Molecular architecture of basement membranes

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.     

Avian type VI collagen : Monoclonal antibody production and immunohistochemical identification as a major connective tissue component of cornea and skeletal muscle

Two monoclonal antibodies have been characterized as being against avian type VI collagen. By competition ELISA, the antibodies bound to the native type VI collagen molecule but not to its separated chains or to any of the other native collagen types tested. By rotary shadowing analysis of complexes of antibody-type VI collagen monomers, one of the antibodies (VI-EC6) has been shown to bind to a site in the triple helical domain of the molecule. The site at which this antibody binds to the dimeric form of type VI collagen is consistent with the previously proposed model for a supramolecular organization of the molecule (Furthmayr et al., Biochem j 211 (1983) 303) in which the monomers are arranged in an antiparallel, slightly staggered overlap. Immunofluorescence analyses of sections of chicken eyes and skeletal muscle demonstrate that type VI collagen is a major component of most stromal matrices.     

Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.     

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.     

Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix

Steroid receptors have been reported to bind to the nuclear matrix. The nuclear matrix is operationally defined as the residual nuclear structure that remains after extraction of most of the chromatin and all soluble and loosely bound componnets. To obtain insight in the molecular mechanism of the interaction of steroid receptors with the nuclear matrix, we studied the binding of several deletion mutants of the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) to the nuclear matrix. Receptor binding was tested for two different nuclear matrix preparations: complete matrices, in which most matrix proteins are retained during the isolation procedure, and depleted matrices, which consist of only a subset of these proteins. The results show that the C-terminal domain of the hAR binds tightly to both depleted and complete matrices. In addition, at least one other domain of the hAR binds to complete matrices but not to depleted matrices. In contrast to the hAR, the hGR binds only to complete matrices. For this interaction both the DNA-binding domain and the C-terminal domain of the hGR are required, whereas the N-terminal domain is not. We conclude that specific protein domains of the hAR and the hGR are involved in binding to the nuclear matrix. In addition, our results indicate that the hAR and the hGR are attached to the nuclear matrix through different molecular interactions.     

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens

A heparan sulfate-rich proteoglycan is on the surface of NMuMG mouse mammary epithelial cells apparently intercalated into their plasma membranes. Mild treatment of the cells with trypsin releases the GAG-bearing region (ectodomain) of this molecule as a discrete proteoglycan which is readily purified. At physiological pH and ionic strength, the ectodomain binds collagen types I, III, and V but not types II, IV, or denatured type I. The proteoglycan binds to a single class of high affinity saturable sites on type I collagen fibrils, sites which are selective for heparin-like glycosaminoglycans. The binding of NMuMG cells to type I collagen duplicates that of their cell surface proteoglycan; cells bind to native but not denatured collagen, and binding is inhibited by heparin but not by other glycosaminoglycans. These binding properties suggest that cell surface heparan sulfate proteoglycans could act as receptors for interstitial collagens and mediate changes in cell behavior induced by collagenous matrices.     

Evidence for distinct sulfhydryl groups associated with steroid- and DNA-binding domains of rat thymus glucocorticoid receptors

We have found that nonactivated and activated forms of the rat thymus glucocorticoid-receptor complex (GRC) will react with reactive sulfhydryl matrices to form covalently immobilized complexes that can subsequently be eluted with reducing agents. The interaction of GRCs with these matrices depends on the nature of both the immobilized sulfhydryl group and the type of leaving group attached. One matrix, agarose CL-4B-diaminoethyl-succinyl-thioethylamine-2-thiopyridyl+ ++ (DSTT), binds total receptor-bound steroid. A second matrix, agarose CL-4B-diaminoethyl-succinyl-cysteinyl-2-thiobenzoic acid (DSCT), binds activated but not nonactivated complexes. The reaction of activated complexes with the DSCT matrix is apparently through a sulfhydryl group located near the DNA binding domain, as soluble DNA interferes with the reaction. This sulfhydryl group(s) appears to be located in a portion of the GRC that is resistant to degradation, since proteolytic digestion of activated GRC to a point where DNA binding is lost results in only a moderate decrease in binding with the DSCT matrix. Purified receptor, covalently labeled with [3H]dexamethasone to the sulfhydryl associated with the steroid binding domain, was able to bind to DSCT matrix, providing evidence for distinct sulfhydryl groups associated with the steroid and DNA binding domains.     

Direct in vitro and in vivo evidence for interaction between Hsp47 protein and collagen triple helix

Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.     

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.     

Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix

The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane- intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin- resistant molecule that binds cells to the sequence RGD and a trypsin- labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.     

Identification of novel and distinct binding sites within tenascin-C for soluble and fibrillar fibronectin

Interactions between fibronectin and tenascin-C within the extracellular matrix provide specific environmental cues that dictate tissue structure and cell function. The major binding site for fibronectin lies within the fibronectin type III-like repeats (TNfn) of tenascin-C. Here, we systematically screened TNfn domains for their ability to bind to both soluble and fibrillar fibronectin. All TNfn domains containing the TNfn3 module interact with soluble fibronectin. However, TNfn domains bind differentially to fibrillar fibronectin. This distinct binding pattern is dictated by the fibrillar conformation of FN. TNfn1-3, but not TNfn3-5, binds to immature fibronectin fibrils, and additional TNfn domains are required for binding to mature fibrils. Multiple binding sites for distinct regions of fibronectin exist within tenascin-C. TNfn domains comprise a binding site for the N-terminal 70-kDa domain of fibronectin that is freely available and a binding site for the central binding domain of fibronectin that is cryptic in full-length tenascin-C. The 70-kDa and central binding domain regions are key for fibronectin matrix assembly; accordingly, binding of several TNfn domains to these regions inhibits fibronectin fibrillogenesis. These data highlight the complexity of protein-protein binding, the importance of protein conformation on these interactions, and the implications for the physiological assembly of complex three-dimensional matrices.     

