Collagens: molecular and antigenic structure.
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Basement Membrane↗
Biomedical subjects
R Timpl
Publications and source records attributed to R Timpl.
Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts.
Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.
Acetaldehyde↗
[Basement membranes--structure, function, pathology].
Basement membranes are extracellular structures with a heterogeneous molecular composition. Several components have been identified and could be localized in specific morphological structures. Type IV collagen is found in the lamina densa, whereas laminin is the major component of the lamina lucida. Small amounts of heparan sulfate proteoglycan are also present in the lamina lucida. In some basement membranes, fibronectin and another glycoprotein, nidogen, could be identified. Epidermal basement membranes contain, in addition, the bullous pemphigoid antigen. Basement membranes are involved in several diseases and play an important part in tumor progression. Antibodies against distinct components of basement membranes have been shown to be useful as diagnostic tools in bullous disorders (e.g., epidermolysis bullosa) and for identifying the extracellular matrix of skin tumors (e.g., neurofibroma, cylindroma, granular cell myoblastoma).
Basement Membrane↗
Nidogen: a new, self-aggregating basement membrane protein.
Nidogen was purified from a mouse tumor basement membrane where it accounted for 2-3% of the total proteins. It was isolated as two forms (A and B) of a monomer (Mr = 80000) each consisting of a single polypeptide chain folded into a globular head connected to a small tail. The B form of the monomer was shown to be capable of aggregating into a nest-like structure (Mr greater than 250000). A smaller form (Mr = 45000) was observed in some of the extracts. The amino acid composition of nidogen was different to that of other basement membrane proteins. It contained about 10% carbohydrate, with N-linked and O-linked oligosaccharide chains in similar proportions. Isoelectrofocussing demonstrated a limited heterogeneity of nidogen with pI in the range 6.5 - 7. Monomeric nidogen failed to interact with other basement membrane components and heparin. Aggregation could be induced by limited proteolysis and was reversed by detergents or high salt concentrations. Together with the observation that most of the nidogen could be solubilized only after destroying the collagenous matrix, the data indicate that aggregation of nidogen reflects an activity involved in matrix assembly. Specific antibodies raised against nidogen did not distinguish between the monomeric and aggregated form of the protein but showed that the fragment was antigenically deficient. These antibodies did not cross-react with collagen type IV, laminin, entactin and heparansulfate proteoglycan. Immunofluorescence staining and absorption studies demonstrated that nidogen is a common component of authentic basement membranes. Larger forms of nidogen (Mr about 100000 and 150000) were found in organ cultures of Reichert's membrane suggesting that it is synthesized in precursor forms.
Animals↗
Immunochemical properties of the aminopropeptide of procollagen type III.
The precursor-specific aminopropeptide of bovine type III procollagen is a strong immunogen in rabbits, guinea pigs and mice and induces antibodies which do not cross-react with type I procollagen. The antibody response is regulated by immune response genes associated with the major histocompatibility complex. Major antigenic determinants were found in the compact, non-collagenous domain (fragment Col 1) located at the N terminus of the aminopropeptide and were destroyed by reduction of disulfide bonds. Minor antigenic determinants independent of disulfide bonds also exist in fragment Col 1 and could be localized on a distinct tryptic peptide. Fragment Col 1 showed a lower affinity for antibody when compared with the intact aminopropeptide which causes a non-parallel shift in radioimmuno-inhibition profiles. Monovalent antibody fragments showed an average tenfold reduction in affinity constant and failed to distinguish between aminopropeptide and fragment Col 1. This indicates that the stronger binding of bivalent antibody by the triple-stranded aminopropeptide is due to multiple interactions with both antibody binding sites which are lost for a single-stranded antigen (Col 1) or with monovalent antibody fragments.
Animals↗
Characterization of protease-resistant fragments of laminin mediating attachment and spreading of rat hepatocytes.
