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.     

Bacterial proteins binding to the mammalian extracellular matrix

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best-characterized bacterial proteins active in these interactions are the mycobacterial fibronectin-binding proteins, the fibronectin- and the collagen-binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A-protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein-protein or on a protein-carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

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.     

Constitutive release of alpha4 type V collagen N-terminal domain by Schwann cells and binding to cell surface and extracellular matrix heparan sulfate proteoglycans

During peripheral nerve development, Schwann cells synthesize collagen type V molecules that contain alpha4(V) chains. This collagen subunit possesses an N-terminal domain (NTD) that contains a unique high affinity heparin binding site. The alpha4(V)-NTD is adhesive for Schwann cells and sensory neurons and is an excellent substrate for Schwann cell and axonal migration. Here we show that the alpha4(V)-NTD is released constitutively by Schwann cells both in culture and in vivo. In cultures of neonatal rat Schwann cells, alpha4(V)-NTD release is increased significantly by ascorbate treatment, which facilitates collagen post-translational modification and collagen trimer assembly. In peripheral nerve tissue, the alpha4(V)-NTD is localized to the region of the outer Schwann cell membrane and associated extracellular matrix. The released alpha4(V)-NTD binds to the cell surface and extracellular matrix heparan sulfate proteoglycans of Schwann cells. Pull-down assays and immunofluorescent staining showed that the major alpha4(V)-NTD-binding proteins are glypican-1 and perlecan. alpha4(V)-NTD binding occurs via a mechanism that requires the high affinity heparin binding site and that is blocked by soluble heparin, demonstrating that binding to proteoglycans is mediated by their heparan sulfate chains.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

Interactions of thrombospondin with endothelial cells: receptor- mediated binding and degradation

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.     

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

Endocardiac infectivity and binding to extracellular matrix proteins of oral Abiotrophia species

Microorganisms of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are members of the oral flora and often isolated from patients with endocarditis, but pathogenicity of oral Abiotrophia species has not been examined yet. In this study, 17 strains isolated from healthy human oral cavities and 7 reference strains (all derived from patients with endocarditis) of Abiotrophia spp. were tested for their abilities to cause infections in damaged heart tissues in catheterized rats and to adhere to extracellular matrix proteins in vitro. The reference strains of A. defectiva and A. adiacens showed high infectivities in the rats. Four oral isolates of these two species showed similarly high infectivities and three had moderate infectivities. Most of 10 oral strains of A. para-adiacens and A. elegans were found to be generally less infective. The highly infective A. adiacens strains showed markedly high fibronectin-binding capacity, suggesting a possible relationship between the fibronectin-binding capacity and damaged heart tissue infectivity of the Abiotrophia species. A. defectiva strains which were also highly infective had moderate levels of binding to fibronectin and other extracellular matrix proteins. Most of A. para-adiacens and A. elegans strains showed low or negligible binding capacities to any extracellular matrix proteins tested.     

Structural requirements for heparin/heparan sulfate binding to type V collagen

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.     

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.     

Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy

Binding of thrombospondin (TSP) to types I-V collagen was examined by direct binding assays using 125I-TSP and by visualization of rotary-shadowed intermolecular complexes in the electron microscope. The binding of TSP was highest to type V collagen in the absence of Ca, while lower but significant levels of binding were observed to all other collagen types in the presence or absence of Ca. Unlike intact TSP, the trimeric collagen-binding domain of TSP composed of 70-kD chains showed no Ca dependence in its binding to type V collagen. Further evidence for binding of TSP to types I and III collagen was obtained by competition studies in which these soluble collagens effectively inhibited binding of 125I-TSP to immobilized type V collagen. The binding of TSP to type V collagen was inhibited by heparin and fucoidin, both high-affinity ligands of TSP's heparin-binding domain. mAb A6.1, which binds to the 70-kD domain of TSP, is also the best of a panel of anti-TSP mAbs at inhibiting the TSP-collagen interaction. Electron microscopy of rotary-shadowed replicas of TSP-collagen complexes revealed that all five types of collagen examined had a binding site for TSP at one end of the pepsinized, triple helical molecule. The specificity of this site was tested by examining the ability of BSA to form a complex with the end of the pepsinized collagens. Rotary-shadowed replicas revealed a low frequency of apparent BSA-collagen complexes, and histograms of these data showed no evidence for the preferential association of BSA with the end of the collagen molecules. In addition to the specific end site, type V collagen had an internal binding site for TSP located about two-thirds of the distance along the length of the collagen molecule from the end site. The internal binding site for TSP on type V collagen is apparently the site responsible for the higher affinity binding of TSP to that protein observed in direct binding assays. The trimeric 70-kD collagen-binding domain of TSP bound to the same sites on the collagens as did intact TSP.     

Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.     

Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix

Summary Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [³H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic. This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan, by a grant from Osaka Eye Bank, Osaka, Japan, and by an intramural research fund of Kinki University. Part of this research was presented at the annual meeting of the Japanese Ophthalmological Society (May 1985) at Kyoto, Japan, and at the annual meeting of the Association for Research in Vision and Ophthalmology (May 1987) at Sarasota, FL.     

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECM components.     

Interaction of Helicobacter pylori with extracellular matrix proteins

Binding of 11 isolates of the gastroduodenal pathogen Helicobacter pylori from three geographical regions to extracellular matrix proteins was investigated. None of the strains bound soluble proteins, but a proportion (27%) bound immobilized laminin, fibronectin, and types I and V collagens. Microaerobic conditions were required to demonstrate reproducible binding. Contrary to previous reports, interstrain variation in the pattern of binding to various proteins was observed, possibly reflecting pathogenic or virulence differences between strains. Binding was strongest to laminin. Purified lipopolysaccharide completely inhibited the binding of H. pylori to laminin indicating that this bacterial surface molecule is involved in the adhesion process.     

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias.     

Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.