Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.     

Mutation of a basic sequence in the laminin alpha2LG3 module leads to a lack of proteolytic processing and has different effects on beta1 integrin-mediated cell adhesion and alpha-dystroglycan binding

A RRKRRQ sequence unique to the LG3 module of the laminin alpha2 chain was previously shown to be sensitive to endogenous proteolysis during the recombinant production of the tandem array alpha2LG1-3. Mutation of RQ surrounding the cleaved peptide bond did not prevent this processing and intracellular degradation. Alanine mutagenesis of three alternate basic residues, however, was shown to prevent the cleavage in alpha2LG1-3, allowing for the alpha2LG3 module to be obtained as a folded, globular fragment. The mutation did not change heparin and sulfatide binding or cell adhesion of alpha2LG1-3 which can be mediated by alpha3beta1 and alpha6beta1 integrins. It did, however, cause a 10-fold reduction in alpha-dystroglycan binding. The data favor the interpretation that binding epitopes for heparin/sulfatides, beta1 integrins and alpha-dystroglycan occupy different parts of the alpha2LG1-3 structure.     

Distinct requirements for heparin and alpha-dystroglycan binding revealed by structure-based mutagenesis of the laminin alpha2 LG4-LG5 domain pair

Laminin-2 (alpha2beta1gamma1) is found in basement membranes surrounding muscle and peripheral nerve cells. Several types of cellular receptors bind to the laminin G-like (LG) domains at the C terminus of the alpha2 chain, the interaction with alpha-dystroglycan (alpha-DG) being particularly important in muscle. We have used site-directed mutagenesis and in vitro binding assays to map the binding sites on the laminin alpha2 chain LG4-LG5 domain pair for alpha-DG, heparin and sulfatides. Calcium-dependent alpha-DG recognition requires the calcium ion in LG4, but not the one in LG5, as well as basic residues in both LG domains. Heparin and sulfatides also bind to basic residues in both LG domains, but there is little overlap in the binding sites for alpha-DG and heparin/sulfatides. The results should prove useful for the molecular dissection of laminin-receptor interactions in vivo.     

Crystal structure and cell surface anchorage sites of laminin alpha1LG4-5

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin alpha1LG4-5 domains and provide a mutational analysis of heparin, alpha-dystroglycan, and galactosylsulfatide binding. The two domains of alpha1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin alpha2 LG4-5 but with a substantially different interdomain angle. Recombinant alpha1LG4-5 binding to heparin, alpha-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to alpha-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands.     

Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.     

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.     

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.     

Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V.

The C-terminal perlecan domain V of about 90 kDa consists of laminin-type G domain modules (LG) (25 kDa) and epidermal growth factor-like modules (EG) (4 kDa) in the tandem arrangement LG1-EG1-EG2-LG2-EG3-EG4-LG3. Several shorter fragments have been prepared by recombinant production in mammalian cells and used to map the single glycosaminoglycan (GAG) substitution site and the binding of several carbohydrate and protein ligands. This identified a Ser3511 residue located in a short link region between EG4 and LG3 as being involved in GAG attachment. Electron microscopy provided evidence that the same substitution exists in tissue forms of perlecan. Heparan sulphate attached to this site was shown to bind to the alpha1LG4 module of laminin-1, indicating a role in basement membrane assembly and cell-matrix interactions. This site is also close to an Asn-Asp bond which is readily cleaved by an endogenous protease that depends on the presence of Asp and the LG2 module. A weak heparin binding site was shown to include the EG2 module, which contains five basic residues. Binding to sulphatides and the alpha-dystroglycan receptor was much stronger and required at least two LG modules. However, single LG modules appear to be sufficient for the interaction with the laminin-nidogen complex, while EG3-4 and some flanking regions are apparently involved in fibulin-2 binding. These observations indicate that a complex modular structure is required for domain V in order to provide a rich repertoire of potential biological functions.     

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.     

Structure and function of laminin LG modules.

Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.     

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.     

Binding of laminin alpha1-chain LG4-5 domain to alpha-dystroglycan causes tyrosine phosphorylation of syntrophin to initiate Rac1 signaling

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser(65). How this signaling is initiated was investigated. Grb2-binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin alpha1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin alpha1 LG4 is known to bind alpha-dystroglycan, this directly implicates alpha-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The alpha-dystroglycan antibody, IIH6, which blocks binding of laminins to alpha-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is alpha-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, beta-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented.     

The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin

Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding.     

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.     

Contributions of the LG modules and furin processing to laminin-2 functions

The alpha2-laminin subunit contributes to basement membrane functions in muscle, nerve, and other tissues, and mutations in its gene are causes of congenital muscular dystrophy. The alpha2 G-domain modules, mutated in several of these disorders, are thought to mediate different cellular interactions. To analyze these contributions, we expressed recombinant laminin-2 (alpha(2)beta(1)gamma(1)) with LG4-5, LG1-3, and LG1-5 modular deletions. Wild-type and LG4-5 deleted-laminins were isolated from medium intact and cleaved within LG3 by a furin-like convertase. Myoblasts adhered predominantly through LG1-3 while alpha-dystroglycan bound to both LG1-3 and LG4-5. Recombinant laminin stimulated acetylcholine receptor (AChR) clustering; however, clustering was induced only by the proteolytic processed form, even in the absence of LG4-5. Furthermore, clustering required alpha(6)beta(1) integrin and alpha-dystroglycan binding activities available on LG1-3, acting in concert with laminin polymerization. The ability of the modified laminins to mediate basement membrane assembly was also evaluated in embryoid bodies where it was found that both LG1-3 and LG4-5, but not processing, were required. In conclusion, there is a division of labor among LG-modules in which (i) LG4-5 is required for basement membrane assembly but not for AChR clustering, and (ii) laminin-induced AChR clustering requires furin cleavage of LG3 as well as alpha-dystroglycan and alpha(6)beta(1) integrin binding.     

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.     

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.     

Perlecan domain V of Drosophila melanogaster. Sequence, recombinant analysis and tissue expression.

The C-terminal domain V of the basement membrane proteoglycan perlecan was previously shown to play a major role in extracellular matrix and cell interactions. A homologous sequence of 708 amino-acid residues from Drosophila has now been shown to be 33% identical to mouse perlecan domain V. It consists of three laminin G-type (LG) and epidermal growth factor-like (EG) modules but lacks the EG3 module and a link region found in mammalian perlecans. Recombinant production of Drosophila perlecan domain V in mammalian cells yielded a 100-kDa protein which was folded into a linear array of three globular LG domains. Unlike the mouse counterpart, domain V from Drosophila was not modified by glycosaminoglycans and endogenous proteolysis, due to the absence of the link region. It showed moderate affinities for heparin and sulfatides but did not bind to chick alpha-dystroglycan or to various mammalian basement membrane proteins. A single RGD sequence in LG3 of Drosophila domain V was also incapable of mediating cell adhesion. Production of a proteoglycan form of perlecan (approximately 450 kDa) in one Drosophila cell line could be demonstrated by immunoblotting with antibodies against Drosophila domain V. A strong expression was also found by in situ hybridization and immunohistology at various stages of embryonic development and expression was localized to several basement membrane zones. This indicates, as for mammalian species, a distinct role of perlecan during Drosophila development.     

Probing the integrin-binding site within the globular domain of laminin-511 with the function-blocking monoclonal antibody 4C7.

In an attempt to elucidate the integrin-binding site within laminin-511 (alpha5beta1gamma1), we mapped the epitope for mAb 4C7, which recognizes the globular (G) domain of the laminin alpha5 chain and inhibits binding of integrin alpha6beta1 to laminin-511, using a series of recombinant laminin-511 mutants with deletions or substitutions in the G domain. Deletion of the LG2-5 modules only partially compromised the 4C7 binding activity, while deletion of all 5 LG modules completely abrogated the activity, indicating that the epitope for 4C7 resides in the LG1 module. In support of this conclusion, 4C7 reactivity was abolished when the LG1 module of laminin-511 was swapped with the corresponding module of laminin-111, but the reactivity was retained after swapping the LG2 or LG3 module. Despite the requirement of LG1 for 4C7 binding, a recombinant LG1 module failed to bind to 4C7 when expressed alone or in tandem with LG2, but exhibited significant 4C7 binding activity when expressed as an array of LG1-3. These results indicate that 4C7 recognizes an epitope in the LG1 module, whose active conformation is stabilized in the context of the LG1-3 modules. Despite their 4C7 binding activities, neither the recombinant LG1-3 fragment nor the LG2 and LG3 swap mutants were capable of binding to integrin alpha6beta1. Thus, the integrin binding activity does not necessarily parallel the 4C7 reactivity, and possibly requires a strictly defined conformation of the LG1 module which can only be attained within an array of the intact LG1-3 modules connected to the preceding coiled-coil domain.     

Heparin binds to the laminin alpha4 chain LG4 domain at a site different from that found for other laminins

We previously reported that the LG4 domain of the laminin alpha4 chain is responsible for high-affinity heparin binding. To specify the amino acid residues involved in this activity, we produced a series of alpha4 LG4-fusion proteins in which each of the 27 basic residues (arginine, R; histidine; lysine, K) were replaced one by one with alanine (A). When the effective residues R1520A, K1531A, K1533A, and K1539A are mapped on a structural model, they form a track on the concave surface of the beta-sandwich, suggesting that they interact with adjacent sulfate groups along the heparin chain. Whereas low-affinity heparin-binding sites of other LG domains have been located at the top of the beta-sheet sandwich opposite the N and C termini, the residues for high-affinity heparin binding of alpha4 LG4 reveal a new topological area of the LG module.