Mapping of binding sites for nidogens, fibulin-2, fibronectin and heparin to different IG modules of perlecan

Perlecan, a major basement membrane proteoglycan, has a complex modular structure designed for the binding of many cellular and extracellular ligands. Its domain IV, which consists of a tandem of immunoglobulin-like modules (IG2-IG15), is rich in such binding sites, which have been mapped to different modules obtained by recombinant production. Heparin/sulfatide binding was restricted to IG5 and shown to depend on four arginine residues that are close in space in beta strands B and E of the C-type IG fold. The nidogen-1 and nidogen-2 isoforms bind to IG3 with high affinity (K(d) approximately 10 nM). This interaction depends on the globular nidogen domain G2 and is crucial for the formation of ternary complexes with laminins. Two loops of IG3 located between beta strands B/C and F/G, which are spatially close, make a major contribution to binding. Fibronectin binding was localized to IG4-5 and fibulin-2 binds to IG2 and IG13-15 with different affinities. This implicates a complex cluster of heterotypic interaction sites apparently important for the supramolecular organization of perlecan in tissues.     

Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies.

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.     

Recombinant domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2 and heparin.

Domain IV of mouse perlecan, which consists of 14 immunoglobulin superfamily (IG) modules, was prepared from recombinant human cell culture medium in the form of two fragments, IV-1 (IG2-9, 100 kDa) and IV-2 (IG10-15, 66 kDa). Both fragments bound to a heparin column, being eluted at ionic strengths either below (IV-2) or above (IV-1) physiological level, and could thus be readily purified. Electron microscopy demonstrated an elongated shape (20-25 nm), and folding into a native structure was indicated by immunological assay and CD spectroscopy. Solid-phase and surface plasmon resonance assays demonstrated strong binding of fragment IV-1 to fibronectin, nidogen-1, nidogen-2 and the laminin-1-nidogen-1 complex, with Kd values in the range 4-17 nM. The latter binding apparently occurs through nidogen-1, as shown by the formation of ternary complexes. Only moderate binding was observed for fibulin-2 and collagen IV and none for fibulin-1 and BM-40. Fragment IV-2 showed a more restricted pattern of binding, with only weaker binding to fibronectin and fibulin-2. None of these activities could be demonstrated for recombinant fragments corresponding to the N-terminal perlecan domains I to III. This indicates a special role for domain IV in the integration of perlecan into basement membranes and other extracellular structures via protein-protein interactions.     

Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1.

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.     

The absence of one or both nidogens does not alter basement membrane composition in adult murine kidney

Nidogen-1 and nidogen-2 are major components of all basement membranes and are considered to function as link molecules between laminin and collagen type IV networks. Surprisingly, the knockout of one or both nidogens does not cause defects in all tissues or in all basement membranes. In this study, we have elucidated the appearance of the major basement membrane components in adult murine kidney lacking nidogen-1, nidogen-2, or both nidogens. To this end, we localized laminin-111, perlecan, and collagen type IV in knockout mice, heterozygous (+/-) or homozygous (-/-) for the nidogen-1 gene, the nidogen-2 gene, or both nidogen genes with the help of light microscopic immunostaining. We also performed immunogold histochemistry to determine the occurrence of these molecules in the murine kidney at the ultrastructural level. The renal basement membranes of single knockout mice contained a similar distribution of laminin-111, perlecan, and collagen type IV compared to heterozygous mice. In nidogen double-knockout animals, the basement membrane underlying the tubular epithelium was sometimes altered, giving a diffuse and thickened pattern, or was totally absent. The normal or thickened basement membrane of double-knockout mice also showed a similar distribution of laminin-111, perlecan, and collagen type IV. The results indicate that the lack of nidogen-1, nidogen-2, or both nidogens, plays no crucial role in the occurrence and localization of laminin-111, collagen type IV, and perlecan in murine tubular renal basement membranes.     

