fatal neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Among these diseases are bovine spongiform encephalopathy (BSE), scrapie of sheep and Creutzfeldt-Jakob disease (CJD) in humans. They occur as sporadic, infectious or genetic disorders and have in common the accumulation of an abnormal, pathogenic isoform of the cellular prion protein PrPc which is converted in a post-translational process into PrPSc concomitant with conformational changes of the protein. During this process PrPc acquires a high beta-sheet content and becomes partially resistant to proteases. The mechanism of this conversion as well as the physiological function of the cellular prion protein PrPc are poorly understood, but studies employing PrP knock-out mice demonstrated that PrPc is required for the development of prion diseases. The involvement of co-factors such as chaperones, receptors or an unknown protein, designated "protein X" in the conversion process are discussed. In a yeast two-hybrid screen we have identified the 37 kDa laminin receptor precursor (LRP) as an interactor of the cellular prion protein and this interaction could be confirmed by co-infection and co-transfection studies in mammalian and insect cells. LRP evolved from the ribosomal protein p40 essential for protein synthesis lacking any laminin binding activity to a cell surface receptor binding laminin, elastin and carbohydrates. The gene encoding 37 kDa LRP/p40 has been identified in a variety of species including the sea urchin Urechis caupo, Chlorohydra viridissima, the archaebacterium Haloarcula marismortui, the yeast Saccharomyces cerevisiae as well as in mammals where it is highly conserved. LRP works as a receptor for alphaviruses and is associated with the metastatic potential of solid tumors where it was first identified. The 37 kDa LRP forms its mature 67 kDa isoform with high laminin binding capacity by an unknown mechanism involving acylation. The multifunctionality of LRP as a ribosomal protein and a cell surface receptor for infectious agents such as viruses and prions might be extended by additional properties.

Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin.

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.

Laminin receptor in lymph node negative breast carcinoma.

Expression of 67 kD laminin binding protein, 67LR, is reported to be associated with invasive and metastatic phenotypes in several types of human malignancies. In mammary carcinomas, however, the biologic role of 67LR has been less clear. The authors explored the potential biologic significance of expression of 67LR in 148 patients with axillary lymph node negative breast carcinoma. Formalin fixed, paraffin embedded histologic sections were immunohistochemically evaluated for 67LR using monoclonal antibody MLuC5. The staining results were correlated with morphologic data as well as with estrogen receptor content and p53 product accumulation. There were statistically significant correlations between positivity for 67LR and lower histologic grade (P = 0.003), lower nuclear grade (P = 0.002), positivity for estrogen receptor (P = 0.003), and lack of p53 abnormality (P < 0.001). Expression of 67LR had no independent effect on the disease free or overall survival of lymph node negative patients with breast carcinoma. Nevertheless, in the subgroup of 67LR positive patients, positivity for estrogen receptor was associated with significantly longer overall survival (P = 0.008). The data from this study suggest that tissue expression of 67LR, as detected by antibody MLuC5, is associated with better differentiated, less aggressive forms of axillary lymph node negative breast carcinoma.

Laminin interactions with the apoproteins of acute-phase HDL: preliminary mapping of the laminin binding site on serum amyloid A

During AA amyloidosis, the major basement membrane components, collagen type-IV (C-IV), entactin, laminin and perlecan codeposit both spatially and temporally with AA fibrils. Our previous work demonstrated that laminin, and collagen type-IV, can associate with high affinity to a mixture of mouse acute-phase serum amyloid A isoforms (apoSAA1, apoSAA2 andapoSAA3). However, laminin also bound to residual HDL from which apoSAAs were extracted. To characterize further laminin binding specificity for acute-phase HDL apolipoproteins, we have systematically isolated the acute-phase apoSAAs, apoA-I, apoA-II and the apoCs (I, II and III) by reverse phase high pressure liquid chromatography (RP-HPLC), and tested their laminin binding activities individually by ELISA. All the apoSAAs tested bound laminin saturably and with high affinity (Kd 2 nM). In addition, apoA-I also showed laminin binding activity (Kd a 4.6 nM). Specific binding for apoA-II and the apo-Cs was not detected. To localize the laminin binding site on apoSAA, we generated defined CNBr fragments of apoSAA I and apoSAA2, purified them by RP-HPLC, and tested their laminin binding activity by ELISA. A 53 residue peptide corresponding to residues 24-76 ofapoSAA2 had the highest laminin binding activity, followed by an 80 residue peptide corresponding to residues 24-103 of apoSAA1, both of which contain a 30 residue sequence that has changed little during evolution. In addition, a 7 residue peptide (residues 17-23), which is common to both apoSAA1 and apoSAA2, also had laminin binding activity. We postulate that laminin facilitates AA amyloidogenesis by sequestering apoSAA and providing a “surface” on which heparan sulfate-dependent fibrillogenic nucleation events can take place.

Critical Factors in Basal Cell Adhesion Molecule/Lutheran-mediated Adhesion to Laminin

Basal cell adhesion molecule (B-CAM) and Lutheran (LU) are two spliceoforms of a single immunoglobulin superfamily protein containing five Ig domains and comprise the sickle (SS) red cell receptor for laminin. We have now analyzed laminin binding to murine erythroleukemia cells transfected with various human B-CAM/LU constructs. B-CAM and LU bound equally well to laminin, indicating that the longer cytoplasmic tail of LU is not required for binding. However, binding of soluble laminin did require the presence of the membrane-proximal fifth immunoglobulin superfamily (IgSF) domain of LU, while deletion of IgSF domains 1, 2, 3, or 4 individually or together did not abrogate laminin binding. Under flow conditions, MEL cells expressing B-CAM, LU, and LU lacking domains 1, 2, 3, or 4 adhered to immobilized laminin with critical shear stresses over 10 dynes/cm2. However, MEL cells expressing LU lacking domain 5 bound to laminin poorly (critical shear stress = 2.3 dynes/cm2). Moreover, expression of only IgSF domain 5 of LU was sufficient to mediate MEL cell adhesion to immobilized laminin (critical shear stress >10 dynes/cm2). Finally, Scatchard analysis showed that SS red cells had an average of 67% more B-CAM/LU than normal red cells, and low density red cells from sickle cell disease patients expressed 40-55% more B-CAM/LU than high density SS red cells. B-CAM/LU copy number thus may also play a role in the abnormal adhesion of SS red cells to laminin.

Shedding of the 67-kD laminin receptor by human cancer cells.

The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis.

De novo design of heterotrimeric coiled coils.

The three-helix bundle is a common structural motif among natural proteins. It has been observed in numerous important proteins, such as fibrinogen, laminin, spectrin, dystrofin, hemagglutinin, and mannose binding proteins. The three-helix bundle is a simple structure in which three alpha-helices pack against each other, with a slight left-handed twist. Because of its simplicity relative to other structural motifs, the three-helix bundle can be conveniently used both to clarify the forces responsible for the protein folding and stability, and for the design of novel proteins. In this paper we describe the design, synthesis, and characterization of three peptides that self-assemble into antiparallel, heterotrimeric coiled coils. The experimental results, obtained from CD spectroscopy and ultracentrifugation equilibrium sedimentation, indicate that the mixture of the three peptides preferentially forms heterotrimers; moreover, these aggregates represent attractive systems for combinatorial design of libraries of pseudo C3 symmetric ligands or binding sites.

Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish.

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity.

Differential expression of laminin receptors in human hepatocellular carcinoma

Background—Laminin receptors are involved in cell-extracellular matrix interactions in malignant cells that show invasion and metastasis. Hepatocellular carcinoma frequently shows early invasion into blood vessels, and intrahepatic and extrahepatic metastases....

LBP-p40 Binds DNA Tightly through Associations with Histones H2A, H2B, and H4

Laminin binding protein precursor p40 (LBP-p40) was long believed to be located exclusively in the cytoplasm. We recently reported localization of epitope-tagged LBP-p40 to the nucleus tightly associated with nuclear structure as well as on ribosomes. In this paper, we analyze the interaction of LBP-p40 with DNA and nuclear proteinsin vitro.LBP-p40 was found to bind to a double-stranded DNA cellulose column at moderate salt. However, when mixed with a high salt nuclear extract, LBP-p40 was eluted from the DNA cellulose column only at higher salt. An LBP-p40 affinity column indicated that both histone H1 and in particular the core histones associate with LBP-p40. Using recombinant core histone molecules fused with glutathioneS-transferase (GST), we demonstrate that histones H2A, H2B, and H4 are capable of interacting with LBP-p40, whereas H3 is not. These results suggest that association of LBP-p40 with histones H2A, H2B, and H4 confers tight binding of LBP-p40 to chromatin DNA in the nucleus.

Fibronectin and laminin binding of urogenital and oral Prevotella species

88 strains of five Prevotella species--P. bivia, P. buccae, P. disiens, P. oralis, and P. oris--were examined for their fibronectin and laminin binding properties with the aid of latex particle agglutination assays. Beside single protein binding activities, all species showed strains that adhered to both fibronectin and laminin. The oral species, P. buccae, P. oralis, and P. oris were found to interact with laminin to a pronouncedly higher extent than with fibronectin. The urogenital species, P. bivia and P. disiens showed comparable activities of binding to fibronectin and laminin, with P. bivia exhibiting higher matrix protein binding rates than P. disiens. Within the oral species group, P. oralis showed a higher percentage of fibronectin and laminin reactive strains than did P. buccae and P. oris. The finding of species-related different binding properties may throw some light on the known differences in clinical relevance and pathogenicity of the urogenital species, P. bivia and P. disiens, but does so only in part concerning the oral species, P. buccae, P. oralis, and P. oris. Moreover, the observed differences in matrix protein binding of Prevotella species may have implications in chemotaxis and opsonization on the one hand and maintenance of colonization activities under antibiotic therapy on the other.