Robert Garrone

Collagen family of proteins.

Collagen molecules are structural macromolecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo‐ and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemets membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril‐associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin‐beaded filaments that may interact with fibrils and cells.—van der Rest, M.; Garrone, R. Collagen family of proteins. FASEB J. 5: 2814‐2823; 1991.

Type IV collagen in sponges, the missing link in basement membrane ubiquity*

Summry— 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 NC1 domain, as are the interruptions in the carboxyl‐terminal end of the triple helical domain. The gene organization of the region coding for the NC1 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.

Evolution of collagens

The extracellular matrix is often defined as the substance that gives multicellular organisms (from plants to vertebrates) their structural integrity, and is intimately involved in their development. Although the general functions of extracellular matrices are comparable, their compositions are quite distinct. One of the specific components of metazoan extracellular matrices is collagen, which is present in organisms ranging from sponges to humans. By comparing data obtained in diploblastic, protostomic, and deuterostomic animals, we have attempted to trace the evolution of collagens and collagen‐like proteins. Moreover, the collagen story is closely involved with the emergence and evolution of metazoa. The collagen triple helix is one of numerous modules that arose during the metazoan radiation which permit the formation of large multimodular proteins. One of the advantages of this module is its involvement in oligomerization, in which it acts as a structural organizer that is not only relatively resistant to proteases but also permits the creation of multivalent supramolecular networks. Anat Rec 268:302–316, 2002.

Binding of tenascin-X to decorin

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.

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.

Human Recombinant α1(V) Collagen Chain HOMOTRIMERIC ASSEMBLY AND SUBSEQUENT PROCESSING

Human embryonic kidney cells (293-EBNA) have been transfected with the full-length human α1 chain of collagen V using an episomal vector. High yields (15 μg/ml) of recombinant collagen were secreted in the culture medium. In presence of ascorbate, the α1(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 °C for the recombinant homotrimer. The major secreted form is a 250-kDa polypeptide (α1FL). N-terminal sequencing and collagenase digestion indicate that α1FL retains the complete N-propeptide but lacks the C-propeptide. However, α1FL might undergo a further N-terminal trimming into a form (α1TH) 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 (α1(V))2α2(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 (α1(V))3 recovered from tissues.

Atomic force microscopy study of the collagen fibre structure

Observations of intact reconstituted and native collagen fibres were performed with the atomic force microscope. The results are compared between the two types of fibres and with those obtained previously with the electron microscope on freeze‐etched or negative stained samples. Some of the findings presented here indicate that the specimens observed in air with the atomic force microscope were still in a hydrated state.

Marine Sponges Discriminate between Food Bacteria and Bacterial Symbionts: Electron Microscope Radioautography and in situ Evidence

This study considered whether marine sponges are selective particle feeders, and whether they are capable of distinguishing between sponge bacterial symbionts and other bacteria. Four species of marine sponges (Aplysina aerophoba, A. cavernicola, Pericharax heteroraphis and Spongia sp.) were fed in situ with tritium-labelled bacteria, either symbionts or other bacteria isolated from seawater. A large proportion of bacterial symbionts passed through the sponge and were expelled in the exhalant current, whereas seawater bacteria disappeared from the incubation water and were retained within the sponge. The seawater bacteria were observed in choanocytes and occasionally in pinacocytes after 30-40 min, whereas symbiont bacteria were rarely observed. Although sponges do have mechanisms to ingest particles such as food, they appear to be unable to consume bacterial symbionts, probably because their identity is masked by capsular sheaths. A specific mechanism for recognition and rejection of self-particulate matter is proposed for sponge epidermal cells.

Control of Heterotypic Fibril Formation by Collagen V Is Determined by Chain Stoichiometry

Although the collagen V heterotrimer is known to be involved in the control of fibril assembly, the role of the homotrimer in fibrillar organization has not yet been examined. Here, the production of substantial amounts of recombinant collagen V homotrimer has allowed a detailed study of its role in homotypic and heterotypic fibril formation. After removal of terminal regions by pepsin digestion, both the collagen V heterotrimer and homotrimer formed thin homotypic fibrils, thus showing that diameter limitation is at least in part an intrinsic property of the collagen V triple helix. When mixed with collagen I, however, various complementary approaches indicated that the collagen V heterotrimer and homotrimer exerted different effects in heterotypic fibril formation. Unlike the heterotrimer, which was buried in the fibril interior, the homotrimer was localized as thin filamentous structures at the surface of wide collagen I fibrils and did not regulate fibril assembly. Its localization at the fibril surface suggests that the homotrimer can act as a molecular linker between collagen fibrils or macromolecules in the extracellular matrix or both. Thus, depending on their respective distribution in tissues, the different collagen V isoforms might fulfill specific biological functions.

Molecular Features of the Collagen V Heparin Binding Site

A heparin binding region is known to be present within the triple helical part of the α1(V) chain. Here we show that a recombinant α1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual α1(V) chains and HepV are eluted at identical NaCl concentrations (0.35m) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of α1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the α1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues.