Evolution of metazoan collagens.
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Animals↗
Biomedical subjects
R Garrone
Publications and source records attributed to R Garrone.
Microfibrillar composition of umbilical cord matrix: characterization of fibrillin, collagen VI and intact collagen V.
Ultrastructural studies made on human umbilical cord revealed that the striated collagen fibrils of the Wharton's jelly matrix are mixed with many microfibrillar structures. Microfibrils were found with a tubular cross-section of 10-12 nm diameter and were organized as beaded filaments characteristic of fibrillin-rich microfibrils. Beads had an average diameter of 25 nm and were spaced at about 50-80 nm. This ultrastructural observation was confirmed by indirect immunofluorescent staining of the jelly matrix using monoclonal antibody to fibrillin. Another constituent of the microfibrillar network was present as typical 100-nm periodic filaments of type VI collagen. Indirect immunofluorescent staining using antibodies to collagen VI showed for the first time that this collagen appeared to be distributed largely in the jelly matrix. In addition, other microfibrils with no specific banding pattern were observed. These microfibrils may constitute an organization of type V collagen different from the one which is generally assembled in heterotypic fibrils with collagen I. Among the latter heterotypic fibrils, type V collagen was studied using an anti-peptide antibody to the most N-terminal non-collagenous region of its alpha 2(V) chain. This antibody recognized a filamentous mesh decorating the bundles of collagen fibrils by immunofluorescent staining. This indicates that at least this part of alpha 2(V) chain may be accessible to the antibody at the surface of the fibrils.
Actin Cytoskeleton↗
Atomic force microscopy and modeling of natural elastic fibrillin polymers.
A central issue in the understanding of Marfan syndrome deals with the functional architecture of fibrillin-containing microfibrils. Fibrillin-rich microfibrils are long extracellular matrix fibrillar components exhibiting a 50 nm periodic beaded-structure with a width of around 20-25 nm after rotary shadowing and a 10-12 nm diameter when observed in ultra-thin sections. They are composed of fibrillin monomers more or less associated with many other components which are, for the most part, poorly characterized up to date. They are known to be elastic but few data have been accumulated to understand their properties. Atomic force microscopy (AFM) allowed us to morphologically differentiate fibrillin-rich microfibrils from other fibrillar components and to investigate the thin structure of these beaded filaments in their native state. They showed, in AFM, a periodic beaded structure ranging from 50 to 60 nm and a width of about 40 nm. The different sizes of fibrillin-containing microfibrils previously observed after rotary shadowing and in ultra-thin sections was resolved with our technique and is revealed to be 10 nm in diameter. Each beaded microfibril appears to be composed of heterogeneous beads connected by 2-3 arms. An orientation of the microfibrils has been shown, and allows us to propose a complementary model of microfibrillar monomer association.
Animals↗
Fibrillin-rich microfibrils: structural modifications during ageing in normal human zonule.
Ageing is marked by ultrastructural and functional changes in most tissues. In part, these changes are caused by a loss of elasticity in the elastic fibers of the extracellular matrix. These fibers are composed of the protein elastin associated with microfibrils of 8 to 12 nm in diameter. Microfibrils contain fibrillins as major constituents. Mutations in fibrillin genes are considered as primary causes of Marfan syndrome, a genetic disorder with pathological manifestations in the cardiovascular and skeletal systems, in addition to dysfunctions in the eye. Fibrillin is also the major protein of the ciliary zonule fibers. During ageing, these fibers become more fragile, and concomitantly, an increased risk for ocular pathologies is observed. We have investigated structural modifications in fibrillin-rich microfibrils during ageing of human ciliary zonule. Observations using light microscopy and transmission electron microscopy after rotary shadowing allowed us to describe the organization of the zonule fibers and their insertion into the ciliary body. Our results emphasize qualitative differences between young and old zonules, which are likely due to modifications in the structure of microfibrils.
Actin Cytoskeleton↗
[Collagen and laminin: which messages for which cells?].
