The role of collagen-derived proteolytic fragments in angiogenesis.

Basement membrane molecules and fragments derived from them are regulators of biological activities such as cell growth, differentiation and migration. This review describes proteolytically derived fragments from the non-collagenous (NC1) domain at the C-terminus of the basement membrane collagens type IV, XV and XVIII, which have been implicated as regulators of angiogenesis. Endostatin is an endogenous collagen XVIII/NC1 derivative, inhibiting endothelial cell proliferation and migration in vitro and tumor-growth in vivo. A homologous NC1 domain fragment of type XV collagen has anti-angiogenic activity as well. Furthermore, NC1 domain fragments of the most abundant basement membrane collagen, type IV collagen, have been shown to inhibit induced vessel growth.     

The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.

Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.     

HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development

A large family of molecular chaperones can be divided into two major groups: general chaperone and substrate-specific chaperone. HSP47 is a collagen-specific molecular chaperone residing in the endoplasmic reticulum (ER). Recent studies revealed that HSP47 is essential molecular chaperone for mouse development and is essential for collagen molecular maturation in the ER. In the absence of HSP47, collagen microfibril formation and basement membrane formation are impaired in mouse embryos because the failure in the molecular maturation of types I and IV collagens, respectively. The tissue-specific expression of HSP47 is always correlated with that of various types of collagens and closely related with the collagen-related diseases including fibrosis in various organs. The importance of HSP47 in the therapeutic strategy for fibrotic diseases as well as for a marker of collagen-related autoimmune diseases will also be discussed.     

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.     

The role of the main noncollagenous domain (NC1) in type IV collagen self-assembly

Type IV collagen incubated at elevated temperatures in physiologic buffers self-associates (a) via its carboxy-terminal (NC1) domain, (b) via its amino-terminal (7S) domain, and (c) laterally; and it forms a network. When examined with the technique of rotary shadowing, isolated domain NC1 was found to bind along the length of type IV collagen to four distinct sites located at intervals of approximately 100 nm each. The same 100-nm distance was observed in domain NC1 of intact type IV collagen bound along the length of the collagen molecules during initial steps of network formation and in complete networks. The presence of anti-NC1 Fab fragments in type IV collagen solutions inhibited lateral association and network formation in rotary shadow images. During the process of self-association type IV collagen develops turbidity; addition of isolated domain NC1 inhibited the development of turbidity in a concentration-dependent manner. These findings indicate that domain NC1 of type IV collagen plays an important role in the process of self-association and suggest that alterations in the structure of NC1 may be partially responsible for impaired functions of basement membranes in certain pathological conditions.     

Demosponge and sea anemone fibrillar collagen diversity reveals the early emergence of A/C clades and the maintenance of the modular structure of type V/XI collagens from sponge to human

Collagens are often considered a metazoan hallmark, with the fibril-forming fibrillar collagens present from sponges to human. From evolutionary studies, three fibrillar collagen clades (named A, B, and C) have been defined and shown to be present in mammals, whereas the emergence of the A and B clades predates the protostome/deuterostome split. Moreover, several C clade fibrillar collagen chains are present in some invertebrate deuterostome genomes but not in protostomes whose genomes have been sequenced. The newly sequenced genomes of the choanoflagellate Monosiga brevicollis, the demosponge Amphimedon queenslandica, and the cnidarians Hydra magnipapillata (Hydra) and Nematostella vectensis (sea anemone) allow us to have a better understanding of the origin and evolution of fibrillar collagens. Analysis of these genomes suggests that an ancestral fibrillar collagen gene arose at the dawn of the Metazoa, before the divergence of sponge and eumetazoan lineages. The duplication events leading to the formation of the three fibrillar collagen clades (A, B, and C) occurred before the eumetazoan radiation. Interestingly, only the B clade fibrillar collagens preserved their characteristic modular structure from sponge to human. This observation is compatible with the suggested primordial function of type V/XI fibrillar collagens in the initiation of the formation of the collagen fibrils.     

Identification and analysis of collagen alpha 1(XXI), a novel member of the FACIT collagen family.

