RESUMEN
Tenascin-X (TN-X) is an extracellular matrix protein whose absence results in an alteration of the mechanical properties of connective tissue. To understand the mechanisms of integration of TN-X in the extracellular matrix, overlay blot assays were performed on skin extracts. A 100 kDa molecule interacting with TN-X was identified by this method and this interaction was abolished when the extract was digested by chondroitinase. By solid-phase assays, we showed that dermatan sulfate chains of decorin bind to the heparin-binding site included within the fibronectin-type III domains 10 and 11 of TN-X. We thus postulate that the association of TN-X with collagen fibrils is mediated by decorin and contributes to the integrity of the extracellular network.
Asunto(s)
Proteoglicanos/metabolismo , Tenascina/química , Tenascina/metabolismo , Animales , Biglicano , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Unión Competitiva/efectos de los fármacos , Western Blotting , Bovinos , Condroitina ABC Liasa/metabolismo , Colágeno/metabolismo , Decorina , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacología , Heparina/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Proteoglicanos/farmacología , Piel/química , Piel/embriología , Extractos de Tejidos/químicaRESUMEN
The Sea URchin Fibrillar (SURF) domain is a four-cysteine module present in the amino-propeptide of the sea urchin 2alpha fibrillar collagen chain. Despite numerous international genome and expressed sequence tag projects, computer searches have so far failed to identify similar domains in other species. Here, we have characterized a new sea urchin protein of 2656 amino acids made up of a series of epidermal growth factor-like and SURF modules. From its striking similarity to the modular organization of fibropellins, we called this new protein fibrosurfin. This protein is acidic with a calculated pI of 4.12. Eleven of the 17 epidermal growth factor-like domains correspond to the consensus sequence of calcium-binding type. By Western blot and immunofluorescence analyses, this protein is not detectable during embryogenesis. In adult tissues, fibrosurfin is co-localized with the amino-propeptide of the 2alpha fibrillar collagen chain in several collagenous ligaments, i.e., test sutures, spine ligaments, peristomial membrane, and to a lesser extent, tube feet. Finally, immunogold labeling indicates that fibrosurfin is an interfibrillar component of collagenous tissues. Taken together, the data suggest that proteins possessing SURF modules are localized in the vicinity of mineralized tissues and could be responsible for the unique properties of sea urchin mutable collagenous tissues.
Asunto(s)
Tejido Conectivo/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , ADN Complementario/análisis , ADN Complementario/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Datos de Secuencia Molecular , Erizos de Mar/genética , Erizos de Mar/metabolismoRESUMEN
Tenascin-X is known as a heparin-binding molecule, but the localization of the heparin-binding site has not been investigated until now. We show here that, unlike tenascin-C, the recombinant fibrinogen-like domain of tenascin-X is not involved in heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at physiological pH, hence a set of recombinant proteins comprising these domains was tested for interaction with heparin. Using solid phase assays and affinity chromatography, we found that interaction with heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us to predict exposed residues that were then submitted to site-directed mutagenesis. In this way, we identified the basic residues within each domain that are crucial for this interaction. Blocking experiments using antibodies against domain 10 were performed to test the efficiency of this site within intact tenascin-X. Binding was significantly reduced, arguing for the activity of a heparin-binding site involving domains 10 and 11 in the whole molecule. Finally, the biological significance of this site was tested by cell adhesion studies. Heparan sulfate cell surface receptors are able to interact with proteins bearing domains 10 and 11, suggesting that tenascin-X may activate different signals to regulate cell behavior.
Asunto(s)
Fibronectinas/química , Heparina/metabolismo , Tenascina/química , Tenascina/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células CHO , Bovinos , Adhesión Celular , Línea Celular , Cromatografía de Afinidad , Cricetinae , Embrión de Mamíferos , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Piel/metabolismo , Tenascina/aislamiento & purificación , TransfecciónRESUMEN
One of the mechanisms involved in the regulation of the fibril diameter is the retention of the N-propeptide. In sea urchin embryo, thin collagen fibrils harbor numerous extensions at their surface, which we have suggested correspond to the large N-propeptide of the 2alpha collagen chain. To investigate the function of the N-propeptide during fibrillogenesis, we engineered constructs coding for the globular region of the 2alpha N-propeptide. To obtain homotrimeric molecules, the N-telopeptide, the central triple helix and the C-propeptide of the 2alpha chain were replaced by human domains of the proalpha1(I) chain. A single restriction site allowed insertion of distinct versions of the minor triple helix of the N-propeptide. Several human cell lines were transfected, and with one of them we were able to produce intact homotrimeric procollagen molecules. Rotary shadowing of these purified molecules indicates the presence of three large 2alpha N-propeptides that are similar to the extensions present at the surface of the sea urchin thin fibrils. This cassette-vector will be useful in determining the respective contributions of the globular and minor triple helical domains of the N-propeptide in the regulation of fibril diameter.