Cells in animal tissues are in contact with a structured set of well-defined proteins that constitute the extracellular matrix. Among these proteins, collagens are ubiquitous in distribution, whereas laminins are found only in basement membranes. In addition to their structural role, collagens and laminins convey messages to cells. A discussion is presented of the structure and diversity of collagens (about 20 types) and laminins (about 12 types). The nature of the cell receptors involved in message delivery (integrins and non-integrins) and the mechanisms possibly responsible for signal transduction are reviewed.
Animals↗
Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing.
Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human alpha1 chain of collagen V using an episomal vector. High yields (15 microgram/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the alpha1(V) collagen is correctly folded into a stable triple helix as shown by electron microscopy and pepsin resistance. Circular dichroism data confirm the triple-helix conformation and indicate a melting temperature of 37.5 degrees C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (alpha1FL). N-terminal sequencing and collagenase digestion indicate that alpha1FL retains the complete N-propeptide but lacks the C-propeptide. However, alpha1FL might undergo a further N-terminal trimming into a form (alpha1TH) corresponding to the main triple-helix domain plus the major part of the NC2 domain. This processing is different from the one of the heterotrimeric (alpha1(V))2alpha2(V) and could have some physiological relevance. Analysis of cell homogenates indicates the presence of a 280-kDa polypeptide that is disulfide-linked through its C-terminal globular domain. This C-propeptide is rapidly cleaved after secretion in the medium, giving the first evidence of a C-terminal processing of recombinant fibrillar collagens. Rotary shadowing observations not only confirm the presence of a globular domain at the N-terminal end of the molecule but reveal the presence of a kink within the triple helix in a region poor in iminoacids. This region could represent a target for proteases. Together with the thermal stability data, these results might explain the low amount of (alpha1(V))3 recovered from tissues.
Ascorbic Acid↗
Characterization of the bovine tenascin-X.
The primary structure of flexilin, an extracellular matrix glycoprotein previously identified in bovine tissues (Lethias, C., Descollonges, Y., Boutillon, M.-M., and Garrone, R. (1996) Matrix Biol. 15, 11-19) was determined by cDNA cloning. The deduced amino acid sequence (4135 residues) reveals that this protein is composed of a succession of peptide motifs characteristic of the tenascin family: an amino-terminal domain containing cysteine residues and heptads of hydrophobic amino acids, 18.5 epidermal growth factor-like repeats, 30 fibronectin type III-like (FNIII) domains, and a carboxyl-terminal fibrinogen-like motif. Sequence analysis indicated that this protein is the bovine orthologue of human tenascin-X. By rotary shadowing, bovine tenascin-X was identified as monomers with a flexible aspect, which are ended by a globule. More FNIII motifs were characterized in the bovine protein than in human tenascin-X. The main difference between the human and bovine tenascin-X is found in the arrangement of the three classes of highly similar FNIII repeat types in the central region of tenascin-X. The bovine FNIII motif b10 exhibits an RGD putative cell attachment site. The functional role of this sequence is corroborated by cell adhesion on purified tenascin-X, which is inhibited by RGD peptides. Moreover, we demonstrate that this RGD site is conserved at the same location in the human molecule.
Amino Acid Sequence↗
Distribution of minor collagens during skin development.
The skin is a tissue containing a large number of collagen types. Several collagens are restricted at the dermo-epidermal junction, contrarily to others present throughout the dermis. However, the distribution of the dermal collagen varies during embryonic development. In this contribution, we have been interested in the collagen types associated with the major collagenous components of the dermis, which are the collagen types I and III. Type V collagen, which is mixed with collagen types I and III to form heterotypic fibrils, has been studied during mouse embryo development. Transcripts of the alpha 1 (V) gene have been localized by in situ hybridization, on flattened cells of the stratum germinativum first, and then only on dermal cells. The expression of the gene decreases at birth, while the expression of the alpha 1(I) gene remains constant, with, however, a ring of high intensity around hair follicles. Other collagen types (VI, and the fibril-associated collagens XII and XIV) have been studied during calf embryonic development by immunofluorescence and ultrastructural immunogold detection. Type VI collagen appears homogeneously distributed throughout the dermis. Type XII collagen is first widely distributed and becomes restricted in the upper, papillary dermis after 6 months of gestation. Type XIV collagen, on the contrary, is first located as a delicate framework around hair follicles (at 19 weeks of gestation), and progressively invades the whole dermis where it appears abundant just before birth. The different functions of all these collagens are discussed in terms of dermis architecture, mechanical properties and physiology.