The FACIT collagens bind to the surface of collagen fibrils linking them with other matrix molecules. Bioinformatics analysis of cDNA clone DKFZp564B052 showed that it resembled the FACIT collagens and was therefore designated collagen alpha 1(XXI). Phylogenetic analyses of the N-terminal NC3 domains of alpha 1(XXI) and other FACIT collagens showed that (i) alpha 1(XXI) clustered with the FACIT collagens; (ii) collagen alpha 1(XXI) arose before the divergence of alpha 1(XII), alpha 1(XIV) and alpha 1(XX); (iii) collagen alpha 1(XIV) derived from the C-terminal region of the NC3 domain of a collagen alpha 1(XII)-like molecule; and (iv) collagen alpha 1(XX) derived from a collagen alpha 1(XIV)-like molecule. This study provides a framework for the evolution of the FACIT collagens which will be of value in linking NC3 domains with their functions.     

Collagen binding by the mannose receptor mediated through the fibronectin type II domain

The macrophage mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of an N-terminal cysteine-rich domain related to the R-type carbohydrate-recognition domain of ricin, a fibronectin type II domain and eight to ten domains related to C-type carbohydrate-recognition domains. The mannose receptor acts as a molecular scavenger, clearing harmful glycoconjugates or micro-organisms through recognition of their defining carbohydrate structures. Cell-adhesion assays, as well as collagen-binding assays, have now been used to show that the mannose receptor can also bind collagen and that the fibronectin type II domain mediates this activity. Neither of the two types of sugar-binding domain in the receptor is involved in collagen binding. Fibroblasts expressing the mannose receptor adhere to type I, type III and type IV collagens, but not to type V collagen, and the adherence is inhibited by isolated mannose receptor fibronectin type II domain. The fibronectin type II domain shows the same specificity for collagen as the whole receptor, binding to type I, type III and type IV collagens. This is the first activity assigned to the fibronectin type II domain of the mannose receptor. The results suggest additional roles for this multifunctional receptor in mediating collagen clearance or cell-matrix adhesion.     

The C-terminus type I collagen is a major binding site for heparin

The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15–1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TC^B was shown to have greater binding affinity for heparin than the N-terminal TC^A fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind.     

Heparin type IV collagen interactions: equilibrium binding and inhibition of type IV collagen self-assembly

Interactions between type IV collagen and heparin were examined under equilibrium conditions with rotary shadowing, solid-phase binding assays, and affinity chromatography. With the technique of rotary shadowing and electron microscopy, heparin appeared as thin, short strands and bound to the following three sites: the NC1 domain, and in the helix, at 100 and 300 nm from the NC1 domain. By solid-phase binding assays the binding of [3H]heparin in solution to type IV collagen immobilized on a solid surface was found to be specific, since it was saturable and could be displaced by an excess of unlabeled heparin. Scatchard analysis indicated three classes of binding sites for heparin-type IV collagen interactions with dissociation constants of 3, 30, and 100 nM, respectively. Furthermore, by the solid-phase binding assays, the binding of tritiated heparin could be competed almost to the same extent by unlabeled heparin and chondroitin sulfate side chains. This finding indicates that chondroitin sulfate should also bind to type IV collagen. By affinity chromatography, [3H]heparin bound to a type IV collagen affinity column and was eluted with a linear salt gradient, with a profile exhibiting three distinct peaks at 0.18, 0.22, and 0.24 M KCl, respectively. This suggested that heparin-type IV collagen binding was of an electrostatic nature. Finally, the effect of the binding of heparin to type IV collagen on the process of self-assembly of this basement membrane glycoprotein was studied by turbidimetry and rotary shadowing. In turbidity experiments, the presence of heparin, even in small concentrations, drastically reduced maximal aggregation of type IV collagen which was prewarmed to 37 degrees C. By using the morphological approach of rotary shadowing, lateral associations and network formation by prewarmed type IV collagen were inhibited in the presence of heparin. Thus, the binding of heparin resulted in hindrance of assembly of type IV collagen, a process previously described for interactions between various glycosaminoglycans and interstitial collagens. Such regulation may influence the assembly of basement membranes and possibly modify functions. Furthermore, qualitative and quantitative changes of proteoglycans which occur in certain pathological conditions, such as diabetes mellitus, may alter molecular assembly and possibly permeability functions of several basement membranes.     

Is there an evolutionary relationship between WARP (von Willebrand factor A-domain-related protein) and the FACIT and FACIT-like collagens?

We suggest that there is an evolutionary relationship between von Willebrand factor A-domain-related protein (WARP), and the fibril-associated collagen with interrupted triple helix (FACIT) and FACIT-like subfamilies of collagens. Data from a comparison of amino acid sequences, domain organisation and chromosomal location are consistent with the hypothesis that WARP and these collagens share a common collagen ancestor. In support of this is the observation that the WARP 3' coding region is GC-rich suggesting that this may represent the remnant of a triple helix protein domain which WARP has 'lost' during evolution.     

Amino acid sequence of the non-collagenous globular domain (NC1) of the alpha 1(IV) chain of basement membrane collagen as derived from complementary DNA

NC1, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NC1 domain of the alpha 1(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NC1 domain to be deduced as well as the C-terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.     

Tissue-specific expression of type XIV collagen--a member of the FACIT class of collagens.

The collagens represent a highly diverse superfamily of extracellular matrix proteins that can be divided into several distinct families. One of the families, called FACIT (fibril-associated collagens with interrupted triple-helices) family, contains molecules that appear to be associated with cross-striated fibrils composed of members of the fibrillar collagen family. We have determined a portion of the primary structure of a recently discovered member of the FACIT family, chicken alpha 1(XIV) collagen, based on cloning and sequencing cDNAs. A synthetic oligopeptide from within the carboxy-terminal non-triple-helical domain of the alpha 1(XIV) chain has been used for generating specific polyclonal antibodies. The antiserum, PS1, recognizes a 220 kDa polypeptide in immunoblots of extracts of chicken skin, tendons, and cartilage. Sequencing of a tryptic peptide generated from purified, immunoreactive material, gives a sequence identical to that derived from cDNA sequencing, providing strong support for the type XIV-specificity of PS1. We have examined the expression of type XIV collagen in developing chick embryos by immunostaining of sections from 12-day-old embryos with PS1 and by Northern blot analysis of RNA from several tissues from both 12- and 17-day-old embryos. The results show that type XIV collagen is prevalent within relatively dense connective tissues such as dermis, tendons, perichondrium, perimysium, the stroma of lungs and liver, and blood vessels.     

New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo

Collagen type IV is a major component of the basal lamina of blood vessels. Six genetically distinct collagen type IV chains have been identified and are distributed in a tissue-specific manner. Here we define a novel function for soluble non-collagenous (NC1) domains of the alpha2(IV), alpha3(IV), and alpha6(IV) chains of human collagen type IV in the regulation of angiogenesis and tumor growth. These NC1 domains were shown to regulate endothelial cell adhesion and migration by distinct alpha(v) and beta(1) integrin-dependent mechanisms. Systemic administration of recombinant alpha2(IV), alpha3(IV), and alpha6(IV) NC1 domains potently inhibit angiogenesis and tumor growth, whereas alpha1(IV), alpha4(IV), and alpha5(IV) showed little if any effect. These findings suggest that specific NC1 domains of collagen type IV may represent an important new class of angiogenesis inhibitors.     

Type XIII collagen and some other transmembrane collagens contain two separate coiled-coil motifs,which may function as independent oligomerization domains

Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1-3) separated by short non-collagenous domains (NC1-4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an alpha-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen alpha chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule.     

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.     

Epithelia, an evolutionary novelty of metazoans

At the point in animal evolution when cells began to adhere to each other they presumably initially functioned as colonies. The formation of an epithelium that enclosed and controlled an internal milieu would have been the first event to distinguish an individual animal from a colony. To better understand when the first epithelium arose and what its characteristics were, we evaluate the morphological, functional, and molecular characters of epithelia in sponges, considered here the extant representatives of the first metazoans. In particular, we show new claudin-like sequences from sponges align most closely with sequences from Drosophila that have a barrier function in septate junctions. We also show that type IV collagen, the main component of the basement membrane (BM), is present in calcareous sponges, and we confirm the presence of type IV-like collagen (spongin short chain collagen) in other sponges. Though in sponges as in other metazoans the epithelium has grades of specialization with varying complexity of junctions and the BM, the main character of a functional epithelium, the ability to seal and control the ionic composition of the internal milieu, is a property of even the simplest sponge epithelium, and therefore the first metazoans likely also had epithelia with these characteristics, which we consider a "true" epithelium.     

Trimerization and triple helix stabilization of the collagen XIX NC2 domain

The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization.     

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the α1, α2, and α3 chains of NC2, a stable heterotrimer with a disulfide bridge between α1 and α3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.     

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.