Binding domain for laminin on type IV collagen

Binding of type IV collagen to laminin was studied by attaching one member of the ligand pair to a solid phase. When laminin was bound to a solid phase, type IV collagen exhibited saturable binding. Digestion of type IV collagen with high concentrations of pepsin destroyed the laminin binding activity. Type IV collagen was also found to bind to fibronectin but the binding activity was not destroyed by pepsin treatment. Rotary shadowing electron microscopy of the pepsin digested type IV collagen indicated that the carboxy terminal end region of about 100 nm is cleaved. Rotary shadowing electron microscopy studies demonstrate that the carboxy terminal end of type IV collagen has a major laminin binding site.     

High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV

Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with a Kd value of 10(-9) M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10(-6) M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin 5 and collagen IV. We postulate that the ability of the NC1 domains to bind with high affinities to laminin 5 and collagen IV facilitates stabilization of the structure of the basement membrane itself and that the NC1-collagen I interaction may be less important for stabilization of the dermal-epidermal junction.     

Heparin type IV collagen interactions: equilibrium binding and inhibition of type IV collagen self-assembly

Interactions between type IV collagen and heparin were examined under equilibrium conditions with rotary shadowing, solid-phase binding assays, and affinity chromatography. With the technique of rotary shadowing and electron microscopy, heparin appeared as thin, short strands and bound to the following three sites: the NC1 domain, and in the helix, at 100 and 300 nm from the NC1 domain. By solid-phase binding assays the binding of [3H]heparin in solution to type IV collagen immobilized on a solid surface was found to be specific, since it was saturable and could be displaced by an excess of unlabeled heparin. Scatchard analysis indicated three classes of binding sites for heparin-type IV collagen interactions with dissociation constants of 3, 30, and 100 nM, respectively. Furthermore, by the solid-phase binding assays, the binding of tritiated heparin could be competed almost to the same extent by unlabeled heparin and chondroitin sulfate side chains. This finding indicates that chondroitin sulfate should also bind to type IV collagen. By affinity chromatography, [3H]heparin bound to a type IV collagen affinity column and was eluted with a linear salt gradient, with a profile exhibiting three distinct peaks at 0.18, 0.22, and 0.24 M KCl, respectively. This suggested that heparin-type IV collagen binding was of an electrostatic nature. Finally, the effect of the binding of heparin to type IV collagen on the process of self-assembly of this basement membrane glycoprotein was studied by turbidimetry and rotary shadowing. In turbidity experiments, the presence of heparin, even in small concentrations, drastically reduced maximal aggregation of type IV collagen which was prewarmed to 37 degrees C. By using the morphological approach of rotary shadowing, lateral associations and network formation by prewarmed type IV collagen were inhibited in the presence of heparin. Thus, the binding of heparin resulted in hindrance of assembly of type IV collagen, a process previously described for interactions between various glycosaminoglycans and interstitial collagens. Such regulation may influence the assembly of basement membranes and possibly modify functions. Furthermore, qualitative and quantitative changes of proteoglycans which occur in certain pathological conditions, such as diabetes mellitus, may alter molecular assembly and possibly permeability functions of several basement membranes.     

Binding of laminin to type IV collagen: a morphological study

A mixture of laminin and type IV collagen was analyzed by rotary shadowing using carbon/platinum and electron microscopy. Laminin was found to form distinct complexes with type IV collagen: one site of interaction is located 140 nm from the COOH-terminal, noncollagenous (NC1) domain and the other is located within the NH2-terminal region. The isolated NC1 fragment of type IV collagen does not appear to interact with laminin, while pepsin-treated type IV collagen, which lacks the NC1 domain, retains its ability to form complexes with laminin. Analysis of the laminin-type IV complexes indicates that laminin binds to type IV collagen via the globular regions of either of its four arms. This finding is supported by experiments using fragment P1 of laminin which lacks the globular regions and which does not bind to type IV collagen in a specific way. In addition, after heat-denaturation of laminin no specific binding is observed.     

The role of the main noncollagenous domain (NC1) in type IV collagen self-assembly

Type IV collagen incubated at elevated temperatures in physiologic buffers self-associates (a) via its carboxy-terminal (NC1) domain, (b) via its amino-terminal (7S) domain, and (c) laterally; and it forms a network. When examined with the technique of rotary shadowing, isolated domain NC1 was found to bind along the length of type IV collagen to four distinct sites located at intervals of approximately 100 nm each. The same 100-nm distance was observed in domain NC1 of intact type IV collagen bound along the length of the collagen molecules during initial steps of network formation and in complete networks. The presence of anti-NC1 Fab fragments in type IV collagen solutions inhibited lateral association and network formation in rotary shadow images. During the process of self-association type IV collagen develops turbidity; addition of isolated domain NC1 inhibited the development of turbidity in a concentration-dependent manner. These findings indicate that domain NC1 of type IV collagen plays an important role in the process of self-association and suggest that alterations in the structure of NC1 may be partially responsible for impaired functions of basement membranes in certain pathological conditions.     