Molecular interactions in the retinal basement membrane system: A proteomic approach

Basement membranes (BMs) are physiologically insoluble extracellular matrix sheets present in all multicellular organisms. They play an important role in providing mechanical strength to tissues and regulating cell behavior. Proteomic analysis of BM proteins is challenged by their high molecular weights and extensive post-translational modifications. Here, we describe the direct analysis of an in vivo BM system using a mass spectrometry (MS) based proteomics approach. Retinal BMs were isolated from embryonic chick eyes. The BM macromolecules were deglycosylated and separated by low percentage gradient SDS PAGE, in-gel digested and analyzed by LC-MS/MS. This identified over 27 extracellular matrix proteins in the retinal BM. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, β2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, β1, γ2, γ3 and collagen IV subunits α5 and α6 were minor constituents. To examine binding interactions that contribute to the stability of the retinal BM, we applied the LC-MS/MS based approach to detect potential BM complexes from the vitreous. Affinity-captured nidogen- and heparin-binding proteins from the vitreous contained > 10 and > 200 proteins respectively. Comparison of these protein lists with the retinal BM proteome reveals that glycosaminoglycan and nidogen binding interactions play a central role in the internal structure and formation of the retinal BM. In addition, we studied the biomechanical qualities of the retinal BM before and after deglycosylation using atomic force microscopy. These results show that the glycosaminoglycan side chains of the proteoglycans play a dominant role in regulating the thickness and elasticity of the BMs by binding water to the extracellular matrix. To our knowledge, this is the first large-scale investigation of an in vivo BM system using MS-based proteomics.     

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.     

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.     

Impaired wound healing in mice lacking the basement membrane protein nidogen 1

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable α-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the β1 and β4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.     

The type XIII collagen ectodomain is a 150-nm rod and capable of binding to fibronectin, nidogen-2, perlecan, and heparin

Type XIII collagen consists of a short N-terminal intracellular domain, a transmembrane domain, and a collagenous ectodomain, and it is found at many sites of cell adhesion. We report on the characterization of recombinant type XIII collagen. The shed ectodomain was purified from insect cell culture medium and shown to form 240-kDa trimers with a T(m) of 42 degrees C. Correct chain association into a triple-helical conformation was confirmed by limited pepsin digestion and CD spectroscopy. Rotary shadowing electron microscopy of the ectodomain revealed it to be a 150-nm rod with two flexible hinges separating 31-, 52-, and 68-nm portions. The rods represent the collagenous domains 1-3, and the hinges coincide with the non-collagenous domains 2 and 3. By using surface plasmon resonance analysis, the ectodomain showed interaction with immobilized fibronectin, nidogen-2, and perlecan with K(D) values in the nanomolar range. The binding sites of type XIII collagen for fibronectin were localized to the collagenous domains, whereas the binding activities for nidogen-2 and perlecan resided in the pepsin-sensitive portions of the ectodomain. Furthermore, the ectodomain bound significantly to heparin, which also inhibited shedding of the ectodomain in insect cell cultures. The results reveal that type XIII collagen is notably distinct in its structure compared with other cell-surface proteins, and the in vitro binding with fibronectin, heparin, and two basement membrane components is indicative of multiple cell-matrix interactions in which this ubiquitously expressed protein participates.     

Structural elucidation of full-length nidogen and the laminin–nidogen complex in solution

Nidogen-1 is a key basement membrane protein that is required for many biological activities. It is one of the central elements in organizing basal laminae including those in the skin, muscle, and the nervous system. The self-assembling extracellular matrix that also incorporates fibulins, fibronectin and integrins is clamped together by networks formed between nidogen, perlecan, laminin and collagen IV. To date, the full-length version of nidogen-1 has not been studied in detail in terms of its solution conformation and shape because of its susceptibility to proteolysis. In the current study, we have expressed and purified full-length nidogen-1 and have investigated its solution behavior using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The ab initio shape reconstruction of the complex between nidogen-1 and the laminin γ-1 short arm confirms that the interaction is mediated solely by the C-terminal domains: the rest of the domains of both proteins do not participate in complex formation.     

Cleavage of nidogen-1 by cathepsin s impairs its binding to basement membrane partners

Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss(-/-) spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases.     

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.     

Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by snake venom metalloproteinases

Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels.     

Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice.