Asunto(s)
Procolágeno/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Microscopía Electrónica , Procolágeno/química , Procolágeno/ultraestructura , Conformación Proteica , Erizos de Mar , Células Tumorales CultivadasRESUMEN
In sea urchin, two fibrillar collagen chains (alpha1 and alpha2) have been characterized by molecular biology while two biochemically detected chains (alpha1 and alpha2) have been reported. Here, to determine the relationship between these results, Western-blotting and Edman degradation sequencing of the amino-termini of pepsinized sea urchin fibrillar collagen chains were performed. The data demonstrate that the 2alpha chain corresponds to the alpha2 chain and is involved in the formation of heterotrimeric molecules [(1alpha)(2)2alpha].
Asunto(s)
Colágeno/química , Erizos de Mar/química , Secuencia de Aminoácidos , Animales , Western Blotting , Colágeno/inmunología , Pepsina A/química , Estructura Cuaternaria de ProteínaRESUMEN
The annelid Alvinella pompejana is probably the most heat-tolerant metazoan organism known. Previous results have shown that the level of thermal stability of its interstitial collagen is significantly greater than that of coastal annelids and of vent organisms, such as the vestimentiferan Riftia pachyptila, living in colder parts of the deep-sea hydrothermal environment. In order to investigate the molecular basis of this thermal behavior, we cloned and sequenced a large cDNA molecule coding the fibrillar collagen of Alvinella, including one half of the helical domain and the entire C-propeptide domain. For comparison, we also cloned the 3' part of the homologous cDNA from Riftia. Comparison of the corresponding helical domains of these two species, together with that of the previously sequenced domain of the coastal lugworm Arenicola marina, showed that the increase in proline content and in the number of stabilizing triplets correlate with the outstanding thermostability of the interstitial collagen of A. pompejana. Phylogenetic analysis showed that triple helical and the C-propeptide parts of the same collagen molecule evolve at different rates, in favor of an adaptive mechanism at the molecular level.
Asunto(s)
Adaptación Fisiológica , Colágeno/química , Colágeno/metabolismo , Calor , Poliquetos/química , Poliquetos/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Colágeno/genética , ADN Complementario/genética , Ambiente , Evolución Molecular , Variación Genética/genética , Datos de Secuencia Molecular , Filogenia , Poliquetos/genética , Procolágeno/química , Procolágeno/genética , Procolágeno/metabolismo , Prolina/análisis , Prolina/genética , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
The use of tobacco plants as a novel expression system for the production of human homotrimeric collagen I is presented in this report. Constructs were engineered from cDNA encoding the human proalpha1(I) chain to generate transgenic tobacco plants expressing collagen I. The recombinant proalpha1(I) chains were expressed as disulfide-bonded trimers and were shown to fold into a stable homotrimeric triple helix. Moreover, the recombinant procollagen was subsequently processed to collagen as it occurs in animals. Large amounts of recombinant collagen were purified from field grown plant material. The data suggest that plants are a valuable alternative for the recombinant production of collagen for various medical and scientific purposes.
Asunto(s)
Nicotiana/genética , Plantas Tóxicas , Procolágeno/genética , Secuencia de Aminoácidos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Procolágeno/química , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestructura , TripsinaRESUMEN
Adhesive properties of tenascin-X (TN-X) were investigated using TN-X purified from bovine skin and recombinant proteins encompassing the RGD sequence located within the tenth fibronectin type-III domain, and the fibrinogen-like domain. Osteosarcoma (MG63) and bladder carcinoma cells (ECV304) cells were shown to adhere to purified TN-X, but did not spread and did not assemble actin stress fibers. Both cell types adhered to recombinant proteins harboring the contiguous fibronectin type-III domains 9 and 10 (FNX 9-10) but not to the FNX 10 domain alone. This adhesion to FNX 9-10 was shown to be mediated by alphavbeta3 integrin, was inhibited by RGD peptides and was strongly reduced in proteins mutated within the RGD site. As antibodies against alphavbeta3 integrin had no effects on cell adhesion to purified TN-X, we suggest that the RGD sequence is masked in intact TN-X. Cell attachment to the recombinant TN-X fibrinogen domain (FbgX) and to purified TN-X was greater for MG63 than for ECV304 cells. A beta1-containing integrin was shown to be involved in MG63 cell attachment to FbgX and to purified TN-X. Although the existence of other cell interaction sites is likely in this huge molecule, these similar patterns of adhesion and inhibition suggest that the fibrinogen domain might be a dominant site in the whole molecule.
