Isolation, Characterization and Biological Evaluation of Jellyfish Collagen for Use in Biomedical Applications

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2–10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications

The Fibrillar Collagen Family

Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral α chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of “whole genome duplication” leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils

Fine structures of sponge cell membranes: comparative study with freeze-fracture and conventional thin section methods.

International audienceFreeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed.Freeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed

Distribution of minor collagens during skin development.

International audienceThe 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.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

Immunolabelling of freeze-fractured tissue: thin sections of rat aorta after labelling of elastic laminae.

International audienceRat aortae were fixed, reduced into fragments, fixed again according to the fracture-label method from Pinto da Silva et al. (1981) and labelled with anti-elastin antibodies. We thus demonstrate that this method, extensively used with lectins, is also usable with antibodies to investigate components of the extracellular matrix.Rat aortae were fixed, reduced into fragments, fixed again according to the fracture-label method from Pinto da Silva et al. (1981) and labelled with anti-elastin antibodies. We thus demonstrate that this method, extensively used with lectins, is also usable with antibodies to investigate components of the extracellular matrix

FREEZE-FRACTURE STUDY OF SPONGE CELL-MEMBRANES AND EXTRACELLULAR-MATRIX - PRELIMINARY-RESULTS

International audiencexx

Rôle d une protéine de la matrice extracellulaire, la ténascine-X, sur l étalement des cellules sur un substrat de collagène I.

La ténascine-X (TNX) est une glycoprotéine de la matrice extracellulaire capable d interagir avec certains collagènes ainsi que des protéoglycanes. Elle possède la structure modulaire caractéristique des membres de la famille des ténascines avec 3 types de domaines : des motifs de type EGF, des domaines similaires au FNIII et, enfin, une extrémité globulaire similaire au fibrinogène. La déficience en TNX chez l homme conduit à une forme récessive du syndrome d Ehlers-Danlos caractérisée par une hypermobilité articulaire et une peau fragile très extensible. Elle est également corrélée à un phénotype invasif plus important dans un modèle de tumeur injectée chez la souris knock-out pour la TNX. Ces données suggèrent un rôle de la TNX à la fois dans l assemblage matriciel et dans la régulation du comportement cellulaire. Ce travail de thèse a permis de caractériser l existence d une interaction directe entre la TNX et le collagène de type I, dépendante de la conformation en triple hélice de ce dernier. L étude de l interaction entre différents types de cellules (fibrosarcome, fibroblastes primaires ) et la TNX a confirmé que la TNX recombinante permet une faible adhérence des cellules en utilisant un récepteur de la famille des intégrines. De plus, il a été démontré par des études morphométriques que la TNX est capable de moduler l étalement cellulaire sur un substrat de collagène I. Cette modulation passe par une modification des structures mises en place par les cellules pour interagir avec leur environnement. Ainsi, la formation des filopodes est augmentée en présence de TNX. Par ailleurs, les adhésions focales adoptent une composition et une localisation singulières en présence de TNX par rapport à notre condition contrôle sur collagène I. La signalisation associée à ces structures est également modulée par la TNX puisqu une diminution de l activité de FAK et de Rac ainsi qu une diminution de la phosphorylation de la paxilline ont pu être mesurées. Ce travail de thèse constitue la première étude des effets cellulaires de cette protéine de la matrice extracellulaire encore méconnue. Il apporte les premières évidences attribuant à la TNX le rôle de protéine matricellulaire capable de moduler les interactions cellule-matrice. Ces propriétés pourraient favoriser le phénotype prométastatique observé chez les souris déficientes en TNX.Tenascin-X (TNX) is a large glycoprotein from the extracellular matrix associated with collagen fibrils and proteoglycans. TNX shares the typical modular structure of the TN family members with EGF, FNIII and Fbg domains. TNX deficiency leads to a recessive human form of Ehlers Danlos syndrome characterized by joint hypermobility, skin fragility and hyperextensible skin. It is also correlated with a more invasive phenotype in tumour-induced TNX-/- mice, suggesting a role in the extracellular matrix assembly and in the modulation of cell behaviour. In this thesis, we identify a direct interaction between TNX and collagen I in a triple-helical conformation. Concerning the interaction between cells and TNX, we demonstrate that TNX allows weak adhesion of numerous cell lines (fibrosarcoma cells, primary fibroblasts...), mediated by at least one integrin receptor. Moreover, using morphometrical analysis, we show that recombinant TNX can modulate cell spreading on collagen I. This modulation occurs through the modification of the cellular structures responsible for the interaction between cells and their surrounding matrix. For example, the formation of filopodia is enhanced when TNX is added to collagen I. In addition, focal adhesions display a modified composition and localisation. The signalling pathways initiated from these adhesion sites are modulated by TNX, i.e. FAK and small GTPase Rac activities are downregulated and paxillin phosphorylation is impaired. Together, the results of this thesis demonstrate that TNX regulates cell-matrix induced signalisation and modifies cell spreading. Hence, TNX can be considered as an authentic matricellular protein. These data constitute promising foundations to understand the prometastatic phenotype observed in TNX-deficient miceLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Expression of type XIV collagen during the differentiation of fetal bovine skin: immunolabeling with monoclonal antibody is prominent in morphogenetic areas.

