Laminin modulates morphogenic properties of the collagen XVIII endostatin domain

We have shown previously that the oligomeric endostatin domain of collagen XVIII (NC1) functioned as a motility-inducing factor regulating the extracellular matrix-dependent morphogenesis of endothelial cells. This motogenic activity gave rise to structures resembling filipodia and lamellipodia and was dependent on Rac, Cdc42, and mitogen-activated protein kinase. Here, we demonstrate that these properties of endostatin are primarily mediated by laminin in the basement membrane and heparan sulfates on the cell surface. The sites of interaction between laminin and oligomeric endostain include the N-terminal regions of all three laminin chains (amino acids 204-1243 of the alpha chain, 932-1161 of the beta chain, and 150-965 of the gamma chain). A monoclonal antibody that blocks the interactions between endostatin and laminin was utilized to inhibit the motogenic activity of endostatin. In parallel, we have engineered selective point mutations and produced recombinant forms that lack binding to heparan sulfates on the cell surface. Our data are consistent with a model of endostatin with two binding sites: one mainly to laminin in the basement membrane and the other to heparan sulfates on the cell surface. The two binding domains on endostatin appear to be separate with the possibility of some overlap between the two sites.     

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.     

Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.     

Sequence and embryonic expression of collagen XVIII NC1 domain (endostatin) in the zebrafish

Endostatin, located in the NC1 domain of the collagen XVIII, is believed to inhibit the migration and proliferation of endothelial cells (Fed. Am. Soc. Exp. Biol. J. 15 (2001) 1044) and to play a role in axon guidance in Caenorhabditis elegans (J. Cell Biol. 152 (2001) 1219). Zebrafish is an attractive vertebrate model to determine the role of endostatin and the entire molecule of collagen XVIII during vertebrate development. Therefore, we have investigated the expression pattern of COL18A1 in zebrafish embryos from the segmentation to the hatching period stages.     

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.     

The ultrastructural composition of basement membranes in vivo

The ultrastructure of basement membranes has a homogeneous appearance. The enormous cell biological importance of basement membranes and their components for cell proliferation, migration and differentiation implies that their composition is more complex than their structure suggests. To elucidate the molecular composition of basement membranes in vivo, we optimised immunogold histochemistry to allow the determination of the molecular arrangement of matrix molecules. Basically, we apply a mild fixation and embed the tissues in the hydrophilic LR-Gold. This preserves the basement membrane with a quality similar to freeze substitution. The application of two antibodies directed toward the C- and N-terminal ends of a molecule and coupled to gold particles of different sizes allows determination of the orientation of a molecule within the basement membrane. We were able to demonstrate that the molecular orientation of the laminin-1 molecule changes in the basement membrane according to cell biological needs. We also showed that ultrastructurally identical basement membranes like the ones of the proximal and distal tubules of the kidney have a differing molecular arrangement. Integrin alpha7 influences the molecular composition of the basement membranes at the myotendinous junction. With the help of double labelling at the ultrastructural level we could show that nidogen-1 is co-localised with laminin-1 and only found in fully developed, mature basement membranes. In general, laminin-1, nidogen-1 and collagen type IV are localised over the entire width of basement membranes, with laminin-1 and nidogen-1 co-localised, in accordance with the current basement membrane models. Incidentally, our investigations warn us, that not every matrix protein found at the light microscopic level as a linear staining pattern underneath an epithelium (basement membrane zone) is a real basement membrane component when investigated at the ultrastructural level. Instead, one and the same molecule, e.g. endostatin, can be a basement membrane component in one organ and a matrix molecule in another.     

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans

Background

Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism.

Methods and Findings

We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia.

Conclusions

This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.     

The vWFA2 domain of type VII collagen is responsible for collagen binding

Type VII collagen (Col7) is the major component of anchoring fibrils and very important for skin integrity. This is emphasized by the Col7 related skin blistering diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita. Structural data that provides insights into the interaction network of Col7 and thus providing a basis for a better understanding of the pathogenesis of the diseases is missing.We proved that the von-Willebrand-factor A like domain 2 (vWFA2) of Col7 is responsible for type I collagen binding. The interaction has a K_D value of 90 μM as determined by SPR and is enthalpy driven as derived from the van’t Hoff equation. Furthermore, a hitherto unknown interaction of this domain with type IV collagen was identified. The interaction of vWFA2 with type I collagen is sensitive to the presence of magnesium ions, however, vWFA2 does not contain a magnesium binding site thus magnesium must bind to type I collagen.A lysine residue has been identified to be crucial for type I collagen binding. This allowed localization of the binding site. Mutational analysis suggests different interaction mechanisms in different species and that these interactions might be of covalent nature.     