In attempts to identify cell binding domains in the basement membrane protein laminin (Mr = 900,000), responsible for the substrate attachment mediating activity of the protein, proteolytic fragments were isolated and compared with respect to their biological activity. The fragments analyzed were generated by digestion of the protein with elastase or pepsin and included the previously described fragments 1 to 4 and three additional fragments, 5, 6, and 7 with molecular weights of 30,000 to 50,000. Fragments 1, 5, and 6 promoted substrate attachment of rat hepatocytes. Fragment 5, and to a lesser extent also fragment 6, but not fragment 1, induced spreading of the cells. Other fragments including the heparin-binding domain were inactive. Structural and immunological analyses indicated that fragment 1 is distinctly different from the other cell-binding fragments, whereas fragment 5 and 6 are similar but not identical. Furthermore, the active fragments were localized to different regions of the three short arms of the cross-shaped laminin molecule. Thus, the data suggest that fragments 1, 5, and 6 represent three separate domains with cell binding capacity. Attachment of hepatocytes to the fragments but not to intact laminin could be inhibited by specific antibodies indicating that the intact protein may contain an additional cell-binding site.
Amino Acids↗
A monoclonal antibody specific for the amino terminal cleavage site of procollagen type I.
A monoclonal mouse IgG1 antibody was produced against the aminopropeptide of dermatosparactic sheep procollagen type I by using the hybridoma technique. Radioimmunoassays demonstrated an apparent affinity constant of 10(8) l X mol-1. The antibody reacted with a 19-amino-acid-long sequence spanning the procollagen N-proteinase cleavage site with stronger binding to structures contributed by the aminopropeptide. The antibody showed strong cross-reactions with similar antigens of bovine, human or chick origin but failed to react with the aminopropeptide of procollagen type III. Incubation of chick or sheep procollagen type I with stoichiometric amounts of antibody blocked the release from procollagen molecules of the aminopropeptide by procollagen N-proteinase. Thus, this antibody seems useful for studying various biological problems encountered in the conversion of procollagen.
Animals↗
Relationship of collagen-tailed acetylcholinesterase with basal lamina components. Absence of binding with laminin, fibronectin, and collagen types IV and V and lack of reactivity with different anti-collagen sera.
In view of their supposed localization in extracellular structures, such as basal lamina, we have investigated the possible interactions of collagen-tailed forms of acetylcholinesterase from Electrophorus and bovine superior cervical ganglion with matrix proteins: laminin, fibronectin and types IV and V collagens. Using binding and sedimentation assays, with iodinated or non-radioactive matrix proteins, we have not observed any significant interaction, in conditions of high or low ionic strength. We also examined whether the collagen tail of acetylcholinesterase asymmetric forms possessed an immunological relationship with known collagen types (I, III, IV, V) from mammalian sources. We found no specific immunoreactivity with any of the 32 sera studied, either with the iodinated Electrophorus or with the native bovine enzyme. We conclude from these negative results that the collagen-like tail of acetylcholinesterase is clearly distinct from the classical types of collagen and that asymmetric forms of the enzyme do not interact specifically with the matrix proteins studied. This does not exclude the possibility of specific interactions with other components, remaining to be identified.
Acetylcholinesterase↗
Monoclonal antibodies used as probes for the structural organization of the central region of fibronectin.
Two monoclonal mouse antibodies against human plasma fibronectin were compared in their reactivity for proteolytic fragments of the antigen by enzyme immunoassay and immunoblotting. These antibodies were shown to react with two different structures within a short segment (about 30 kDa) located about one-third away from the C-terminus of the fibronectin chains.
Animals↗
Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta.
Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains.
Amino Acids↗
Electron-microscopical approach to a structural model of intima collagen.
Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.
Chemical Phenomena↗
Serum and urine analysis of the aminoterminal procollagen peptide type III by radioimmunoassay with antibody Fab fragments.
