Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain

Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering.     

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.     

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.     

High and low affinity heparin-binding sites in the G domain of the mouse laminin alpha 4 chain

G domains of the mouse laminin alpha 1 and alpha 4 chains consisting of its five subdomains LG1-LG5 were overexpressed in Chinese hamster ovary cells and purified by heparin chromatography. alpha 1LG1-LG5 and alpha 4LG1-LG5 eluted at NaCl concentrations of 0.30 and 0.47 m, respectively. In solid phase binding assays with immobilized heparin, half-maximal concentrations of 14 (alpha 1LG1-LG5) and 1.4 nm (alpha 4LG1-LG5) were observed. N-Glycan cleavage of alpha 4LG1-LG5 did not affect affinity to heparin. The affinity of alpha 4LG1-LG5 was significantly reduced upon denaturation with 8 m urea but could be recovered by removing urea. Chymotrypsin digestion of alpha 4LG1-LG5 yielded high and low heparin affinity fragments containing either the alpha 4LG4-LG5 or alpha 4LG2-LG3 modules, respectively. Trypsin digestion of heparin-bound alpha 4LG1-LG5 yielded a high affinity fragment of about 190 residues corresponding to the alpha 4LG4 module indicating that the high affinity binding site is contained within alpha 4LG4. Competition for heparin binding of synthetic peptides covering the alpha 4LG4 region with complete alpha 4LG1-LG5 suggests that the sequence AHGRL1521 is crucial for high affinity binding. Introduction of mutation of H1518A or R1520A in glutathione S-transferase fusion protein of the alpha 4LG4 module produced in Escherichia coli markedly reduced heparin binding activity of the wild type. When compared with the known structure of alpha 2LG5, this sequence corresponds to the turn connecting strands E and F of the 14-stranded beta-sheet sandwich, which is opposite to the proposed binding sites for calcium ion, alpha-dystroglycan, and heparan sulfate.     

Identification of homologous biologically active sites on the N-terminal domain of laminin alpha chains.

Laminin, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha, beta, and gamma chains. To date, five different alpha chains have been identified. N-terminal domain VI in the alpha1 chain has various biological activities. Here we screened biologically active sequences on domain VI of the laminin alpha2, alpha3, and alpha5 chains using a large number of overlapping peptides. HT-1080 human fibrosarcoma cell attachment to the peptides was evaluated using peptide-coated plastic plates and peptide-conjugated Sepharose beads. We identified four cell adhesive sequences from laminin alpha2 chain domain VI, two sequences from the alpha3 chain, and two sequences from the laminin alpha5 chain. Sequences homologous to A13 (RQVFQVAYIIIKA, alpha1 chain 121-133) on all the alpha chains (FQIAYVIVKA, alpha2 chain 130-139; GQLFHVAYILIKF, alpha3 chain 96-108; FHVAYVLIKA, alpha5 chain 74-83) showed strong cell attachment activity. A5-16 (LENGEIVVSLVNGR, alpha5 chain 147-160) showed the strongest cell attachment activity in the plate assay, and the homologous peptide in the alpha3 chain promoted similar strong cell attachment activity. A5-16 and its homologous peptide in the alpha2 chain promoted moderate cell attachment, while the homologous peptide to A5-16 in the alpha1 chain did not show activity. A2-7 (SPSIKNGVEYHYV, alpha2 chain 108-120) showed cell attachment activity only in the plate assay, but homologous sequences in the alpha1, alpha3, and alpha5 chains did not promote activity. A2-7 promoted endothelial cell sprouting from aortic rings in vitro and melanoma colonization to murine lungs in vivo. However, none of the homologous peptides of A2-7 promoted experimental pulmonary metastasis by B16-BL6 melanoma cells. These results indicate that there are chain-specific active sites in domain VI of the laminin alpha chains, some of which contain conserved activities.     

Identification of an active site on the laminin alpha4 chain globular domain that binds to alphavbeta3 integrin and promotes angiogenesis

Angiogenesis is important for wound healing, tumor growth, and metastasis. The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in endothelial cell basement membranes. It mediates endothelial cell adhesion by binding with its receptors such as alphavbeta3 integrin and participates in new blood vessel formation. In this study, we found the recombinant laminin alpha4LG modules (rLG1-3, rLG1, and rLG2) mediate HUVECs adhesion. The attachment of HUVECs to the rLG2 was specifically inhibited by a function-blocking monoclonal antibody LM609 specific for alphavbeta3 integrin. Using deletion mutants of the alpha4LG2 revealed the HUVECs-adhesion site is located in amino acids 1121-1139. A synthetic G(1121-1139) peptide could be attached by HUVECs at same efficiency with the rLG2 and promoted angiogenesis in CAM. In conclusion, we have identified a new alphavbeta3 integrin-interacting peptide within laminin alpha4 G domain. This suggests that G(1121-1139) peptide-containing proteins may perform their biological functions by interacting with alphavbeta3 integrin.     

Galactosyl transferase-dependence of neurite outgrowth on substratum-bound laminin

The cell surface enzyme beta 1-4 galactosyl transferase (galtase) has been implicated in a number of cellular events involving adhesion and recognition, among them migration of neural crest and mesenchymal cells as well as initiation and elongation of neurites from PC12 cells. Results presented here demonstrate that reagents that specifically alter galtase activity modulate the rate of neurite outgrowth from chick dorsal root ganglia on substrata coated with the large extracellular matrix glycoprotein, laminin (LN), a known substrate for galtase activity. Not all neurites responded equally to reagent addition, and in every experiment a subset of neurites was ostensibly unaffected by reagent, even at the highest concentration tested. Those neurites that were affected demonstrated an ability to adapt to the continued presence of reagent and resume normal elongation. These results support the hypothesis that cell surface galtase activity plays an important role in mediating neurite elongation and suggest further that differential expression of galtase at the nerve growth cone might contribute to axonal guidance through glycoconjugate-rich environments in vivo.     

Identification of active regions for neurite outgrowth activity of neurocrescin

We previously identified and cloned a neurite outgrowth promoting protein, Neurocrescin (NC), from the extract of the chick denervated leg muscles. In this study, we explored the active region of NC for neurite outgrowth. Using the deletion mutants of NC, we tested their neurite outgrowth activity in the cultured telencephalic neurons of E5 chick embryos. We found three regions which independently had significant neurite outgrowth activity comparable with that of the extract of the chick denervated leg muscles. These regions were not homologous to any well-known active sites such as the laminin active region, IKVAV. In parallel, searching the endogenous deletion mutants of NC in the rat brain, we cloned a mutant in which the region including the larger part of one of the three active regions was deleted. The neurite outgrowth activity of the mutant was significantly lower than that of normal NC. These results suggest the physiological significance of these active regions.     

Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain

Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val(2718)-Arg-Lys-Arg-Leu(2722), Tyr(2738)-Tyr(2739), Tyr(2789)-Ile-Lys-Arg-Lys(2793) and Val(2817)-Glu-Arg-Lys(2820), on the mouse laminin alpha1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg(90)-Glu-Leu-Ser-Glu-Asp(95) on the omega loop, and also the involvement of Pro(40)-Pro-Val(42), Arg(46) (linked to Glu(94) by a salt bridge) and the hexapeptide Asp(61)-Ala-Thr-Thr-Phe-Gln(66). Epitope analysis, using CLiPS technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro(40)-Pro-Met-Gly-Pro-Arg-Arg-Phe(48) (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro(78)-Gly-Phe-Glu-Gly-Thr-Glu(84) and Pro(88)-Asn-Arg-Glu-Leu-Ser-Glu-Asp(95). Antibodies that recognized other features of the PAS area but not the Arg(90)-Gly-Leu-Ser-Glu-Asp(95) motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse.     

Structure of the C-terminal laminin G-like domain pair of the laminin alpha2 chain harbouring binding sites for alpha-dystroglycan and heparin

The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency.     

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival-potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity-purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin-binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell-binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod-like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin-binding domain of laminin is therefore responsible for its effects on neurons.     

Quantitative effects of laminin concentration on neurite outgrowth in vitro

Recent studies indicate that mediation of neurite outgrowth by the glycoprotein laminin may be a significant factor in the outgrowth of neurites to their targets during embryogenesis. To further characterize the possible role of this extracellular matrix molecule during development, we have systematically measured several features of outgrowth by neonatal rat sympathetic neurons on different concentrations of laminin. Individual neurons, obtained by mechanical dissociation of superior cervical ganglia (SCG), were cultured at low density on laminin substrates ranging from 0.01 to 1.0 microgram/cm2. Outgrowth characteristics were subsequently analyzed for noninteracting cells in both fixed and live cultures. Data obtained from neurons fixed after 11 hr of culture showed approximately twofold increases in neurite initiation and outgrowth, and a twofold decrease in branching for a corresponding 100-fold increase in adsorbed laminin concentration. In time-lapse videomicroscopy observations, the root-mean square speed of growth cone movement increased from 60 to 90 microns/hr over the same range in concentration, while the persistence time remained constant at 0.10 hr. In general, neurite outgrowth parameters were relatively insensitive to changes in laminin concentration, supporting the idea that laminin is a permissive rather than an "instructive" substrate during development. Data obtained from fixed cultures were examined in terms of probability models to suggest possible mechanisms contributing to the dose-dependent effects observed.     

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.     

Structural and functional analysis of the recombinant G domain of the laminin alpha4 chain and its proteolytic processing in tissues

The C-terminal G domains of laminin alpha chains have been implicated in various cellular and other interactions. The G domain of the alpha4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, alpha4LG1-3 and alpha4LG4-5. The recombinant fragments were shown to fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity for the alpha-dystroglycan receptor when compared with the G domains of the laminin alpha1 and alpha2 chains. Yet antibodies to alpha4LG1-3 but not to alpha4LG4-5 clearly inhibited alpha(6)beta(1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the alpha4LG3 and alpha4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the alpha4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha4LG4-5, indicating their loss from tissues. Immunogold staining demonstrated an association of the alpha4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin alpha2 chains appear primarily associated with various basement membranes.     

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.     

Modification of the laminin alpha 4 chain by chondroitin sulfate attachment to its N-terminal domain

The N-terminal domain of laminin alpha 4 chains corresponds to a short rod-like structure which after recombinant production was found to be modified by chondroitin sulfate. Substitution occurred mainly to a single serine in its N-terminal ASGDG sequence. A similar yet partial modification was also demonstrated for the alpha 4 chain present in extracts of adult mouse tissues. Antibodies to the fragment were useful to demonstrate a relatively high content of alpha 4 in several tissues and for the immunolocalization in various blood vessels, some basement membranes and interstitial regions.     

Weaver granule neurons are rescued by calcium channel antagonists and antibodies against a neurite outgrowth domain of the B2 chain of laminin

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 microM), also rescued the weaver granule neurons. High concentrations of MK-801 (10- 20 microM), a glutamate receptor antagonist and voltage-gated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 microM) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-D-aspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons.