Structure and function of the laminin-nidogen complex.
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Amino Acid Sequence↗
Biomedical subjects
M Aumailley
Publications and source records attributed to M Aumailley.
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.
Animals↗
Down-regulation of alpha 3(VI) chain expression by gamma-interferon decreases synthesis and deposition of collagen type VI.
Treatment of cultured human skin fibroblasts with increasing doses of gamma-interferon produces a distinct reduction of steady-state levels of the alpha 3 chain of collagen VI mRNA by about 60% but not of the alpha 1 and alpha 2 chain mRNAs. A similar decrease was also observed for collagen I and III mRNA while fibronectin mRNA remained at the same level. The decrease in alpha 3(VI) mRNA is accompanied by a reduced synthesis of collagen VI and by a reduced deposition of both collagen VI and fibronectin in urea-insoluble form in the cell matrix. No other gamma-interferon effects were observed for fibronectin biosynthesis. Immunoprecipitation of metabolically labeled collagen VI demonstrated a strongly reduced synthesis (by 65-80%) of intracellular alpha 3(VI) chains with no decrease found for alpha 1(VI) and alpha 2(VI) chains. All three chains were, however, found to be reduced in the culture medium. Pepsin treatment of immunoprecipitated collagen VI showed similar chain ratios for material in the culture medium obtained in the absence or presence of gamma-interferon. It indicates that correctly assembled heterotrimers of the composition [alpha 1(VI) alpha 2(VI) alpha 3(VI)] are formed and secreted also in the absence of an equivalent alpha 3(VI) chain synthesis but at a reduced rate. The data support previous predictions from sequence analyses [Chu et al. (1988) J. Biol. Chem. 263, 18,601-18,606] that collagen VI molecules composed of all three constituent chains are more stable than other assembly alternatives.
Actins↗
The high-affinity binding of laminin to cells. Assignation of a major cell-binding site to the long arm of laminin and of a latent cell-binding site to its short arms.
The laminin proteolytic fragments 1 (derived from the intersection of the short arms of the cruciform laminin molecule) and 8 (derived from the laminin long arm) bind to distinct receptors on HT-1080 human fibrosarcoma cells; both fragments are shown here to inhibit the high-affinity binding of laminin to these cells. Inhibition of binding between fragment 8 and laminin was competitive, whereas that between fragment 1 and laminin was noncompetitive. This indicates that laminin and fragment 8 most probably share the same cellular receptors, whereas laminin and fragment 1 bind to distinct receptors, inhibition being due to steric hindrance. Surprisingly, fragment 1-4 (corresponding to the complete short arms of laminin) neither bound to HT-1080 cells nor inhibited the binding of laminin or fragment 1. After treatment of fragment 1-4 with pepsin, however, the smaller subfragment 1 was liberated, which could then bind to the cells, and so was shown to block the binding of laminin and fragment 1. We conclude that native laminin bound to HT-1080 cells via the fragment-8-binding site near the end of its long arm. Although these cells also have distinct receptors for the short arm fragment 1, this receptor-binding site was not used as it appeared to be latent within the native laminin molecule.
Binding Sites↗
Regulation of collagen VI expression in fibroblasts. Effects of cell density, cell-matrix interactions, and chemical transformation.
Collagen VI expression was studied in cultured human skin fibroblasts and mouse 3T3 cells using cDNA probes specific for alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. A 2-3-fold increase of these mRNAs was observed when fibroblasts were grown at increasing densities while only minimal changes occurred for the mRNA levels of collagens I and III, fibronectin, and beta-actin. Changes in mRNA correlated well with an increased production of corresponding proteins as determined by immunological assays. A comparable increase of alpha 1(VI) and alpha 2(VI) but not of alpha 3(VI) chain mRNAs was found for fibroblasts grown in a three-dimensional collagen gel after gel contraction. These conditions resulted, however, in a decrease of steady-state levels of collagens I and III and actin mRNAs. Transformation of 3T3 cells by phorbol ester did not change collagen VI mRNAs but caused a 3-5-fold reduction in mRNA levels for the other extracellular matrix proteins. These data strongly imply different regulatory mechanisms for the expression of collagen VI compared with collagens I and III and fibronectin. The differences may be correlated to changes in cell shape and reflect the requirement for collagen VI as a cell-binding protein.
Actins↗
Domains of laminin with growth-factor activity.