A radioimmunoassay based on antibody Fab fragments was developed for the aminoterminal peptide Col 1-3 of bovine type III procollagen. This assay does not distinguish the intact aminopropeptide Col 1-3 from its globular fragment Col 1. Parallel inhibition profiles were observed with human serum and urine allowing the simultaneous quantitative determination of intact and fragmented antigens in these samples. Most of the material has a size similar to that of fragment Col 1 indicating that the aminopropeptide is degraded under physiologic conditions. The concentration of aminopeptide in normal sera was in the range 15-63 ng/ml. Daily excretion was found to be in the range 30-110 micrograms. More than 50% of patients with alcoholic hepatitis and liver cirrhosis showed elevated serum levels of aminopropeptide by the Fab assay. Elevated concentrations were detected more frequently with an antibody radioimmunoassay which measures mainly the intact form of the aminopropeptide. It is suggested that analysis of patients material by both assays could improve their diagnostic application.
Adolescent↗
Collagen fibril formation during embryogenesis.
Chicken embryo skin of different ages and adult skin were labeled with antibodies against the amino propeptide and carboxyl propeptide of type I collagen and processed for indirect immunoelectron microscopy by the ferritin technique. The results indicate that the formation of thin collagen fibrils involves polymerization of pN-collagen. Fibrils that are thicker than 35-40 nm do not appear to contain the amino propeptide. How fibrils increase in size is not clear, but growth may involve mechanisms such as lateral aggregation of subfibril structures or fusion of thin fibrils. Carboxyl propeptides were localized near or in contact with thin collagen fibrils, but they did not appear to be arranged in a periodic manner along the fibrils. In experiments using antibodies against the amino propeptides of type III collagen, fibrils 20-40 nm in diameter were also labeled in a periodic fashion. pN-Collagen chains were extracted from embryonic skin and identified by NaDodSO4/polyacrylamide gel electrophoresis and by immunoblotting. The presence of significant amounts of pN-collagen in skin from 10- and 12-day chicken embryos agreed well with the labeling of amino propeptides by immunoelectron microscopy. This study provides evidence for the role of the amino propeptide in collagen fibrillogenesis in embryonic skin.
Animals↗
In vivo and in vitro observations on laminin production by Schwann cells.
Recent reports on the dystrophic mouse mutant suggest that the prominent extracellular matrix component of peripheral nerve tissues plays an important role in peripheral nerve development. We have examined the disposition of two prominent extracellular matrix components, fibronectin and laminin, both in mature peripheral nerve in vivo and in an in vitro system that allows study of Schwann cells in various functional states. In frozen sections of whole nerve, staining with antibodies to fibronectin and laminin shows that fibronectin stains throughout the endoneurium while laminin staining is restricted to regions known to contain basal lamina, particularly the basal lamina of each ensheathing Schwann cell. Tissue culture studies indicate that fibronectin staining at the light microscopic level is a reliable marker for fibroblasts (and not Schwann cells) in culture; conversely, antibodies to laminin stain components related to the Schwann cell surface but not components related to fibroblasts. Unexpectedly, Schwann cells in culture produce laminin at all stages in development, whether in contact with axons or not. As Schwann cells in culture begin to ensheathe axons, punctate regions of laminin on their surfaces become confluent. After ensheathment is completed, a continuous line of staining is found in the region of the Schwann cell basal lamina. It has been established that Schwann cells produce a basal lamina only when in contact with axons. Therefore, the production of laminin appears to be necessary but not sufficient for basal lamina formation. The constancy of laminin production by Schwann cells provides a reliable basis for distinguishing between Schwann cells and fibroblasts in tissue culture.
Animals↗
Transferrin as a fetal growth factor: acquisition of responsiveness related to embryonic induction.
