Identification of biologically active sites in laminin an extracellular matrix protein

Laminin-1, a major component of basement membranes, has multiple biological activities including promotion of cell adhesion, spreading, migration, growth, neurite outgrowth and tumor metastasis. Several active sites on laminin-1 have been identified previously. We modified these biologically active peptides to enhance their activities. The multimeric YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides assembled on a branched lysine core were found to strongly enhance the activity of YIGSR in inhibiting tumor growth and metastasis. We also found the all-D-configuration peptide segment containing the IKVAV (Ile-Lys-Val-Ala-Val) sequence had similar biological activities to the native all-L-peptide in vitro and in vivo. These results suggest that these modified compounds are potentially useful for clinical applications. We have identified new active sequences from the laminin alpha 1 chain carboxyl-terminal globular domain (G domain). Using a systematic screening for cell binding sites with 113 overlapping synthetic peptides, we found five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. AG-10 and AG-32 were found to interact with integrins. AG-73 caused metastases of B16-F10 mouse melanoma cells to the liver colonization in mice. Additionally AG-73 was found to promote neurite outgrowth. Moreover, this peptide inhibited laminin mediated acinar-like development of a human submandibular gland cell line. The AG-73 domain on laminin-1 could be one of the most important biologically active sites. These active peptides may useful for study of the molecular mechanism of laminin-receptor interactions and for development of therapeutic reagents for tumor metastasis and angiogenasis.     

Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line

The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin alpha1 and alpha2 chains. The alpha1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous alpha2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-beta1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-beta1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both beta1-integrins and syndecan-1.     

The use of a combinatorial library method to isolate human tumor cell adhesion peptides

Tumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the 'one-bead-one-peptide' concept was incubated with live human prostate cancer cells for 90 min at 37 degrees C. The peptide bead coated with a monolayer of cells was then isolated for microsequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the alpha 6 beta 1 integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are alpha 6-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1,500,000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The alpha 6 integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVS-VNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.     

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy

Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583-20590; Nomizu et al. [1996] FEBS Lett. 396:37-42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730 and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin alpha2 chain-specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide-treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.     

Cell binding sequences in mouse laminin alpha1 chain

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.     

Neural crest cell interactions with laminin: structural requirements and localization of the binding site for alpha 1 beta 1 integrin

We have identified the sites of neural crest cell interaction with laminin in vitro by examining their ability to attach to and migrate on proteolytic fragments of the molecule and the ability of fragment-specific antibodies to inhibit these interactions. The binding site on laminin was localized to the E8 domain on the long arm of laminin, as well as the T8' fragment within this domain, but not the E1', E3, or E4 fragments. Only subfragments containing the carboxy-terminal rod-like portion of the A chain plus the corresponding B1 and B2 chains retained the attachment-promoting activity of the parent E8 fragment. In addition, interactions required maintenance of the triple-stranded and alpha-helical coiled-coil structure of this domain. Reduction and alkylation of laminin and the E8 and T8 fragments significantly reduced neural crest cell attachment and migration. An antiserum against chick alpha 1 integrin reduced migration and adhesion of neural crest cells on an intact laminin-nidogen complex, the E8 fragment, and all its active subfragments. Furthermore, we observed that neural crest cells modified laminin substrata prepared in the absence of divalent cations. Early stable attachment to these substrata was mediated by an integrin other than alpha 1, whereas later attachment and migration were mediated by alpha 1 integrins. Our results suggest that neural crest cells selectively bind to the B1-A-B2 mid-portion (T8') of the E8 domain of laminin, requiring structural integrity of this region and that they modify laminin substrata as a result of prolonged cell-matrix interactions.     

The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.     