Previous studies have shown that inhibition of nidogen-laminin binding interferes with basement membrane stabilization in various mouse organ cultures while no overt phenotype has been observed following inactivation of the nidogen-1 gene in mice. We have now used recombinant mouse nidogen-1 and nidogen-2 in order to evaluate a possible compensation between the two isoforms in the knock-out mice. Essentially, a comparable in vitro binding of nidogens-1 and -2 to the same laminin gamma1 chain structure and to several other basement membrane proteins has been revealed. Quantitative radioimmuno-assays have demonstrated high concentrations of nidogen-1 exceeding those of laminin gamma1 and nidogen-2 by factors of 5 and 20-50, respectively, in tissue extracts of wild-type mice. A three- to sevenfold increase in nidogen-2 was observed in heart and muscle of mice with nidogen-1 deficiency and confirmed by a similar increase in the intensity of immunogold staining of these tissues. However, a few of the tissues from mice with the gene knock-out still contained some nidogen-1-like immunoreactivity (1% of wild-type). Furthermore, both nidogen isoforms showed a similar distribution in various organs during embryonic development which, however, as shown previously, changed in some adult tissues. The data support the nidogen-2 compensation hypothesis to explain the limited phenotype observed following elimination of the nidogen-1 gene.     

Basement membranes in skin: unique matrix structures with diverse functions?

The view of extracellular matrix (ECM) has evolved from a merely scaffolding and space filling tissue element to an interface actively controlling cellular activities and tissue functions. A highly specialized form of ECM is the basement membrane (BM), an ubiquitous sheet-like polymeric structure composed of a set of distinct glycoproteins and proteoglycans. In this review we are largely focusing on function and assembly of BM in skin (1) at the dermo-epidermal interface and (2) in the resident micro-vasculature. The role of the non-polymeric components perlecan and particularly nidogen is exemplified by reviewing experiments based on genetic approaches and adequate experimental skin models in vivo and in vitro. While in mice total deficiency of one of these components is eventually developmentally lethal, the severity of the defects varies drastically between tissues and also the skin models recapitulating BM formation in vitro. There is accumulating evidence that this relies on the mechanical properties, the molecular composition of the BM, the adjacent ECM or connective tissue, the dynamics of molecular assembly, and ‘minor’ tissue-specific modifier or adapter components. Though the role of nidogen or perlecan is still remaining a controversial issue, the statements ‘being essential for BM/or not’ should be consequently referred to the developmental, tissue, and functional (e.g., repair) context.     

Extracellular matrices of the avian ovarian follicle. Molecular characterization of chicken perlecan

In egg-laying species, such as the chicken, the mode of transport of lipoprotein particles from the capillary plasma to endocytic receptors on the oocyte surface is largely unknown. Here we show by molecular characterization that the large prominent heparan sulfate proteoglycan of extracellular matrices, termed perlecan or HSPG2 (the product of the hspg2 gene), is a component of ovarian follicles that may participate in this process. However, although normally a major HSPG of basement membranes or basal laminae, in chicken follicles, perlecan is absent from the membranous structure between the theca interna and granulosa cell layers, which to date has been considered a bona fide basement membrane. Rather, the protein is localized in the extracellular matrix of theca externa cells, which produce this HSPG. Furthermore, in chicken testes, perlecan is localized in the peritubular spaces but in less organized fashion than the classical basement membrane components, agrin and laminin. All five domains and structural hallmarks of chicken perlecan (4071 residues) have been conserved in its mammalian counterparts. We have produced the recombinant domain II (containing low density lipoprotein (LDL) receptor-like binding repeats) of chicken perlecan and demonstrate its capacity to bind LDL and very low density lipoprotein (VLDL), apolipoprotein B-containing lipoproteins ultimately destined for uptake into oocytes via members of the low density lipoprotein receptor family. Binding to perlecan heparan sulfate side chains may facilitate the interaction of lipoproteins with domain II. Based on the current results and on domain-domain interactions revealed by recent ultrastructural investigations of the LDL receptor, nidogen, and laminin (Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002) Science 298, 2353-2358 and Takagi, J., Yang, Y., Liu, J. H., Wang, J. H., and Springer, T. A. (2003) Nature 424, 969-974), we propose a novel role of perlecan in mediating plasma-to-oocyte surface transport of VLDL particles.     

Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.     

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.     

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)