Asunto(s)
Adhesión Celular/fisiología , Integrinas/metabolismo , Tenascina/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Línea Celular , Humanos , Integrinas/química , Cinética , Ratones , Oligopéptidos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Piel/citología , Tenascina/química , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Tenascina/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Tenascina/genética , Tenascina/metabolismoRESUMEN
The time of appearance and location of three distinct collagen gene transcripts termed 1 alpha, 2 alpha, and 3 alpha, were monitored in the developing S. purpuratus embryo by in situ hybridization. The 1 alpha and 2 alpha transcripts of fibrillar collagens were detected simultaneously in the primary (PMC) and secondary (SMC) mesenchyme cells of the late gastrula stage and subsequently expressed in the spicules and gut associated cells of the pluteus stage. The 3 alpha transcripts of the basement membrane collagen appeared earlier than 1 alpha and 2 alpha, and were first detected in the presumptive PMC at the vegetal plate of the late blastula stage. The PMC exhibited high expression of 3 alpha at the mesenchyme blastula stage, but during gastrulation the level of expression was reduced differentially among the PMC. In the late gastrula and pluteus stages, both PMC and SMC expressed 3 alpha mRNA, and thus at these stages all three collagen genes displayed an identical expression pattern by coincidence. This study thus provides the first survey of onset and localization of multiple collagen transcripts in a single sea urchin species.
Asunto(s)
Colágeno , Regulación del Desarrollo de la Expresión Génica , Erizos de Mar , Animales , Membrana Basal/fisiología , Colágeno/biosíntesis , Colágeno/genética , Hibridación in Situ , Erizos de Mar/embriología , Erizos de Mar/genética , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Anélidos/genética , Colágeno/química , Colágeno/genética , Filogenia , Secuencia de Aminoácidos , Animales , Anélidos/química , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Secuencia Conservada , ADN Complementario , Desoxirribonucleasa EcoRI/metabolismo , Evolución Molecular , Humanos , Invertebrados/química , Datos de Secuencia Molecular , Alineación de Secuencia , VertebradosRESUMEN
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.
Asunto(s)
Colágeno/biosíntesis , Colágeno/química , Fosfopéptidos/química , Procolágeno , Precursores de Proteínas/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Colágeno/inmunología , Colágeno Tipo I , Escherichia coli , Ratones , Microscopía Electrónica , Datos de Secuencia Molecular , Péptidos/inmunología , Fosfopéptidos/inmunología , Procolágeno N-Endopeptidasa/metabolismo , Proteínas Recombinantes/inmunología , Erizos de MarRESUMEN
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.
Asunto(s)
Colágeno/análisis , Poríferos/química , Secuencia de Aminoácidos , Animales , Membrana Basal/química , Clonación Molecular , ADN Complementario/aislamiento & purificación , Genoma , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Filogenia , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Colágeno/genética , Cisteína/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Clonación Molecular , Colágeno/química , Datos de Secuencia Molecular , Erizos de Mar , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Colágeno/genética , Erizos de Mar/genética , Secuencia de Aminoácidos , Animales , Evolución Biológica , ADN Complementario , Humanos , Datos de Secuencia Molecular , Erizos de Mar/embriología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We have completed the analysis of a genomic clone, G238, that contains most of the coding region of the sponge COLF1 fibrillar collagen gene. The main triple helical domain is encoded by 31 exons. Except for the 5' junction exon and the two last 3' exons (126 and 18 base pairs), all these exons are related to a 54-bp unit and begin with an intact glycine codon. A good correlation can be made between this sponge gene and a vertebrate fibrillar collagen gene, revealing the high conservation of the members of this family during evolution. The reconstitution of an ancestral collagen gene can be made by considering all the exon/intron junctions of these genes. We suggest that such an ancestral gene arose from multiple duplications of a 54-bp exon and a (54 + 45)-bp module.
Asunto(s)
Evolución Biológica , Colágeno/genética , Genes , Modelos Genéticos , Poríferos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Colágeno/química , Exones , Intrones , Datos de Secuencia Molecular , Familia de Multigenes , Estructura Secundaria de Proteína , Erizos de Mar/genética , Vertebrados/genéticaRESUMEN
We isolated several overlapping cDNAs from Strongylocentrotus purpuratus coding for a nonfibrillar collagen chain structurally homologous to the vertebrate type IV collagen chains and arbitrarily termed 3 alpha chain. The deduced amino acid sequence of the sea urchin polypeptide includes a 28-residue signal peptide, a 14-residue amino-terminal non-collagenous segment, a triple-helical domain of 1390 residues containing 23 imperfections, and a 226-residue carboxyl-terminal non-collagenous region. Comparison of the sea urchin amino- and carboxyl-terminal non-collagenous domains with those of the vertebrate type IV collagen chains indicated a high level of sequence identity to the alpha 1 (IV) and alpha 5 (IV) chains. This evolutionary relationship was further strengthened by the analysis of the genomic organization of the 5' portion of the sea urchin gene, which also provided the composition of some of the upstream sequences. In addition, this work demonstrated that our gene product is identical to that encoded by the partial cDNA clone recently isolated by others (Wessel, G. M., Etkin, M., and Benson, S. (1991) Dev. Biol. 148, 261-272) who demonstrated its involvement in the biomineralization process of cultured mesenchyme cells.