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY- D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine- containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N- glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin- sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.     

Sphingosine inhibits attachment of murine Lewis lung carcinoma cells to laminin and type IV collagen

The effect of sphingosine (SPH) on the adhesive properties of Lewis lung carcinoma (3LL) cells was investigated using plastic precoated with the extracellular matrix proteins, laminin, fibronectin, or type IV collagen. Treatment of 3LL cells with SPH (0.5-10 microM) resulted in a dose-dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on attachment to fibronectin. Phorbol 12-myristate 13-acetate (PMA) selectively enhanced attachment of 3LL cells to laminin and collagen. The inhibitory effect of SPH on attachment to both proteins was competitively antagonized by PMA. These results suggest that SPH acts as a negative effector for cell attachment to laminin and collagen, and that the cell attachment process to both proteins might be regulated in part by protein kinase C.     

Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration

Laminin and type IV collagen were compared for the ability to promote aortic endothelial cell adhesion and directed migration in vitro. Substratum-adsorbed IV promoted aortic endothelial cell adhesion in a concentration dependent fashion attaining a maximum level 141-fold greater than controls within 30 min. Aortic endothelial cell adhesion to type IV collagen was not inhibited by high levels (10(-3) M) of arginyl-glycyl-aspartyl-serine. In contrast, adhesion of aortic endothelial cells on laminin was slower, attaining only 53% of the adhesion observed on type IV collagen by 90 min. Type IV collagen when added to the lower well of a Boyden chamber stimulated the directional migration of aortic endothelial cells in a concentration dependent manner with a maximal response 6.9-fold over control levels, whereas aortic endothelial cells did not migrate in response to laminin at any concentration (.01-2.0 X 10(-7) M). Triple helix-rich fragments of type IV collagen were nearly as active as intact type IV collagen in stimulating both adhesion and migration whereas the carboxy terminal globular domain was less active at promoting adhesion (36% of the adhesion promoted by intact type IV collagen) or migration. Importantly, aortic endothelial cells also migrate to substratum adsorbed gradients of type IV collagen suggesting that the mechanism of migration is haptotactic in nature. These results demonstrate that the aortic endothelial cell adhesion and migration is preferentially promoted by type IV collagen compared with laminin, and has a complex molecular basis which may be important in angiogenesis and large vessel repair.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.     

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.     

Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen

The globular domain of type IV collagen from bovine glomerular basement membrane was solubilized by collagenase digestion. Components of this domain include several monomer-size and structurally related dimer-size polypeptides. The monomer-size polypeptides were resolved into three fractions (M1, M2, and M3) with slightly different mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced Mr = 24,500-28,300). Chemical and immunochemical studies indicate that each is a distinct component. M2 is reactive with antibodies from patients with Goodpasture syndrome. The molecular weight by sedimentation equilibrium was 32,000 for M2 and 28,000 for M1. The dimers were characterized as two classes, D1 and D2. D1 consists of two sets of nonreactive components (D1a-d and D1a,c) whereas D2 contains one set of four components (D2a-d), each of which is reactive with Goodpasture sera. Chemical and immunochemical studies indicate that a monomer-dimer relationship exists between M1 and D1 and between M2 and D2. The origin of M3 remains undetermined. Rabbit antibodies to type IV collagen alpha chains react with M1 and M2, and antibodies to M1 and M2 react with type IV collagen alpha chains, which provides additional evidence for the localization of the Goodpasture antigen to one of the chains of type IV collagen.     

Functional differences in the interactions of glycosylation-deficient cell lines with fibronectin, laminin, and type IV collagen

Fibronectin isolated from the conditioned medium of monolayer cultures of baby hamster kidney (BHK) cells and several ricin-resistant (Ric) mutants derived from them express differences in N-glycosylation. The asparagine-linked oligosaccharides of BHK cell-derived fibronectin consist largely of complex chains, whereas hybrid and/or high-mannose chains are present in the fibronectins of mutant cell lines. The fibronectins exhibiting different glycosylation patterns are incorporated to similar extents into the cell-layer of human skin fibroblasts. In contrast, mutant cells retain significantly less endogenously produced fibronectin than BHK cells and also incorporate less human cellular fibronectin into a pericellular matrix. In vitro adhesion assays show that mutant cells attach to and spread relatively poorly on fibronectin-or type IV collagen-coated substrata but interact as well as do BHK cells with a laminin substratum. These results indicate that asparagine-linked oligosaccharides of fibronectin are not required for the binding and incorporation of the molecule into cell layers, but, as constituents of other cellular glycoproteins, they do modulate the ability of BHK cells to interact with some matrix components.     

Immunofluorescent localization of type IV collagen and laminin during endochondral bone differentiation and regulation by pituitary growth hormone

Vascularization and the influence of growth hormone on this process were studied during endochondral bone differentiation. Vascular invasion was monitored by immunofluorescent localization of two vascular basement membrane proteins, type IV collagen and laminin, a recently described glycoprotein. In addition, endothelial cell invasion was identified by localization of Factor VIII. New bone formation was induced by subcutaneous implantation of a coarse powder of demineralized rat bone matrix. On days 1 through 9, no vascular elements were detected in the plaque. Mesenchymal cells appeared on day 3, proliferated, and differentiated into cartilage on day 7, while the capillaries proliferated at the periphery of the plaque. Beginning on day 9 with capillary incursion into the center of the plaque, type IV collagen, laminin, and Factor VIII were localized in the invading vascular endothelial cells. Type IV collagen and laminin appeared synchronously in the capillary basement membranes and later in the endothelial lining of cavernous sinusoids. Their distribution pattern was identical. The vascular invasion was prominent by day 14. In hypophysectomized rats, cartilage differentiated normally but vascularization was delayed and reduced. Bone formation was scanty as indicated by ⁴⁵Ca incorporation. Administration of bovine growth hormone to hypophysectomized recipients restored vascularization and bone formation to the level observed in controls.     

Subunit structure and assembly of the globular domain of basement-membrane collagen type IV

The globular domain of collagen IV was solubilized by collagenase digestion from a mouse tumor, human placenta and bovine aorta and was purified by chromatographic methods. The materials show a unique, mainly non-collagenous amino acid composition and contain small amounts of glucosamine and galactosamine. The globular structures with Mr = 170 000 appear as a hexameric assembly originating from two collagen IV molecules. Subunits of this assembly are two different dimers Da and Db (Mr about 56 000) and monomers (Mr = 28 000). Their N-terminal amino acid sequences start with short triple-helical sequences, which overlap with the C-terminal triple helix of the alpha 1(IV) and alpha 2(IV) chain, demonstrating that the globule originates from the C terminus of collagen IV. Dimers arise from monomers by disulfide cross-linking (form Db) and/or formation of non-reducible cross-links (form Da). Reduction under non-denaturing conditions causes partial dissociation of the globule and of collagen IV dimers, indicating that reducible cross-links are formed between monomers of two different collagen IV molecules. Dissociation of the hexamer into the subunits can be achieved with 8 M urea, sodium dodecyl sulfate or in the pH range 2.5-4. The latter indicates that carboxyl groups are essential for association. Mixtures of the subunits (monomers and dimers) or purified dimers reassemble in neutral buffer into hexamers as shown by ultracentrifugation and electron microscopy. Reconstituted hexamers, however, dissociate in a much broader pH range than the native globules. Circular dichroic spectra indicate that the structure is more completely refolded from acid-treated than from urea-treated material. These data suggest that globules originating from monomers (as existing in single collagen IV molecules) are stabilized by the adjacent triple helix. Covalent cross-link formation stabilizes the globular structure and allows reconstitution in stoichiometric proportions.     

Expression and localization of laminin 5, laminin 10, type IV collagen,and amelotin in adult murine gingiva

The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules—laminin 5 (γ2 chain), type IV collagen (α1 chain), and laminin 10 (α5 chain)—and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

Immuno-electron-microscopic localization of laminin and collagen type IV in normal and denervated tooth pulp of the cat

Summary The distribution of laminin-like immunoreactivity in adult normal and denervated cat mandibular tooth pulps was studied by the use of fluorescence microscopy and pre-embedding immunogold electron microscopy. Immunoreactivity to collagen IV was also assessed in order to distinguish basement membranes. In normal pulps, light-microscope laminin-like immunoreactivity was strong along blood vessels and Schwann cell sheaths, and a faint immunoreactivity was seen also in the odontoblast layer. Electron microscopy confirmed the laminin-like immunoreactivity of endothelial and Schwann cell basement membranes at all pulpal levels. In the odontoblast layer and the predentine, nerve-like structures lacking basement membranes but possessing strong membrane laminin-like immunoreactivity were encountered. In addition, a clear-cut laminin-like immunoreactivity of plasma membranes of the somata and processes of odontoblasts was seen. Observations on denervated pulps as well as pulps in which nerve regeneration had taken place did not reveal any changes in the pattern of laminin-immunoreactivity in basement membranes or odontoblasts. Distribution of collagen IV-like immunoreactivity was very similar to laminin-like immunoreactivity in basement membranes of blood vessels and Schwann cells, and appeared unaffected by denervation. The odontoblasts and nerve-like profiles in the odontoblast layer were devoid of collagen IV-like immunoreactivity. We propose that odontoblast-associated laminin could be of significance as guidance for regenerating terminal pulpal nerve fibers to appropriate targets.     

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.