Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.     

Localization of a major nidogen-binding site to domain III of laminin B2 chain

The large pepsin fragments P1 and P1X, which comprise most of the rod-like domains III of the three short arms of laminin from the mouse Engelbreth-Holm-Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70-80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine-rich repeats with similarity to epidermal growth factor showed substantial nidogen-binding activity. Further degradation of this component to an active 28-kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine-rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.     

Dissection of laminin by cathepsin G into its long-arm and short-arm structures and localization of regions involved in calcium dependent stabilization and self-association

Native laminin-nidogen complex isolated from mouse Engelbreth-Holm-Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment C1-4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8-9 (about 350 kDa) contains the entire triple-coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C-terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment C1-4 and only this fragment exhibited strong Ca2+ dependent self-association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment C1-4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8-9 may be employed for exploring the properties and possible functions of the upper long-arm region which so far has not been available as a fragment.     

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.     

Urokinase binding to laminin-nidogen. Structural requirements and interactions with heparin.

Recently we have shown that heparin and related sulfated polyanions are low-affinity ligands of the kringle domain in the amino-terminal region (ATF) of human urokinase (u-PA), and proposed that this may facilitate loading of u-PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin-nidogen) for u-PA binding, and found that laminin-nidogen is also a ligand of the u-PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin-nidogen showed that there are u-PA binding sites in fragment E4 of laminin as well as in nidogen. The long-arm terminal domain of laminin (fragment E3), which contains a heparin-binding site, competed for binding of u-PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u-PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy-terminal domain. Direct binding assays confirmed that u-PA binds to nidogen through a site in the u-PA ATF. We conclude that u-PA binds to laminin-nidogen by interactions involving the ATF region of u-PA, the E4 domain of laminin and the rod or amino-terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u-PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.     

Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alternative model for the internal structure of laminin

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.     

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.     

Use of fragments of hirudin to investigate thrombin-hirudin interaction

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.     

A single EGF-like motif of laminin is responsible for high affinity nidogen binding.

A major nidogen binding site of mouse laminin was previously localized to about three EGF-like repeats (Nos 3-5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3-5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3-5 and nidogen could be efficiently converted into a covalent complex by cross-linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3-5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3-5 was indicated from its protease resistance and the complete loss of cross-reacting epitopes as well as of nidogen-binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF-like repeats 3-4 and 4-5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen-binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28-residue signal peptide. The data support a previously proposed dumb-bell model of nidogen by demonstrating a large N-terminal globular domain (641 residues), five EGF-like repeats constituting the rod-like domain (248 residues) and a smaller C-terminal globule (328 residues). Two more EGF-like repeats interrupt the N-terminal and terminate the C-terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta-hydroxylation, two N-linked carbohydrate acceptor sites and, within one of the EGF-like repeats an Arg-Gly-Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C-terminal globule but not yet precisely localized.     

Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin

Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.     

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.     

The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites

The molecular interactions of laminin with several tumor cell lines and skin fibroblasts were investigated by radioligand binding studies and cell attachment assays using laminin, the laminin-nidogen complex, and laminin fragments as substrates and also domain-specific antibodies as inhibitors of cell attachment. The majority of cells showed a dual binding pattern for fragments 1 and 8 which originate from short-arm or long-arm structures of laminin, respectively. Both of these fragments in solution bind to suspended cells with high affinity (KD = 1-10 nM), with the receptor numbers for each fragment depending on the cell type. Competition studies and independent variation of receptor numbers demonstrated that the cell-binding structures on each fragment are different, implicating the existence of two distinct cellular receptors for laminin. The ability of these fragments to act as substrates for cell adhesion correlated with the presence of high affinity binding sites on the cells. However, only antibodies to fragment 8 were able to block cell adhesion to laminin, despite the presence of binding sites for fragment 1. A few cells had very low numbers of high affinity receptors for either fragment 1 or 8. The latter cell type was used to demonstrate that complex formation between laminin and nidogen, which binds to fragment 1 structures, reduces the potential of laminin for cell binding.     

Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on fibronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment P1, corresponding to the center of the cross, and only poorly on fragment E8, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment P1, and fibronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular massess of 180 and 135 kDa. The latter is recognized by an antiserum to integrin beta 1 subunit. The bound laminin receptor could be displaced by EDTA, but not by Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to P1. The expression of the 180-kDa alpha subunit of the laminin receptor at the cell surface was increased 10-fold after NGF treatment. The effect of NGF is specific since the amount of a 150-kDa fibronectin-binding integrin alpha subunit remained unchanged. Moreover, the increased expression of the 180/135 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment P1. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on laminin.     

Evidence for coiled-coil alpha-helical regions in the long arm of laminin.

Three new laminin fragments, E8, E9 and 25K with mol. wt. 50 000-280 000, were prepared from a limited elastase digest of laminin and from tissue extracts. They were similar with respect to their rod-like structure, a high alpha-helix content, the assembly from two chain segments and immunological cross-reactivity. Two of the fragments (E8 and E9) possess in addition globular domains which lack alpha-helices. Chemical, immunological and physical data together with sequence analysis strongly indicate that the alpha-helical segments are assembled in coiled-coil structures which are located in the rod of the long arm of laminin. These data give new insights into the overall structure of the protein.     

Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes.

The presence of laminin in authentic basement membranes was examined at the level of a large pepsin-resistant fragment P1. This strongly antigenic fragment has been recently isolated from a mouse tumour basement membrane. By using antibodies to mouse laminin P1 for identification it was possible to isolate a homologous fragment P1 (Mr about 250 000) and a related component Pa (Mr about 70 000--90 000) from pepsin digests of human placenta and kidney. The fragments were in half-cystine (90--130 residues/1000) and carbohydrate and showed strong binding to concanavalin A. Reduction of disulphide bonds produced several smaller peptide chains, indicating a complex pepsin cleavage. Immunological assays demonstrated partial antigenic identity between laminin fragments obtained from mouse and human tissue, and suggested that fragment Pa may originate from a protein not completely identical with laminin. The results showed that laminin is an abundant component of tissue rich in basement membranes, which has been previously suggested by immunohistological studies.     

Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.     

Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV

The laminin-nidogen complex and purified nidogen both bind collagen IV but not other collagens, as shown by solid-state ligand-binding and inhibition assays. Laminin purified from the dissociated complex and a variety of laminin proteolytic fragments failed to bind collagen IV. Complexes formed in solution between nidogen or laminin-nidogen and collagen IV were visualized by rotary shadowing which identified one major binding site about 80 nm away from the C-terminus of the collagen triple helix. A second, weaker binding site may exist closer to its N-terminus. Binding sites of nidogen were assigned to its C-terminal globular domain which also possesses laminin-binding structures. A more diverse collagen-IV-binding pattern was observed for the laminin nidogen complex, whereby interactions may involve both nidogen and short-arm structures of laminin.