International audienceType XIV collagen belongs to the subclass of fibril-associated collagens with interrupted triple helices, which are composed of alternative triple helical and non-collagenous domains. Structural data show that these molecules interact with collagen fibrils and suggest that they might interact with cells. We have investigated the expression of type XIV collagen in bovine skin during development. Fetuses from 9 to 37 weeks were examined. Anti-type XIV collagen monoclonal antibody was produced, characterized, and used for immunofluorescence detection of the molecule. The localization of immunolabeling was analyzed by comparison with light and electron microscopic observations. In 9-week-old fetus, no type XIV collagen was found in the skin. From 19 weeks to birth, extensive immunofluorescence was observed on bundles of collagen fibrils in deep dermis. As shown by electron microscopy, this area exhibited bundles of collagen fibrils and cells with an abundant rough endoplasmic reticulum. In the upper dermis, a delicate fibrillar network of type XIV collagen was revealed by immunofluorescence around growing hair follicles at 19 and 24 weeks. Double labeling for type XIV collagen and fibronectin shows a more restricted pattern of expression of type XIV collagen in this area. The electron microscopic examination of skin of fetuses at these stages shows that the whole upper dermis is composed by a loose connective tissue containing scattered small bundles of collagen fibrils. Type XIV collagen was synthesized in the upper dermis between 24 weeks and birth. From this study, it appears that type XIV collagen expression is distinct from that of fibrillar collagens, at least during some developmental events. The prominent localization of type XIV collagen around growing hair follicles suggests a role for this molecule in epithelial-mesenchymal interactions.Type XIV collagen belongs to the subclass of fibril-associated collagens with interrupted triple helices, which are composed of alternative triple helical and non-collagenous domains. Structural data show that these molecules interact with collagen fibrils and suggest that they might interact with cells. We have investigated the expression of type XIV collagen in bovine skin during development. Fetuses from 9 to 37 weeks were examined. Anti-type XIV collagen monoclonal antibody was produced, characterized, and used for immunofluorescence detection of the molecule. The localization of immunolabeling was analyzed by comparison with light and electron microscopic observations. In 9-week-old fetus, no type XIV collagen was found in the skin. From 19 weeks to birth, extensive immunofluorescence was observed on bundles of collagen fibrils in deep dermis. As shown by electron microscopy, this area exhibited bundles of collagen fibrils and cells with an abundant rough endoplasmic reticulum. In the upper dermis, a delicate fibrillar network of type XIV collagen was revealed by immunofluorescence around growing hair follicles at 19 and 24 weeks. Double labeling for type XIV collagen and fibronectin shows a more restricted pattern of expression of type XIV collagen in this area. The electron microscopic examination of skin of fetuses at these stages shows that the whole upper dermis is composed by a loose connective tissue containing scattered small bundles of collagen fibrils. Type XIV collagen was synthesized in the upper dermis between 24 weeks and birth. From this study, it appears that type XIV collagen expression is distinct from that of fibrillar collagens, at least during some developmental events. The prominent localization of type XIV collagen around growing hair follicles suggests a role for this molecule in epithelial-mesenchymal interactions

Tracing the evolution of vertebrate fibrillar collagens from an ancestral alpha chain.

International audienceFrom considerations of gene structure, phylogenetic analysis, modular organisation of related proteins and fibril shapes, we suggest a model for the evolution of contemporary vertebrate fibrillar collagens from a common ancestral alpha chain.From considerations of gene structure, phylogenetic analysis, modular organisation of related proteins and fibril shapes, we suggest a model for the evolution of contemporary vertebrate fibrillar collagens from a common ancestral alpha chain

Flexilin: a new extracellular matrix glycoprotein localized on collagen fibrils.

International audienceWe 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.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