Laminin and fragments (1, 1-4) containing the inner rod-like segments from its short arms, which consist of cysteine-rich, "EGF-like" repeats, stimulated thymidine incorporation in cultured cells possessing EGF receptors but had no effect on a cell line lacking this receptor. The response was comparable to that of EGF concerning effective concentrations, magnitude, time dependence, and synergistic enhancement by insulin. Other fragments (4 and 8) were inactive. Laminin and its active fragments could not compete with the binding of EGF to cells. There was no correlation between growth promotion and attachment of cells to a high affinity binding site present on laminin fragment 8. The data indicate that mitogenic effects induced by laminin and EGF proceed in some steps via related pathways and that different domains of laminin are involved in growth promotion and in adhesion and spreading of cells.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains.
Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded alpha 2(VI) and alpha 3(VI) chains but not on the alpha 1(VI) chain. The interactions with the chains were inhibited by low concentrations (10-100 microM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data indicate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.
Amino Acid Sequence↗
Biochemistry of basement membranes.
Basement membranes are thin (20 to 300 nm) extracellular matrices with a ubiquitous occurrence in the body. They consist mainly of collagenous and noncollagenous glycoproteins, are formed early during embryonic development, and in mature tissues they compartmentalize various types of cells and tissue structures. The close apposition to cells is the most remarkable feature of basement membranes, which can either surround cells completely (muscle, fat, nerve axons) or separate them from underlying stroma in a polarized fashion (epithelium, endothelium). These cell contacts are mediated by cellular receptors and specific matrix components and have profound effects on polarization, differentiation, and proliferation of cells and on the control of their migratory behavior. In addition, basement membranes represent barriers regulating filtration of macromolecules and penetration by cells. The supramolecular organization of basement membranes is still insufficiently known. In transmission electron microscopy cross-sections show a two-layered morphology that includes an electron dense (lamina densa) and an electron lucent (lamina rara) zone, the latter being closer to the cells. Other specialized basement membranes such as those in renal glomeruli appear more complex and contain two laminae rarae. Here, presumably epithelial and invading endothelial cells each produce their own basement membrane, which then fuse together to form the filtration units of the glomerulus. Further structural and functional study of authentic basement membranes has been limited because these membranes comprise only a small fraction of tissues and most of their components are notoriously insoluble. Rodent tumors that produce large amounts of basement membrane material and the use of recombinant DNA technology in the past decade have paved the way for a more precise biochemical characterization of several basement membrane proteins. These components include collagen type IV, the cell-binding protein laminin, several proteoglycans, and other proteins. In the following pages we will briefly review the biochemical properties of the basement membranes and discuss their possible functions and contributions to supramolecular structures. For more extensive discussions, including certain biologic and pathologic aspects of basement membrane function, we refer the reader to several recent reviews.
Basement Membrane↗
Serum type III procollagen peptide levels in coronary artery disease (a marker of atherosclerosis).
The known shift in collagen synthesis from procollagen type I to type III in patients with atherosclerosis, suggested measurement of serum procollagen III peptide (PIIIP) levels in patients with coronary artery disease (CAD). Two groups of patients were studied: group I--thirty-six patients with CAD (male, mean age 56.9 +/- 7.5 years, hospitalized for coronary angiography. Risk factors included 16 patients with high blood pressure, four diabetics, 31 smokers and 15 with hypercholesterolaemia. Five patients had no significant lesions, seven had one vessel with over 50% stenosis, 10 had two vessels and 14 had three vessels. Group II--35 patients (male, mean age 39.4 +/- 13.3 years), with normal physical examination and ECG according to WHO criteria, formed the control group: the risk factors included nine patients with high blood pressure, 14 smokers and one with hypercholesterolaemia. Procollagen III peptide levels were determined by radioimmunoassay. In group I, PIIIP levels were 26.8 +/- 16 ng ml-1 vs. 10.4 +/- 3.2 for group II. Sixty-one per cent of group I had pathological levels of PIIIP with an absence of correlation with the severity of atherosclerosis or risk factors. Only 2.8% of patients in group II had pathological levels. Procollagen III peptide determination would appear to be a sensitive, specific and predictive test for atherosclerosis in patients with CAD.
Adult↗
Molecular and clinical aspects of connective tissue.
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Collagen↗
Low production of procollagen III by skin fibroblasts from patients with Ehlers-Danlos syndrome type IV is not caused by decreased levels of procollagen III mRNA.
The Ehlers-Danlos syndrome type IV represents a heterogeneous group of diseases, the molecular defect of which seems to reside in a defective synthesis and processing of collagen III. Here we present data concerning both protein and mRNA levels of collagens I and III in cell cultures established from skin of nine patients and six controls. All patients investigated were characterized by a reduced amount of procollagen III in fibroblast cultures. However, the levels of collagen III propeptides, measured by a radioimmunoassay in culture medium of fibroblasts from the patients, were either decreased or in the range of control levels. Using hybridization with cDNA probes specific for pro alpha 1 (I) and pro alpha 1 (III) collagen chains we determined the ratios of procollagens I and III mRNA. Although synthesis of procollagen III was markedly reduced in fibroblasts of all patients studied, no parallel decrease of procollagen III specific mRNA was found. However, two distinctive groups of patients were identified, one with an unaltered ratio of procollagen I/III mRNA and the other which had an even higher proportion of collagen III mRNA.
Cells, Cultured↗
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.
Animals↗
Laminin-nidogen complex. Extraction with chelating agents and structural characterization.
Large quantities of intact laminin-nidogen complex could be extracted from a mouse tumor basement membrane with a physiological buffer containing EDTA. Analysis of the purified complex demonstrated that the two proteins occur in an equimolar ratio and that anchoring of these complexes to the extracellular matrix requires divalent cations. Reversible dissociation of the complex was achieved with 2 M guanidine X HCl and has been used for purification of the individual components. Electron microscopy and binding studies using laminin fragments demonstrated that nidogen interacts specifically with the center of the cross-shaped laminin molecule as represented by the short-arm structure fragment 1. The complex was also useful to confirm and refine a previously proposed dumb-bell structure of nidogen and to prepare and characterize the cell-binding fragment 8 from the long arm of laminin.
Animals↗
Collagen studies in congenital cutis laxa.
A case of congenital cutis laxa (CCL) of unclear mode of inheritance associated with multiple pulmonary artery branch stenosis was extensively investigated to assess possible correlations between clinical, ultrastructural, and biochemical features. Light microscopy revealed that elastic fibers were absent in the papillary dermis, while hypoplastic elsewhere. Transmission electron microscopy showed a poor elastin matrix content in some elastic fibers, variable diameters of collagen fibrils, and abundant glycogen granules in most dermal cells. Measurement of collagen fibril diameters, using an image analyzer, was carried out in the patient and two age- and site-matched controls. A bimodal distribution was found in the upper reticular dermis of the patient. In vitro analysis of collagen in skin fibroblast cultures of the patient showed increased collagen synthesis with a balanced production of type I and type III procollagens. Our study confirms that CCL represents a disorder both of collagen and elastic connective tissue.
Cells, Cultured↗
[Metabolism of collagen in coronary patients].
The alterations observed in connective tissue of the arterial wall and dermis in atherosclerosis incited us to investigate collagen metabolism in patients with coronary disease. We first studied collagen metabolism in fibroblast cultures, then measured serum levels of type III procollagen aminoterminal peptide. Fibroblast collagen metabolism was investigated in 12 consecutive patients of less than 45 years of age presenting with coronary disease and coronary atherosclerosis was found to be preserved in patients with atherosclerosis, but less type III type I procollagen was synthesized (14.6 +/- 6.6% versus 22.3 +/- 4.3%). This abnormality, found in 83% of our coronary patients, seemed to be unrelated to risk factors or to the severity of atherosclerosis. Serum type III procollagen aminoterminal peptide was assayed comparatively in 36 patients with coronary atherosclerosis confirmed at coronary-ventriculography and in 35 patients free from coronary disease as defined by the W.H.O. criteria. Serum levels of this peptide were significantly higher in patients with coronary atherosclerosis (26.76 +/- 16 ng/ml) than in controls (10.43 +/- 3.18 ng/ml). 61% of coronary patients had a peptide level higher than the normal value (17 ng/ml). No correlation was found between this rise in type III procollagen aminoterminal peptide and the severity of coronary lesions or the importance of risk factors. Thus, collagen metabolism is altered in coronary patients, and this alteration can be detected by a peripheral marker. However, the use of this marker to diagnose the presence or evaluate the course of atherosclerosis requires clarification.
Adult↗
Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor.
A novel acidic glycoprotein, BM-40, with Mr = 40,000, was purified from the basement-membrane-producing mouse EHS tumor and characterized with regard to its unique chemical and antigenic properties. It was obtained from the tumor in a neutral salt-soluble form or as a component requiring extraction with 6M guanidine X HCl. This protein could also be identified in many other tissue extracts and cell and tissue cultures. The most intact form of BM-40 consists of a single polypeptide chain which undergoes limited proteolysis during extraction and purification. BM-40 exists in most tissues in stoichiometric amounts compared to other basement membrane proteins (laminin, nidogen) and is secreted by various teratocarcinoma and epithelial cells. It can be visualized by immunofluorescence in the extracellular matrix of the EHS tumor and Reichert's membrane. Other tissues which contain extractable BM-40 were negative in immunofluorescence.
Animals↗
Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen.
Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.
Animals↗