Syndecan binding sites in the laminin alpha1 chain G domain

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.     

Significant role of laminin-1 in branching morphogenesis of mouse salivary epithelium cultured in basement membrane matrix

Mouse submandibular epithelium shows branching morphogenesis in mesenchyme-free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin-1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1-nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt-like matrix was formed on the epithelial surface in the LN1-nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV-including peptide (2097-2108), AG10 (2183-2194), AG32 (2370-2381) or AG73 (2719-2730) from the alpha1 chain, or YIGSR-including peptide (926-933) from the beta1 chain. Only AG73 (RKRLQVQLSIRT from the alpha1 chain carboxyl-terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1-nidogen can support the branching morphogenesis of submandibular epithelium even if LN1-nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.     

Identification of biologically active sequences in the laminin alpha 4 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.     

Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain

The G domain of the laminin alpha chains consists of five homologous G modules (LG1-5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin alpha5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalian expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These results suggest that the LG4-5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4-5 modules to heparin with K(D) values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated that N-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin alpha5 chain.     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

Identification of a heparin binding site and the biological activities of the laminin alpha1 chain carboxy-terminal globular domain

The carboxy-terminal globular domain (G-domain) of the laminin alpha1 chain has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. In this study, we defined the potential sequences originating from the G-domain of laminin alpha1 chain which possess these functional activities. A series of peptides were synthesized from the G-domain, termed LG peptides (LG-1 to LG-6) and were tested for their various biological activities. In the direct [3H] heparin binding assays, LG-6 (residues 2,335-2,348: KDFLSIELVRGRVK) mediated high levels of [3H]heparin binding, and this peptide also directly promoted cell adhesion and spreading, including B16F10, M2, HT1080, and PC12 cells. The peptide LG-6 also promoted the neurite outgrowth of PC12 cells, mouse granule cells, and chick telencephalic cells. An anti-peptide LG-6 antibody inhibited laminin-1 and peptide LG-6-mediated cell adhesion and neurite outgrowth. Furthermore, an anti-integrin alpha2 antibody also inhibited the cell adhesion activity. These results suggest that peptide LG-6 plays a functional role as a heparin binding site in the G-domain of the laminin alpha1 chain, and this sequence was thus concluded to play a crucial role in regulating cell adhesion and spreading and neurite out-growth which is related to integrin alpha2.     

Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity

It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F1 and F2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.     

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.     

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides

Laminins, heterotrimeric glycoproteins in the basement membrane, are involved in diverse biological activities. So far, five alpha, three beta, and three gamma chains have been identified, and at least 15 laminin isoforms exist composed of various combinations of the different three chains. The major cell-surface receptors for laminins are integrins and proteoglycans, such as dystroglycans and syndecans. Previously, we reported that synthetic peptide A4G82 (TLFLAHGRLVFM, mouse laminin alpha4 chain residues 1514-1525) showed strong cell attachment and syndecan binding activities. On the basis of the crystal structure of the LG module and sequence alignment, A4G82 is located in the connecting loop region between beta-strands E and F in the laminin alpha4 chain LG4 module. Here, we have focused on the structural importance of this E-F loop region for the biological activity of the alpha4 chain LG4 module. To determine the importance of the loop structure, we synthesized peptide A4G82X (cyclo-A4G82X, Cys-TLFLAHGRLVFX-Cys, X= norleucine), which was cyclized via disulfide bridges at both the N- and C-termini. The cyclic peptides derived from A4G82X inhibited the heparin binding activity of the alpha4 chain G domain and promoted HT-1080 cell attachment better than the corresponding linear peptides. We determined FLAHGRLVFX as a minimal sequence of cyclo-A4G82X important for cell adhesion and heparin binding using a series of truncated peptides. Moreover, HT-1080 cell attachment to the cyclic peptides was more efficiently blocked by heparin than cell attachment to the linear peptides. Furthermore, the cyclic peptides showed significantly enhanced syndecan-2-mediated cell attachment activity. These results indicate that the activity of A4G82 is highly conformation-dependent, suggesting that the E-F loop structure is crucial for its biological activity.     

Dystroglycan binding to laminin α1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.     

A unique sequence of the laminin alpha 3 G domain binds to heparin and promotes cell adhesion through syndecan-2 and -4

Laminin-5, consisting of the alpha 3, beta 3, and gamma 2 chains, is localized in the skin basement membrane and supports the structural stability of the epidermo-dermal linkage and regulates various cellular functions. The alpha chains of laminins have been shown to have various biological activities. In this study, we identified a sequence of the alpha 3 chain C-terminal globular domain (LG1-LG5 modules) required for both heparin binding and cell adhesion using recombinant proteins and synthetic peptides. We found that the LG3 and LG4 modules have activity for heparin binding and that LG4 has activity for cell adhesion. Studies with synthetic peptides delineated the A3G75aR sequence (NSFMALYLSKGR, residues 1412--1423) within LG4 as a major site for both heparin and cell binding. Substitution mutations in LG4 and A3G75aR identified the Lys and Arg of the A3G75aR sequence as critical for these activities. Cell adhesion to LG4 and A3G75aR was inhibited by heparitinase I treatment of cells, suggesting that cell binding to the A3G75aR site was mediated by cell surface heparan sulfate proteoglycans. We showed by affinity chromatography that syndecan-2 from fibroblasts bound to LG4. Solid-phase assays confirmed that syndecan-2 interacted with the A3G75aR peptide sequence. Stably transfected 293T cells with expression vectors for syndecan-2 and -4, but not glypican-1, specifically adhered to LG4 and A3G75aR. These results indicate that the A3G75aR sequence within the laminin alpha 3 LG4 module is responsible for cell adhesion and suggest that syndecan-2 and -4 mediate this activity.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Glandular-like morphogenesis of the human submandibular tumor cell line A253 on basement membrane components

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor.     

Cell adhesive sequences in mouse laminin beta1 chain

Laminin-1, a major component of the basement membrane, consists of three different chains, alpha1, beta1, and gamma1. We sought to identify cell adhesive sequences from the mouse laminin beta1 chain by testing HT-1080 fibrosarcoma and B16-F10 melanoma cells for binding to 187 overlapping synthetic peptides which covered the entire chain. Fourteen peptides showed cell adhesive activities with either peptide-conjugated Sepharose beads or peptide-coated plates or both. Additional cells, including neuronal, endothelial, and salivary gland cells, showed biological responses in a cell type-specific manner. B-7, B-133, and B-160 showed the most potent cell attachment. Cell binding on three peptides (B-34, B-133, and B-160) was inhibited by EDTA. Cell adhesion to 11 of the 12 active peptides was inhibited to varying degrees by heparin. Of the 17 active peptides identified in the laminin beta1 chain in this and other studies, 8 are clustered on the amino terminal globular domain, suggesting a possible important role in cell binding for this domain that may be multifunctional. These data demonstrate that the laminin beta1 chain has multiple active sites for cell adhesion, some of which are cell-type specific.     

Cell surface glypicans are low-affinity endostatin receptors

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.     

A novel synthetic peptide from the B1 chain of laminin with heparin- binding and cell adhesion-promoting activities

Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]heparin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.     

Identification of neurite outgrowth active sites on the laminin alpha4 chain G domain

The laminin alpha4 chain is widely distributed in various mesodermal tissues, including the perineurium of peripheral nerves, dorsal root ganglion (DRG), skeletal muscle, and capillaries, and plays important roles in synaptic specialization at the neuromuscular junction and in microvascular formation. The C-terminal globular domain (G domain) of the laminin alpha4 chain was previously found to be critical for heparin binding and cell attachment activity. Here, we focused on neurite outgrowth activity of the laminin alpha4 chain G domain. We found that the recombinant alpha4 chain G domain protein (rec-alpha4G) promoted neurite outgrowth of rat pheochromocytoma PC12 cells. When 114 overlapping synthetic peptides that covered the entire G domain were tested for neurite outgrowth activity, nine peptides were active, but the 105 remaining peptides did not exhibit activity. Three of the nine active peptides, A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), and A4G107 (VIRDSNVVQLDV), strongly promoted neurite outgrowth of PC12 cells. A4G107 was found to form amyloid-like fibrils in Congo red, X-ray, and electron microscopy analyses. We also synthesized cyclic peptides to evaluate their conformational requirements. Cyclic peptide A4G82X (cyc-A4G82X;TLFLAHGRLVFX, where X is norleucine) significantly enhanced neurite outgrowth activity, but the rest of the cyclic peptides eliminated the activity. The A4G82 sequence is located on the loop region, suggesting that the activity of A4G82 is required for a loop conformation. These peptides also exhibited neurite outgrowth activity with dorsal root ganglion (DRG) explants and with DRG cells from E14.5 mouse embryos, indicating that they are active in both neuronal cell lines and native neuronal cells. Taken together, the data suggest that the peptides from the laminin alpha4 chain G domain promote neurite outgrowth activity via a specific conformation.     

Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

Laminin alpha1 chain LG4 module promotes cell attachment through syndecans and cell spreading through integrin alpha2beta1

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.