A network model for the organization of type IV collagen molecules in basement membranes

Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.     

Interaction of skeletal muscle cells with collagen type IV is mediated by perlecan associated with the cell surface

We have previously shown that the expression of perlecan, a heparan sulfate proteoglycan localized on the myoblast surface, is down-regulated during terminal differentiation of skeletal muscle myoblasts (Larraín et al. [1997] Exp. Cell Res. 234:405-412). In this study, we have evaluated the biochemical characteristics of perlecan, its association with the myoblast surface, and its involvement in C(2)C(12) myoblast adhesion to different substrates. Perlecan associated with myoblasts was solubilized by Triton X-100, whereas heparin, high salt, and RGD peptides were unable to solubilize perlecan. Pre-incubation of myoblasts with [(35)S]-Na(2)SO(4), followed by solubilization with Triton X-100 and immunoprecipitation with antibodies against murine perlecan, demonstrated that this proteoglycan present on the cell surface has a heterogeneous size profile with a K(av) value of 0.45, determined by Sepharose CL-4B chromatography. Myoblasts were found to adhere with decreasing affinities to collagen type IV, type I, laminin, fibronectin, perlecan, and matrigel. We found that cell adhesion to collagen type IV was inhibited by blocking this substrate with exogenous perlecan prior to cell plating, whereas no effect was observed for laminin. Furthermore, adhesion of myoblasts to collagen type IV was inhibited by the perlecan core protein obtained by treatment of perlecan with heparitinase, as well as by pre-incubation of the cells with antibodies against murine perlecan. These data support the idea that skeletal muscle cells interact with collagen type IV through the perlecan core protein present on the surface of undifferentiated myoblasts.     

Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.     

Distinct antitumor properties of a type IV collagen domain derived from basement membrane

Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.     

Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes

Type XV and type XVIII collagens are classified as part of multiplexin collagen superfamily and their C-terminal parts, endostatin and restin, respectively, have been shown to be anti-angiogenic in vivo and in vitro. The alpha1(XV) and alpha1(XVIII) collagen chains are reported to be localized mainly in the basement membrane zone, but their distributions in blood vessels and nonvascular tissues have yet to be thoroughly clarified. In the present study, we raised monoclonal antibodies against synthetic peptides of human alpha1(XV) and alpha1(XVIII) chains and used them for extensive investigation of the distribution of these chains. We came to the conclusion that nonvascular BMs contain mainly one of two types: subepithelial basement membranes that contained type XVIII in general, or skeletal and cardiac muscles that harbored mainly type XV. But basement membranes surrounding smooth muscle cells in vascular tissues contained one or both of them, depending on their locations. Interestingly, continuous capillaries contained both type XV and type XVIII collagens in their basement membranes; however, fenestrated or specialized capillaries such as glomeruli, liver sinusoids, lung alveoli, and splenic sinusoids expressed only type XVIII in their basement membranes, lacking type XV. This observation could imply that different functions of basement membranes in various tissues and organs use different mechanisms for the endogenous control of angiogenesis.     

Coating electrospun collagen and gelatin fibers with perlecan domain I for increased growth factor binding

Electrospun natural polymer membranes were fabricated from collagen or gelatin coated with a bioactive recombinant fragment of perlecan, a natural heparan sulfate proteoglycan. The electrospinning process allowed the facile processing of a three-dimensional, porous fibril (2-6 microm in diameter) matrix suitable for tissue engineering. Laser scanning confocal microscopy revealed that osteoblast-like MG63 cells infiltrated the depth of the electrospun membrane evenly without visible apoptosis. Tissue engineering scaffolds ideally mimic the extracellular matrix; therefore, the electrospun membrane must contain both structural and functional matrix features. Fibers were coated, after processing, with perlecan domain I (PlnDI) to improve binding of basic fibroblast growth factor (FGF-2), which binds to native heparan sulfate chains on PlnDI. PlnDI-coated electrospun collagen fibers were ten times more effective than heparin-BSA collagen fibers at binding FGF-2. Because FGF-2 modulates cell growth, differentiation, migration and survival, the ability to effectively bind FGF-2 to an electrospun matrix is a key improvement in creating a successful tissue engineering scaffold.     

The epidermal basement membrane is a composite of separate laminin- or collagen IV-containing networks connected by aggregated perlecan, but not by nidogens

The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.     

Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo

Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain.     

Drosophila type XV/XVIII collagen, Mp, is involved in Wingless distribution

Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo.     

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV)

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.