Specific attachment and migration of human astrocytoma cells on human but not murine laminin

Attachment sites and biological functions of laminin isolated from murine EHS sarcoma have been well studied. Recently several variants of laminin including human placental laminin have been shown to be distinct from EHS-laminin. This study was undertaken to determine attachment, proliferation, and migration phenomena of human astrocytoma cell lines to human and murine sarcoma EHS-laminin. Using short-term attachment assays human placental laminin was shown to be the better substrate for cell adhesion. EHS-laminin mediated approximately 30-50% of the effect observed on human laminin. The astrocytoma cells expressed beta 1, beta 3, and beta 4 subunit mRNA as determined by RT-PCR. Anti-beta 1 antibodies blocked adhesion to EHS-laminin, but antibodies against beta 1, beta 4, and alpha v subunits were all ineffective in blocking adhesion to human laminin. A migration assay showed that astrocytoma cells on human laminin dispersed from a central seeding area, while cells on EHS-laminin remained where they were seeded. The pattern of dispersion could not be accounted for by changes in growth rates of astrocytoma cells on the different proteins, since both cell lines grew equally well on the two laminins. We conclude that unique epitopes on human laminin are recognized by novel receptors on human astrocytoma cells which confer a migratory phenotype to the cells.     

Identification of integrin-dependent and -independent cell adhesion domains in COOH-terminal globular region of laminin-5 alpha 3 chain

Laminin-5 is an isoform of laminin that consists of alpha 3, beta 3, and gamma 2 chains and has potent cell adhesion- and cell migration-promoting activities. In this study, five subdomains in the COOH-terminal globular (G) domain of human laminin alpha 3 chain were individually expressed in Escherichia coli, and their biological activities were investigated. Recombinant G2, G4, and G5 domains promoted adhesion to plastic plates of HT1080 fibrosarcoma cells, A431 epidermoid carcinoma cells, and ECV304 vascular endothelial cells. For the cell adhesion activity, the G2 domain required a divalent cation and heat-sensitive conformation more strongly than G4 and G5. The cell adhesion to G2 but not G4 and G5 was effectively inhibited by an anti-integrin alpha 3 antibody. A cell adhesion sequence of 22 amino acids, alpha 3G2A, that was homologous to the integrin alpha 3 beta 1-binding sequence GD-6 of laminin alpha 1 chain was identified within the G2 structure. The cell adhesion to alpha 3G2A peptide was also inhibited by the anti-integrin alpha 3 antibody. The cell adhesion to G2, alpha 3G2A, G4, and G5 was strongly inhibited by heparin, but that to native laminin-5 was inhibited less effectively. Moreover, G5 potently stimulated chemotactic migration of rat liver epithelial cells in Boyden chambers, but G2 and G4 did not. These results indicate that the G domain of laminin alpha 3 contains multiple cell binding sites with different mechanisms and different functions. The G2 domain seems to recognize integrin alpha 3 beta 1, whereas G4 and G5 may interact with heparin-like molecules on cell surface.     

Organ-specific adhesion of neuroblastoma cells in vitro: correlation with their hepatic metastasis potential

Neuroblastoma (NB) is the most common solid malignant tumor found in pediatric patients and the liver is one of the major sites of metastasis. To investigate the organ specificity of metastatic distribution, the adherence behavior of tumor cells was studied. The data presented are based on studies using a metastatic murine cell line C1300. In vivo, not only intrasplenic but also intravascular injection of C1300 NB cells consistently results in hepatic metastasis formation in syngeneic A/J mice. An in vitro assay was used in which C1300 NB cell attachment to cryostat sections of liver, spleen, brain, kidney and lung obtained from normal A/J mice was measured to compare organ-specific adhesion. A good correlation was found between their metastatic potential in the liver and the adhesion to the liver sections; C1300 NB cells adhered preferentially to liver cryostat sections. Enzyme assays indicated that cell surface glycoproteins were involved in cell adhesion. An adhesion assay with extracellular matrix proteins demonstrated that C1300 NB cells adhered preferentially to vitronectin and fibronectin, and the adherence was strongly inhibited by Arg-Gly-Asp (RGD)-containing peptides. Furthermore, adhesion of C1300 NB cells to liver cryostat sections could be blocked by the synthetic peptide GRGDS. This indicates that the interaction between RGD-containing matrix adhesion protein and cells has an important role for the specific adhesion of C1300 NB cells. The results suggested that tumor cell adhesion to liver cryostat sections could provide a useful tool in the study of host-tumor interactions in the metastasis of NB.     

Secular trend in body height since the Neolithic period

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.     

Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma.     

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.     

MIA ("melanoma inhibitory activity"). Biological functions and clinical relevance in malignant melanoma]

The protein MIA was identified and isolated from the tissue culture supernatant of melanoma cells in vitro by its ability to inhibit thymidine incorporation by melanoma cell lines. After purification and partial sequencing of the peptide, a fragment of the MIA cDNA was cloned by RT-PCR. This cDNA fragment was used to screen phage libraries and subsequently fully encoding human and murine MIA cDNA and genomic DNA clones were obtained. The MIA gene spans a region of approximately 2 kb and is divided into 4 exons. Mapping the MIA gene revealed that the human gene is located on chromosome 19 and the murine gene on chromosome 7. The MIA open reading frame spans 131 (human) or 130 (murine) amino acids. The first 24 (human) or 23 (murine) amino acids represent a signal sequence directing the secretion of MIA into the extracellular compartment. The mature, secreted MIA consists of 107 amino acids and its MW is approximately 11 kDa. Preliminary structural data suggests that MIA is a small globular protein stabilized by two intramolecular disulfide bonds. Expression studies of protein und mRNA levels indicate that MIA is expressed specifically by malignant melanoma cells and chondrocytes. This points to a highly restricted expression pattern which is controlled by the MIA promoter. In addition, MIA provides a clinically useful parameter in patients with malignant melanoma. Enhanced values were measured in the serum of all patients with metastatic melanoma (stage III and IV). In vitro and in vivo experiments using recombinant MIA protein revealed that MIA specifically inhibits attachment of melanoma cells to fibronectin and laminin. Further analysis indicated a direct binding between MIA and the matrix proteins. This finding provides an explanation for the capability of MIA to inhibit proliferation of melanoma cells in vitro. Our studies suggest a putative function of MIA in regulated detachment of melanoma cells from the extracellular matrix which is an important step in metastasis.     

Effect of olive oil on immune function in middle-aged men

Autocrine motility factor is a tumor-secreted cytokine which regulates cellular growth and motility by a receptor-mediated pathway. In the accompanying report (Part I of II), it was demonstrated that high (K1735-M1) and low (K1735-C1.11) metastatic murine melanoma cells display distinct adhesion and spreading characteristics which correlate with their differential spontaneous and stimulated migrations on the extracellular matrix components fibronectin, laminin and collagen IV. These parameters were further related to discrete profiles of focal adhesion plaque integrity and reorganization. Here we describe unique migration patterns observed in these murine melanoma cells which reflect differences in degradation and/or remodeling of the cellular substratum. These profiles of matrix interaction were influenced distinctly by autocrine motility factor and dictated by both substrate composition and cellular phenotype. Since activation of the autocrine motility factor receptor stimulates invasion of a reconstituted basement membrane and enhances experimental metastasis by high- but not low-metastatic K1735 cells, differences in the invasive phenotypes of these cells may be due in part to their differential responses to external stimuli coupled with internal propensities toward either matrix degradation and migration (high-metastatic cells) or matrix remodeling and stasis (low-metastatic cells).     

Inhibition of tumor cell adhesion to lymph nodes by laminin-related peptide and neuraminidase

BACKGROUND: Adhesion to lymph nodes, rather than growth stimulation, accounted for preferential colonization of lymph nodes by a metastatic B16 melanoma. We investigated these adhesive interactions. METHODS: Four classes of molecules were tested for inhibition of melanoma adhesion to cryostat sections of lymph node. RESULTS: Calcium chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetra ace tic acid completely inhibited adhesion (50% adhesion, half-maximal inhibition, at 1 to 3 mmol/L). Cytochalasin B, which impairs contractile microfilaments, inhibited adhesion (60% adhesion at .001 mmol/L, 28% at .01 mmol/L). Colchicine, which disaggregates microtubules, had a similar effect (20% at .01 mmol/L, lowest dose tested). Trypsin slightly increased adhesion (125% adhesion at 10 micrograms/ml). Neuraminidase, which removed sialic acid residues, inhibited it (50% adhesion at 5 micrograms/ml). Gly-arg-gly-asp-ser, a peptide with a cell binding sequence of fibronectin, did not consistently inhibit adhesion (69% adhesion at 0.1 mg/ml, 83% adhesion at 1 mg/ml) or substantially differ from gly-arg-gly-glu-ser-pro (59% adhesion at 0.1 mg/ml, 90% adhesion at 1 mg/ml). In contrast, a peptide with a cell binding region of laminin (tyr-ile-gly-ser-arg) inhibited adhesion (50% adhesion at .05 mg/ml). CONCLUSIONS: Tumor cell-lymph node adhesion is a calcium-dependent process, requiring a functional cytoskeleton, that is mediated by both sialic acid moieties and trypsin-resistant, laminin-related, adhesion molecules.     

Sickle cell adhesion to laminin: potential role for the alpha5 chain

Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the alpha1, alpha2, beta1, and gamma1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin alpha5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin alpha5 chain.     

Laminin localization in enterocytic basement membrane of rat small bowel grafts. A light and electron microscopic study

In vitro laminins stimulate numerous biological effects, such as cell migration, proliferation, attachment and differentiation. In vitro laminins influence immunocompetent cells and in vivo possibly play an important role in graft rejection. To establish how laminins could be involved in the regulation of acute rejection of small bowel allografts (with and without immunosuppression), we investigated laminin distribution in rat small bowel allografts four days after transplantation, i.e., before the onset of histological signs of rejection, using antibodies against alpha1, beta1, gamma1 chain of laminin-1. In immunosuppressed allografts, the ultrastructure of the enterocytic basement membrane appeared normal, but no laminin staining was seen in this membrane, although basement membranes of intramural blood vessels and muscle cells were normally stained. In non-operated immunosuppressed rats, laminin staining was clearly reduced in the enterocytic basement membrane, demonstrating that cyclosporin A is able to affect this membrane. Since only rats in which laminin is altered survive, this laminin alteration in the enterocytic basement membrane presumably plays an important role in overcoming the acute rejection.     

A peptide derived from a neurite outgrowth-promoting domain on the gamma 1 chain of laminin modulates the electrical properties of neocortical neurons

Laminins form a family of large multidomain glycoproteins of the extracellular matrix. The cellular distribution of laminin immunoreactivity in the adult mammalian central nervous system suggests an important role for laminins in mature brain function in addition to their role during brain development. To characterize the effects of this group of extracellular matrix molecules on mature brain function, intracellular recording techniques were applied to in vitro slice preparations of the rat neocortex. The experiments show that a peptide homologous to the C-terminal part of the gamma 1 chain of laminin modulates the electrical activity of pyramidal neurons in the adult neocortex of the rat. The peptide is part of the neurite outgrowth-promoting domain of the gamma 1 chain on the E8 fragment of laminin and it displays the neurite outgrowth-promoting activity of the native laminin molecule. Perfusion of in vitro brain slices with the peptide increased the input resistance of the neuronal membrane. In addition, a rise in inward rectification could be observed. These events were accompanied by a strong increase in direct excitability of the treated neurons. Immunohistochemistry techniques were applied to sections of the adult rat neocortex and hippocampus to demonstrate the presence of both the neurite outgrowth-promoting domain and the native laminin in the adult brain. An antiserum raised against the neurite outgrowth-promoting domain on the gamma 1 chain of laminin, which also recognized the free synthetic peptide, showed immunoreactivity on neurons. In addition, a population of glial fibrillary acidic protein-positive astrocytes in the hippocampus displayed immunoreactivity for this antibody. These results were confirmed by using several antibodies directed against the whole laminin-1 molecule. Neurons in the neocortex and hippocampus, as well as astrocytes in the hippocampus, demonstrated immunoreactivity for antibodies directed against the whole laminin-1 molecule. The results suggest that laminins containing the gamma 1 chain have the potential to modulate neuronal activity. This effect may be mediated either by direct cell-cell contact from surrounding cells, or through the neuronal expression of laminin or laminin-like molecules which are inserted into the neuronal cell membrane.