Actin Cytoskeleton↗
Cloning of an annelid fibrillar-collagen gene and phylogenetic analysis of vertebrate and invertebrate collagens.
Arenicola marina possesses cuticular and interstitial collagens, which are mostly synthesised by its epidermis. A cDNA library was constructed from the body wall. This annelid cDNA library was screened with a sea-urchin-collagen cDNA probe, and several overlapping clones were isolated. Nucleotide sequencing of these clones revealed an open reading frame of 2052 nucleotides. The translation product exhibits a triple helical domain of 138 Gly-Xaa-Yaa repeats followed by a 269-residue-long C-terminal non-collagenous domain (C-propeptide). The triple helical domain exhibits an imperfection that has been previously described in a peptide produced by cyanogen bromide digestion (CNBr peptide) of A. marina interstitial collagen. This imperfection occurs at the same place in the interstitial collagen of the vestimentiferan Riftia pachyptila. This identifies the clone as coding for the C-terminal part of a fibrillar collagen chain. It was called FAm1alpha, for fibrillar collagen 1alpha chain of A. marina. The non-collagenous domain possesses a structure similar to carboxy-terminal propeptides of fibrillar pro-alpha chains. Only six conserved cysteine residues are observed in A. marina compared with seven or eight in all other known C-propeptides. This provides information on the importance of disulfide bonds in C-propeptide interactions and in the collagen-assembly process. Phylogenetic studies indicate that the fibrillar collagen 1alpha chain of A. marina is homologous to the R. pachyptila interstitial collagen and that the FAm1alpha gene evolved independently from the other alpha-chain genes. Complementary analyses indicate that the vertebrate fibrillar collagen family is composed of two monophyletic subgroups with a specific position of the collagen type-V chains.
Amino Acid Sequence↗
Collagen fibrillogenesis during sea urchin development--retention of SURF motifs from the N-propeptide of the 2alpha chain in mature fibrils.
The sea urchin 2alpha fibrillar collagen chain has a unique amino-propeptide structure with several repetitions of a still unknown 140-145-amino-acid, four-Cys module called SURF (for sea urchin fibrillar module). To follow the expression of the amino-propeptide of the 2alpha chain and assign a function to this domain, we have overproduced in Escherichia coli several recombinant proteins corresponding either to the amino-propeptide or to the amino-telopeptide. Monoclonal and/or polyclonal antibodies against these recombinant proteins allowed us to observe a similar tissue distribution during the first stages of development. A signal is first observed at the prism stage as intracellular spots in mesenchymal cells. In plutei, immunofluorescence staining is observed around the skeleton spicules and as a thin meshwork surrounding the mesenchymal cells. At the ultrastructural level, and using antibodies against the amino-propeptide, gold particles are observed at the surface of 25 nm thin periodic fibrils. By rotary shadowing, these fibrils show a brush-bottle aspect, exhibiting at their surface numerous periodically distributed thin rods ended by a small globule. These data indicate that the amino-propeptide is maintained during fibrillogenesis. As previously suggested, the retention of the amino-propeptide could play an important role in regulation of the fibril growth. We propose that the important region of this amino-propeptide in the widely encountered 25-nm-diameter fibrils is the short triple-helical segment. The globular part of the amino-propeptide will not only restrict the fibril growth but also interact with other neighbouring components and playing, as suspected from our immunofluorescence studies, a function during the spiculogenesis of the sea urchin embryo.