Asunto(s)
Colágeno/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Colágeno/genética , ADN , Humanos , Datos de Secuencia Molecular , Erizos de Mar , Homología de Secuencia de AminoácidoRESUMEN
The extracellular matrix constitutes a highly organized intercellular medium. In multicellular animals, it plays important functions for cell cohesion and for the modulation of cell differentiation and behaviour as well. All the investigations conducted in non-vertebrate species have shown that the extracellular matrix is present at the onset of the multicellular life and throughout the animal kingdom. The collagen fibrils are the most constant element. Recent data on the structure of fibrillar collagen molecules and on the organization of the corresponding genes, obtained in sponges and sea-urchins have shown the remarkable conservation of these fibrillar collagens during evolution. This even emphasize their very likely fundamental function. These results, associated with data provided by morphological and biochemical informations obtained in cnidarians suggest that these primitive fibrillar collagens are the direct precursors of some vertebrate minor fibrillar collagens such as type V. Other collagens, with interrupted triple helix, are more variable and their characterization in sponges leads to consider these non-fibrillar collagens as precursors of basement membrane collagens, of fibril-associated collagens (the FACITs collagens), of the so-called "epithelial" collagens. They were probably used as sticking devices, anchoring the animal to its substratum, or as covering layers (cuticles, sheaths), and even as skeletons (i.e. the bath sponge). Adhesive molecules of higher animals ensure the mediation of the interactions between cells (via their membrane receptors of the integrin type) and the fibrous network of the extracellular matrix. It is the same situation at the beginning of the evolution of the multicellular animals where fibronectin, tenascin and then laminin are present. Proteoglycans too are components of primitive extracellular matrices. At last, only collagen mineralized by calcium phosphate (in bone) and elastin (in elastic fibers and laminae) seem to be restricted to vertebrates.
Asunto(s)
Matriz Extracelular/química , Animales , Colágeno/química , Proteínas de la Matriz Extracelular/química , Filogenia , Proteoglicanos/químicaRESUMEN
We isolated overlapping cDNAs from embryonic libraries of the sea urchin Strongylocentrotus purpuratus coding for a fibrillar procollagen (2 alpha chain) with a predicted molecular mass of about 320 kDa. The deduced primary structure of the echinoid chain consists of a 265-amino acid carboxyl-propeptide, a triple helical domain made of 337 uninterrupted Gly-X-Y repeats, and an unusually long amino-propeptide. Aside from a 10-cysteine globular region, a collagenous sequence, and a nonhelical segment, this protein domain includes a novel 4-cysteine motif repeated several times. Interestingly, preliminary evidence indicates that different combinations of the 4-cysteine repeats are encoded by alternatively spliced transcripts. Irrespective of this, the sea urchin 2 alpha procollagen chain represents the longest fibrillar molecule identified to date by cDNA cloning experiments in both vertebrate and invertebrate organisms.
Asunto(s)
Procolágeno/genética , Empalme del ARN , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Embrión no Mamífero/fisiología , Biblioteca de Genes , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Poli A/genética , Poli A/aislamiento & purificación , Conformación Proteica , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero , Mapeo Restrictivo , Erizos de Mar/embriología , Erizos de Mar/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
We isolated several overlapping cDNA clones covering the 4242 nucleotides of a Strongylocentrotus purpuratus transcript that codes for a fibrillar procollagen chain. The sea urchin polypeptide includes a 124-amino acid long amino pre-propeptide, a 1064-amino acid alpha-chain inclusive of 338 uninterrupted Gly-X-Y repeats, and a 226-residue carboxyl-propeptide. The distribution of the highly conserved cysteines within the last domain together with the structural configuration of the amino-propeptide and the organization of the corresponding coding region, strongly suggest that the sea urchin gene is evolutionarily related to the vertebrate pro-alpha 2(I) collagen. This work, therefore, represents the first report of the complete primary structure of an invertebrate fibrillar procollagen chain. It also provides a new insight into the evolution of the amino-propeptide, the most divergent among the major protein domains of fibrillar procollagen chains.