The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor

PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, we found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bound PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibited the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I did not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, did not bind perlecan. Confocal immunohistochemistry showed that PRELP outlines basement membranes with a location adjacent to perlecan. We also found that PRELP binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualized a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, we propose a function of PRELP as a molecule anchoring basement membranes to the underlying connective tissue.     

Constitutive Dimerization of Glycoprotein VI (GPVI) in Resting Platelets Is Essential for Binding to Collagen and Activation in Flowing Blood

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ～29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ～39 and ～44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.     

Steady-state nuclear localization of exportin-t involves RanGTP binding and two distinct nuclear pore complex interaction domains

Vertebrate tRNA export receptor exportin-t (Xpo-t) binds to RanGTP and mature tRNAs cooperatively to form a nuclear export complex. Xpo-t shuttles bidirectionally through nuclear pore complexes (NPCs) but is mainly nuclear at steady state. The steady-state distribution of Xpo-t is shown to depend on its interaction with RanGTP. Two distinct Xpo-t NPC interaction domains that bind differentially to peripherally localized nucleoporins in vitro are identified. The N terminus binds to both Nup153 and RanBP2/Nup358 in a RanGTP-dependent manner, while the C terminus binds to CAN/Nup214 independently of Ran. We propose that these interactions increase the concentration of tRNA export complexes and of empty Xpo-t in the vicinity of NPCs and thus increase the efficiency of the Xpo-t transport cycle.     

Desmin-binding specificities of two desmin CNBr fragments that correspond to the headpiece domain and Helix 1B

D88 and D109, two cyanogen bromide fragments of desmin which essentially correspond to the amino terminal headpiece domain and Helix 1B, respectively, bind to intact desmin with different topological specificities. D88, the headpiece domain fragment, binds only to the headpiece of intact desmin. In contrast, D109, which encompasses Helix 1B and most of the linker L10 binds to desmin even when its headpiece is removed. Additionally, these fragments only bind desmin if they are present during filament assembly; they do not bind pre-assembled desmin IF or tetramers. These observations suggest that, while alpha-helical coiled-coil interaction between rod domains provides the major driving force behind IF protein dimer formation, homophilic binding of head domains of these proteins may provide an additional stabilizing force and/or specify axial registration in certain IF proteins.     

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.     

Molecular basis of organization of collagen fibrils

The collagen fibrils are formed by self-assembly of individual collagen molecules, but the mechanism that drives their orderly packing during fibril formation is not clearly defined. To identify structural determinants critical for the D-periodic alignment of collagen molecules we employed three sets of genetically engineered collagen II variants: (i) a set in which domains corresponding to the specific D periods have been purposely deleted, (ii) a set of collagen variants consisting of tandem repeats of a specific D period, and (iii) a set lacking definite fragments of the D4 period. All collagen variants were analyzed for their ability to assemble into D-periodic fibrils. Even though all genetically engineered collagen variants differ significantly from the wild-type collagen II, most of them were able to form filamentous structures. The D-periodic banding pattern, an indication of the staggered arrangement of collagen monomers, however, occurred only when the D1, D4, and D0.4 domains of interacting collagen monomers could potentially cluster together to form a triad through telopeptide-mediated binding. Our results identify a critical step in the formation of collagenous matrices and provide experimental evidence for the active involvement of the N-terminal and C-terminal regions of fibrillar collagens in this process.     

Crystal structure and function of human nucleoplasmin (npm2): a histone chaperone in oocytes and embryos

Human Npm2 is an ortholog of Xenopus nucleoplasmin (Np), a chaperone that binds histones. We have determined the crystal structure of a truncated Npm2-core at 1.9 ? resolution and show that the N-terminal domains of Npm2 and Np form similar pentamers. This allowed us to model an Npm2 decamer which may be formed by hydrogen bonds between quasi-conserved residues in the interface between two pentamers. Interestingly, the Npm2 pentamer lacks a prototypical A1-acidic tract in each of its subunits. This feature may be responsible for the inability of Npm2-core to bind histones. However, Npm2 with a large acidic tract in its C-terminal tail (Npm2-A2) is able to bind histones and form large complexes. Fluorescence resonance energy transfer experiments and biochemical analysis of loop mutations support the premise that nucleoplasmins form decamers when they bind H2A-H2B dimers and H3-H4 tetramers simultaneously. In the absence of histone tetramers, these chaperones bind H2A-H2B dimers with a single pentamer forming the central hub. When taken together, our data provide insights into the mechanism of histone binding by nucleoplasmins.     

Liquid crystallinity in condensed type I collagen solutions. A clue to the packing of collagen in extracellular matrices.

We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.