Amino Acid Sequence↗
Differential expression of collagens XII and XIV in human skin and in reconstructed skin.
Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.
Adult↗
The evolution of fibrillar collagens: a sea-pen collagen shares common features with vertebrate type V collagen.
The extracellular matrix of marine primitive invertebrates (sponges, polyps and jellyfishes) contains collagen fibrils with narrow diameters. From various data, it has been hypothesized that these primitive collagens could represent ancestral forms of the vertebrate minor collagens, i.e., types V or XI. Recently we have isolated a primitive collagen from the soft tissues of the sea-pen Veretillum cynomorium. This report examines whether the sea-pen collagen shares some features with vertebrate type V collagen. Rotary shadowed images of acid-soluble collagen molecules extracted from beta-APN treated animals, positive staining of segment-long-spacing crystallites precipitated from pepsinized collagen, Western blots of the pepsinized alpha1 and alpha2 chains with antibodies to vertebrate types I, III and V collagens, and in situ gold immunolabeling of ECM collagen fibrils were examined. Our results showed that the tissue form of the sea-pen collagen is a 340-nm threadlike molecule, which is close to the vertebrate type V collagen with its voluminous terminal globular domain, the distribution of most of its polar amino-acid residues, and its antigenic properties.
Amino Acid Sequence↗
Type IV collagen in sponges, the missing link in basement membrane ubiquity.
Basement membrane structures, or their main component, type IV collagen, have been detected in all multicellular animal species, except sponges. We cancel this exception by the demonstration of type IV collagenous sequences in a new marine sponge species by cDNA and genomic DNA studies. One of these sequences is long enough to demonstrate the specific characteristics of type IV collagen chains. The 12 cysteines are at conserved positions in the carboxyl-terminal non-helical NCl domain, as are the interruptions in the carboxyl-terminal end of the triple helical domain. The gene organization of the region coding for the NCl domain is similar to that of the human genes COL4A2, COL4A4 and COL4A6. An additional, shorter sequence suggests the presence of a second chain. The expected tissue localization of this collagen has been confirmed using polyclonal antibodies raised against a sponge recombinant protein. These results demonstrate that type IV collagen is representated in all animal phyla. It is actually the only known ubiquitous collagen and it has at least two different alpha chains in all the species where it has been characterized.
Amino Acid Sequence↗
Flexilin: a new extracellular matrix glycoprotein localized on collagen fibrils.
We have immunopurified and characterized a new glycoprotein of the extracellular matrix, using a monoclonal antibody obtained after immunization with fibril-associated collagens extracted from bovine tendon. In polyacrylamide gels, the protein migrates at about 350 kDa molecular mass. The protein is insensitive to bacterial collagenase, and no disulfide-linked aggregates could be detected; sugars were stained with periodic acid-Schiff's reagent. Amino acid analysis and sequencing of tryptic peptides failed to detect any similarity with known proteins. By rotary shadowing experiments, the protein was observed as flexible, unbranched structures, approximately 150 nm long, with a small globule at one end. Investigation of the tissue distribution of the protein in fetal bovine tissues by immunofluorescence resulted in labeling in extracellular matrices with loosely packed collagen fibrils, such as the peritendineum, embryonic skin and kidney glomeruli; cornea, cartilage matrix and bone were not labeled. Ultrastructural immunolocalization in dermis and in mesangium of glomeruli showed that the protein always occurred in the vicinity of collagen fibrils. In view of its tissue distribution and molecular shape, we postulate that this protein is important in the properties of the extrafibrillar environment. By reference to its shape as observed by rotary shadowing, we propose the name 'flexilin' for this extracellular matrix glycoprotein.
Amino Acid Sequence↗
Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.
A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.
Animals↗
Human corneal fibrillogenesis. Collagen V structural analysis and fibrillar assembly by stromal fibroblasts in culture.
PURPOSE: The stroma of the developing cornea is a highly organized extracellular matrix formed essentially by uniform, small-diameter collagen fibrils with constant interfibrillar spacing. Unlike the fibrillogenesis of chicken cornea, the assembly and maturation of human corneal fibrils have been poorly investigated. In the current study, the authors aimed to ascertain the heterotypic organization (collagens I and V) of the human corneal fibrils at the supramolecular level. To gain more insight into the molecular structure of collagen V, its cellular source, and its role in fibrillogenesis, the authors used cultured human corneal fibroblasts. METHODS: The structure of human corneal stroma after brief homogenization of the tissue was analyzed by immunogold labeling using specific polyclonal antibodies and rotary shadowing. Biochemical, electron microscopic, and immunolabeling approaches were used to investigate the collagen fibril formation and the extracellular matrix synthesis using human corneal fibroblasts grown in culture as a model system. RESULTS: The authors showed that in human corneal stroma, collagen I is distributed uniformly along the striated fibrils, in contrast to collagen V, which could be identified only at sites at which the fibrils partially were disrupted. Rotary shadowing observations of the homogenate revealed that collagen VI, a major component of the human cornea, was associated closely with the collagen fibril surface. Corneal fibroblasts synthesize and deposit a collagenous matrix with fibrils resembling those of the human cornea in appearance and collagen composition. Biochemical data indicate that a high concentration (20% to 30%) of collagen V is synthesized by stromal fibroblasts and that collagen V molecules are processed similarly to matrix forms in which the extension peptides are retained on the molecules. CONCLUSIONS: The heterotypic nature (collagens I and V) of human corneal fibrils was determined. Results indicate that human corneal fibroblasts synthesize the major collagen types in human cornea (collagens I, V, and VI) and express all the posttranslational equipment for correct collagen molecular assembly and processing in a manner that closely resembles the situation in situ, offering the opportunity for more detailed study of this process, which is essential for optical transparency.
Adult↗
Characterization of two genes coding for a similar four-cysteine motif of the amino-terminal propeptide of a sea urchin fibrillar collagen.
We report the characterization of the 5' region of the gene coding for the 2 alpha fibrillar collagen chain of the sea urchin Paracentrotus lividus. This sequence analysis identified the intron/exon organization of the region of the gene coding for the signal peptide, the cysteine-rich domain and the 12 repeats of the four-cysteine module of the unusually long amino-propeptide. This still unknown four-cysteine motif is generally encoded by one exon, which confirms that the distinct amino-propeptide structures of the fibrillar collagens arise from the shuffling of several exon-encoding modules. Moreover, Southern-blot analysis of the sea urchin genome and sequencing of selected genomic clones allowed us to demonstrate that several sea urchin genes could potentially code for the four-cysteine module. Curiously, one of these genes lacks the exons coding for four repeats of this motif while, in another gene, the same exons are submitted to an alternative splicing event.
Amino Acid Sequence↗
Identification of a cell lineage-specific gene coding for a sea urchin alpha 2(IV)-like collagen chain.
We report the isolation of several overlapping cDNAs from an embryonic library of Strongylocentrotus purpuratus coding for a novel sea urchin collagen chain. The conceptual amino acid translation of the cDNAs indicated that the protein displays the structural features of a vertebrate type IV-like collagen alpha chain. In addition to a putative 31-residue signal peptide, the sea urchin molecule contains a 14-residue amino-terminal non-collagenous segment, a discontinuous 1,477-amino acid triple helical domain, and a 225-residue carboxyl-terminal domain rich in cysteines. The amino- and carboxyl-terminal non-collagenous regions of the echinoid molecule are remarkably similar to the 7 S and carboxyl-terminal non-collagenous (NC1) domains of the alpha 1 and alpha 2 chains of vertebrate type IV collagen. The sequence similarity and distinct structural features of the 7 S and NC1 domains strongly suggest that the sea urchin polypeptide is evolutionarily related to the alpha 2(IV) class of collagen chains. Finally, in situ hybridizations revealed that expression of this collagen gene is restricted to the mesenchyme cell lineage of the developing sea urchin embryo.
Amino Acid Sequence↗