Collagens - structure, function and biosynthesis.

The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramolecular processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutical use of members of this protein family discussed in other chapters of this issue

Mini-Collagens in Hydra Nematocytes

We have isolated and characterized four collagen-related c-DNA clones (N-COL 1, N-COL 2, N-COL 3, N-COL 4) that are highly expressed in developing nematocytes in hydra. All four c-DNAs as well as their corresponding transcripts are small in size (600-1,000 bp). The deduced amino acid sequences show that they contain a central region consisting of 14 to 16 Gly-X-Y triplets. This region is flanked amino-terminal by a stretch of 14-23 proline residues and carboxy-terminal by a stretch of 6-9 prolines. At the NH2- and COOH-termini are repeated patterns of cysteine residues that are highly conserved between the molecules. A model is proposed which consists of a central stable collagen triple helix of 12-14 nm length from which three 9-22 nm long polyproline II type helices emerge at both ends. Disulfide linkage between cysteine- rich segments in these helices could lead to the formation of oligomeric network structures. Electrophoretic characterization of nematocyst extracts allows resolution of small proline-rich polypeptides that correspond in size to the cloned sequences

Recombinant Collagen Engineered to Bind to Discoidin Domain Receptors Functions as a Receptor Inhibitor

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities

Collagens in avian neural crest development: distribution in vivo and migration-promoting ability in vitro

This study examines the spatiotemporal distribution of collagen (Col) types I-V and IX during neural crest development in vivo and their ability to support neural crest cell movement in vitro. Col I, III and IV were widespread throughout the embryo, including the neural crest migratory pathways, whereas Col II, V and IX preferentially localized to regions from which migrating neural crest cells were absent. Col I-IV and IX occurred both in association with basement membranes and within interstitial matrices, whereas Col V only was detected in juxtaposition to basement membranes. Although initially distributed throughout the rostrocaudal extent of the somitic sclerotome, Col I and III rearranged to the caudal portion with progressive neural crest cell migration through the rostral portion of the sclerotome. This rearrangement does not occur in neural crest-ablated embryos, suggesting that it is a direct consequence of neural crest cell migration. The perinotochordal matrix, avoided by neural crest cells, contained a metameric Col II/IX immunoreactivity along the rostrocaudal axis which alternated with that of Col I and III. In contrast, Col IV and V were not observed in this matrix, but lined the basement membranes of the notochord and ventrolateral neural tube. To determine their functional significance for neural crest cell migration in vivo, purified collagens were tested for their ability to promote neural crest cell motility in vitro. Neural crest cell migration on isolated collagens was most pronounced on Col I and IV, whereas Col II, V and the triple-helical fragment of Col VII were unable to support cell motility. Substrata created by copolymerization of Col I and fibronectin, or Col I and laminin-nidogen, supported cell motility better than Col I alone, whereas both Col V and a cartilage-type chondroitin sulfate proteoglycan reduced cell movement on Col I. Fibronectin bound to pre-immobilized monomeric Col I, II or V had a reduced ability to support neural crest cell movement when compared to fibronectin alone. A similar reduction was seen for Col IV bound to the low density heparan sulfate proteoglycan from the EHS mouse tumor. The results demonstrate that Col I-IX are differentially distributed in the early avian embryo. During neural crest development several of these collagens undergo dynamic reorganizations that correlate with the migration of neural crest cells. Furthermore, various collagens possess distinct abilities to support neural crest cell migration in vitro, and their migration-promoting activity can be modulated by their conformation and/or association with other matrix components

A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction

Background: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen- 2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction

The cuticle

The nematode cuticle is an extremely flexible and resilient exoskeleton that permits locomotion via attachment to muscle, confers environmental protection and allows growth by molting. It is synthesised five times, once in the embryo and subsequently at the end of each larval stage prior to molting. It is a highly structured extra-cellular matrix (ECM), composed predominantly of cross-linked collagens, additional insoluble proteins termed cuticlins, associated glycoproteins and lipids. The cuticle collagens are encoded by a large gene family that are subject to strict patterns of temporal regulation. Cuticle collagen biosynthesis involves numerous co- and post-translational modification, processing, secretion and cross-linking steps that in turn are catalysed by specific enzymes and chaperones. Mutations in individual collagen genes and their biosynthetic pathway components can result in a range of defects from abnormal morphology (dumpy and blister) to embryonic and larval death, confirming an essential role for this structure and highlighting its potential as an ECM experimental model system

Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: identification and characterization of a novel serine protease inhibitor.

The nematode Caenorhabditis elegans represents an excellent model in which to examine nematode gene expression and function. A completed genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of nematode genes. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. Many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterized bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed

Immunohistochemical localization of collagen types I and VI in human skin wounds

A total of 74 human skin wounds were investigated and collagen types I and VI were localized in the wound area by immunohistochemistry. Collagen type I appeared in the form of ramifying string-like structures after approximately 5–6 days, but positive reactions in the form of a spot-like staining around isolated fibroblasts also occurred in a skin wound aged 4 days. Collagen VI was detectable after a post-infliction interval of at least 3 days showing a strongly positive reacting network associated with fibroblasts in the wound area. Both collagens appeared almost constantly after a wound age of 6–7 clays and could also be found in wounds aged a few months. Therefore, although a positive reaction for collagen type I in the form of string-like and ramifying structures around wound fibroblasts indicates a wound age of at least 5–6 days, a spot-like positive staining for collagen type I cannot exclude a wound age of at least 4 days. A positive staining for collagen type VI represents a post-infliction time of 3 days or more. The almost constant appearance of these collagen types suggests that negative results in a sufficient number of specimens indicate a wound age of less than 6–7 days, but cannot completely exclude longer post-infliction intervals. Since collagen type I and VI are also found in the granulation/scar tissue of lesions with advanced wound age, the immunohistochemical analysis of these proteins provides no further information for an age determination of older skin wounds

Mesenchymal Differentiation and Organ Distribution of Established Human Stromal Cell Lines in NOD/SCID Mice

Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo. Copyright (C) 2001 S, Karger AG, Basel

Transcriptomic analysis of Ascaris suum larvae during their hepatopulmonary migration

Common roundworms are important intestinal nematodes of man (Ascaris lumbricoides) and pig (Ascaris suum). During the first stages of the infection, the larvae of these parasites undergo a hepatopulmonary migration. This migration is likely to require tightly regulated transcriptional changes in the parasite. We explored this aspect in Ascaris suum by characterizing the transcription profiles of infective L3s from eggs, liver- and lung-L3s and intestinal L4s by next generation sequencing approach. When the most abundant transcripts per life stage were investigated, results showed that in the egg-L3s, transcripts associated with the regulation of translation and transcription, mainly ribosomal proteins, were most abundant. From the liver-L3s onwards, high transcription levels were seen for cuticle collagens, indicating the growth of the larvae during their migration. Interestingly, the type of highly expressed cuticle collagens in the intestinal L4s differed with those present in the liver- and lung-L3s. Apart from collagens, potentially important molecules for host-parasite interaction like C-type lectin-4 and Mucin-5 were in the top 5 of most abundant transcripts in the lung-L3. Unfortunately, a great number of transcripts that are specific for certain larval stages did not show any homology to other proteins within the NCBI database, suggesting that many biologically interesting molecules from this parasite are still to be investigated