Differentiation of the metanephric mesenchyme, which is triggered by an inductive tissue interaction, has been shown to proceed in a chemically defined medium containing transferrin. Here, we report that neither transferrin-depleted serum nor a chemically defined medium devoid of transferrin promote differentiation and that activity can be restored by the addition of transferrin. It thus appears that we have identified the serum factor required for kidney differentiation. Transferrin seems to affect differentiation by stimulating cell proliferation. We show by using an organ-culture model system that only mesenchymes induced to differentiate by the 24-hr tissue interaction respond to transferrin by proliferation and differentiation, whereas uninduced mesenchymes remain unresponsive. The inductor tissue used is not responsive to transferrin. Thus, the data suggest that the short-range cell-mediated tissue interaction acts by making the nephrogenic mesenchyme responsive to the long-range mediator, which is transferrin. Transferrin is suggested to be an important circulating growth factor required for proliferation during embryogenesis.
Animals↗
Adhesion, growth, and matrix production by fibroblasts on laminin substrates.
Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates. These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production.
Antibodies↗
Neural crest cell migration: requirements for exogenous fibronectin and high cell density.
Cells of the neural crest participate in a major class of cell migratory events during embryonic development. From indirect evidence, it has been suggested that fibronectin (FN) might be involved in these events. We have directly tested the role of FN in neural crest cell adhesion and migration using several in vitro model systems. Avian trunk neural crest cells adhered readily to purified plasma FN substrates and to extracellular matrices containing cellular FN. Their adhesion was inhibited by antibodies to a cell-binding fragment of FN. In contrast, these cells did not adhere to glass, type I collagen, or to bovine serum albumin in the absence of FN. Neural crest cell adhesion to laminin (LN) was significantly less than to FN; however, culturing of crest cells under conditions producing an epithelioid phenotype resulted in cells that could bind equally as well to LN as to FN. The migration of neural crest cells appeared to depend on both the substrate and the extent of cell interactions. Cells migrated substantially more rapidly on FN than on LN or type I collagen substrates; if provided a choice between stripes of FN and glass or LN, cells migrated preferentially on the FN. Migration was inhibited by antibodies against the cell-binding region of FN, and the inhibition could be reversed by a subsequent addition of exogenous FN. However, the migration on FN was random and displayed little persistence of direction unless cells were at high densities that permitted frequent contacts. The in vitro rate of migration of cells on FN-containing matrices was 50 microns/h, similar to their migration rates along the narrow regions of FN-containing extracellular matrix in migratory pathways in vivo. These results indicate that FN is important for neural crest cell adhesion and migration and that the high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are necessary for effective directional migration.
Animals↗
Biosynthesis of type IV collagen by cultured rat Schwann cells.
We have obtained evidence that rat Schwann cells synthesize and secrete type IV procollagen. Metabolic labeling of primary cultures of Schwann cells plus neurons and analysis by SDS PAGE revealed the presence of a closely spaced pair of polypeptides in the medium of these cultures that (a) were susceptible to digestion by purified bacterial collagenase, (b) co-migrated with type IV procollagen secreted by rat parietal endoderm cells, and (c) were specifically immunoprecipitated by antibodies against mouse type IV collagen. Limited pepsin digestion of metabolically labeled medium or cell layers produced a pepsin-resistant fragment characteristic of pro-alpha 1(IV) chains. Removal of neuronal cell bodies from the cultures immediately before labeling did not reduce the amount of type IV procollagen detected in the medium. This indicated that Schwann cells, not neurons, were responsible for synthesis of type IV procollagen. We believe type IV procollagen is a major constituent of the Schwann-cell extracellular matrix based upon (a) its presence in a detergent-insoluble matrix preparation, (b) its presence in the cell layer of the cultures in a state in which it can be removed by brief treatment with bacterial collagenase or trypsin, and (c) positive immunofluorescence of Schwann cell-neuron cultures with anti-type-IV collagen antibodies. Secretion of type IV procollagen was substantially reduced when Schwann cells were maintained in the absence of neurons. This observation may account for the previously reported finding that Schwann cells assemble a basal lamina only when co-cultured with neurons (Bunge, M. B., A. K. Williams, and P. M. Wood, 1982, Dev. Biol., 92:449).
Animals↗