The C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.     

Structural correlation between collagen VI microfibrils and collagen VI banded aggregates

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.     

Reprint of "Structural correlation between collagen VI microfibrils and collagen VI banded aggregates" [J. Struct. Biol. 154 (2006) 312-326

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.     

Heterogeneity of Collagen VI Microfibrils: STRUCTURAL ANALYSIS OF NON-COLLAGENOUS REGIONS*

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.     

Fibrillin,a new 350-kD glycoprotein,is a component of extracellular microfibrils

A new connective tissue protein, which we call fibrillin, has been isolated from the medium of human fibroblast cell cultures. Electrophoresis of the disulfide bond-reduced protein gave a single band with an estimated molecular mass of 350,000 D. This 350-kD protein appeared to possess intrachain disulfide bonds. It could be stained with periodic acid-Schiff reagent, and after metabolic labeling, it contained [3H]glucosamine. It could not be labeled with [35S]sulfate. It was resistant to digestion by bacterial collagenase. Using mAbs specific for fibrillin, we demonstrated its widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule. Electron microscopic immunolocalization with colloidal gold conjugates specified its location to a class of extracellular structural elements described as microfibrils. These microfibrils possessed a characteristic appearance and averaged 10 nm in diameter. Microfibrils around the amorphous cores of the elastic fiber system as well as bundles of microfibrils without elastin cores were labeled equally well with antibody. Immunolocalization suggested that fibrillin is arrayed periodically along the individual microfibril and that individual microfibrils may be aligned within bundles. The periodicity of the epitope appeared to match the interstitial collagen band periodicity. In contrast, type VI collagen, which has been proposed as a possible microfibrillar component, was immunolocalized with a specific mAb to small diameter microfilaments that interweave among the large, banded collagen fibers; it was not associated with the system of microfibrils identified by the presence of fibrillin.     

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.     

Substrate-dependent morphology of supramolecular assemblies: fibrillin and type-VI collagen microfibrils

Substrate hydrophobicity/hydrophilicity has previously been shown to affect the morphology and biological function of isolated proteins. We have employed atomic force microscopy to investigate substrate dependent morphologies of two biochemically distinct native supramolecular assemblies: fibrillin and type-VI collagen microfibrils. These morphologically heterogeneous microfibrillar systems are found in many vertebrate tissues where they perform structural and cell-signaling roles. Fibrillin microfibrils adsorbed to a hydrophilic mica substrate adopted a diffuse morphology. Fibrillin microfibrils adsorbed to mica coated with poly-L-lysine or to borosilicate glass substrates had a more compact morphology and a directional asymmetry to the bead, which was not present on mica alone. Intermediate morphologies were observed along a substrate gradient. The classical double-beaded appearance of type-VI collagen microfibrils was evident on mica coated with poly-L-lysine and on glass. On hydrophilic mica, morphology was severely disrupted and there was a major conformational reorganization along the whole collagen microfibril repeat. These observations of substrate dependent conformation have important implications for the interpretation of data from in vitro protein interaction assays and cellular signaling studies. Furthermore, conformational changes may be induced by local charge environments in vivo, revealing or hiding binding sites.     

The effect of ascorbate on the synthesis of minor (non-interstitial) collagens by cultured bovine aortic smooth muscle cells

Ultrastructural and biochemical studies were carried out on bovine aortic smooth muscle cells cultured in the presence or absence of ascorbate. In its absence, electron microscopic examination of cultures revealed that the extracellular components consisted primarily of microfibrils. Morphologically identifiable collagen fibrils were only observed in the matrix upon ascorbate supplementation. Smooth muscle cells grown in ascorbate-free media synthesized large amounts of type VI collagen. The identity of the latter was confirmed by ion exchange chromatography, slab gel electrophoresis, and amino acid analysis. Addition of ascorbate resulted in a stimulation of type I collagen production, levels of the type III remained constant, and types V and VI were decreased. Since, in the absence of ascorbate, smooth muscle cells are known to synthesize predominantly elastin, the present data support the contention that the type VI collagen and the microfibrillar component of elastic tissue are either identical or similar.     

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.     

Molecular assembly, secretion, and matrix deposition of type VI collagen

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.     

Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils

Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.     

The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.     

Immunochemistry, genuine size and tissue localization of collagen VI

Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple-helical and globular structures. Antisera prepared against a constituent-chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross-react with other collagen types I-V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide-bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers. Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross-striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.     

An analysis by rotary shadowing of the structure of the mammalian vitreous humor and zonular apparatus

An electron microscopic analysis of human and bovine vitreous humor after rotary shadowing showed the presence of both collagen fibrils and an extensive loose network of hyaluronan molecules. No interaction between the collagen fibrils and the hyaluronan molecules was observed under the conditions used for rotary shadowing. Periodic "struts" were present on the surface of the collagen fibrils. These struts showed an organization the same as that previously observed for type IX collagen on the surface of collagen fibrils from chicken cartilage and vitreous. However, the knob of the noncollagenous NC4 domain of cartilage type IX collagen was not observed at the ends of the struts in a manner identical to that of chicken vitreous humor. Zonular fibrils were dissected out from bovine eyes and shown by rotary shadowing to contain a beaded fibril which is similar in morphology to the "elastin-associated" microfibrils of many connective tissues. Experiments in which the zonular fibrils were stretched and fixed prior to rotary shadowing showed that the distance between each bead is variable and can be accounted for by the bowing out of overlapping filaments which connect each bead.     

Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo.

Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide 'maps', were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].     

Kinked collagen VI tetramers and reduced microfibril formation as a result of Bethlem myopathy and introduced triple helical glycine mutations.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the dominantly inherited disorder, Bethlem myopathy. Glycine mutations that interrupt the Gly-X-Y repetitive amino acid sequence that forms the characteristic collagen triple helix have been defined in four families; however, the effects of these mutations on collagen VI biosynthesis, assembly, and structure have not been determined. In this study, we examined the consequences of Bethlem myopathy triple helical glycine mutations in the alpha1(VI) and alpha2(VI) chains, as well as engineered alpha3(VI) triple helical glycine mutations. Although the Bethlem myopathy and introduced mutations that are toward the N terminus of the triple helix did not measurably affect collagen VI intracellular monomer, dimer, or tetramer assembly, or secretion, the introduced mutation toward the C terminus of the helix severely impaired association of the mutant alpha3(VI) chain with alpha1(VI) and alpha2(VI). Association of the three chains was not completely prevented, however; and some non-disulfide bonded tetramers were secreted. Examination of the secreted Bethlem myopathy and engineered mutant collagen VI by negative staining electron microscopy revealed the striking finding that in all the cell lines a significant proportion of the tetramers contained a kink in the supercoiled triple helical region. Collagen VI tetramers from all of the mutant cell lines also showed a reduced ability to form microfibrils. These results provide the first evidence of the biosynthetic consequences of collagen VI triple helical glycine mutations and indicate that Bethlem myopathy results not only from the synthesis of reduced amounts of structurally normal protein but also from the presence of mutant collagen VI in the extracellular matrix.     

The morphology of adsorbed extracellular matrix assemblies is critically dependent on solution calcium concentration.

The adsorption of proteins to surfaces may alter their biological properties. Understanding and controlling these interactions is important in ultrastructural, biochemical and cellular studies. We have previously demonstrated that both the morphology and biological function of extracellular matrix assemblies such as fibrillin and type VI collagen microfibrils are influenced by surface chemistry. In this study we have employed atomic force microscopy to determine if the morphology of extracellular matrix microfibrils is influenced by solution chemistry. Microfibrils were adsorbed to mica or poly-L-lysine modified mica (mica-PLL) in the presence of 31 microM-1000 microM Ca(2+). Although both microfibrillar species adsorbed to mica and mica-PLL at all calcium concentrations, maximal adsorption was observed on mica at 125-250 microM. On mica surfaces fibrillin microfibril morphology varied continuously with calcium concentration from laterally diffuse assemblies at high concentrations to compact assemblies at low concentrations. In contrast, distinct type VI collagen microfibril morphologies were observed at high, intermediate and low calcium concentrations. Similar calcium dependent microfibrillar morphologies were evident on mica-PLL. Therefore physiologically relevant concentrations of solution calcium, independent of surface charge, profoundly influenced both the adsorbed amount and morphology of native extracellular assemblies. These studies highlight the importance not only of surface chemistry but also of solute composition and concentration in influencing the morphology and hence biological function of adsorbed proteins.     

Dilated capillaries, disorganized collagen fibers and differential gene expression in periodontal ligaments of hypomorphic fibrillin-1 mice

The periodontal ligaments (PDLs) are soft connective tissue between the cementum covering the tooth root surface and alveolar bone. PDLs are composed of collagen and elastic system fibers, blood vessels, nerves, and various types of cells. Elastic system fibers are generally formed by elastin and microfibrils, but PDLs are mainly composed of the latter. Compared with the well-known function of collagen fibers to support teeth, little is known about the role of elastic system fibers in PDLs. To clarify their role, we examined PDLs of mice underexpressing fibrillin-1 (mgR mice), which is one of the major microfibrillar proteins. The PDLs of homozygous mgR mice showed one-quarter of the elastic system fibers of wild-type (WT) mice. A close association between the elastic system fibers and the capillaries was noted in WT, homozygous and heterozygous mgR mice. Interestingly, capillaries in PDLs of homozygous mice were dilated or enlarged compared with those of WT mice. A comparable level of type I collagen, which is the major collagen in PDLs, was expressed in PDL-cells of mice with three genotypes. However, multi-oriented collagen fiber bundles with a thinner appearance were noted in homozygous mice, whereas well-organized collagen fiber bundles were seen in WT mice. Moreover, there was a marked decrease in periostin expression, which is known to regulate the fibrillogenesis and crosslinking of collagen. These observations suggest that the microfibrillar protein, fibrillin-1, is indispensable for normal tissue architecture and gene expression of PDLs.     

Interaction of DNA with connective tissue matrix proteins reveals preferential binding to type V collagen

The interaction of DNA with type I to VI collagens and laminin was studied in vitro in systems in which the connective tissue components were immobilized, as well as when in solution. In studies on immobilized components, significant binding of DNA was observed only for type V collagen, and the binding of radiolabeled DNA to this component could be effectively inhibited in a concentration-dependent manner by the addition of unlabeled DNA. Similar results were observed in solution assays in which it was observed that DNA binding to type V collagen was dependent on the native triple-helical conformation of the collagen. The preferential binding of DNA to native type V collagen may be due to the relative basicity of type V collagen chains, as well as the unique spatial arrangement of amino acid side chains in the native molecules. The data are of potential clinical relevance in that binding of DNA to type V collagen may represent at least one component of the mechanism whereby DNA and its immune complexes are deposited in connective tissues in certain pathologic conditions.