Extracellular matrix components synthesized by human amniotic epithelial cells in culture.
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Amnion↗
Biomedical subjects
R Timpl
Publications and source records attributed to R Timpl.
Characterization of antibodies to basement membrane (type IV) collagen in immunohistological studies.
Antisera were produced in rabbits and guinea pigs against basement membrane (type IV) collagens which were extracted from a mouse tumor with acetic acid and from human placenta after limited digestion with pepsin. The antisera were specific for type IV collagen and did not crossreact with collagens type I, II and III from interstitial connective tissue, with collagen type V (AB2) from placenta and with a non-collagenous protein (laminin) obtained from basement membranes. Purified antibodies against both human and type IV collagen reacted in indirect immunofluorescence tests with the mouse tumor matrix and with authentic basement membranes in various human and mouse tissues. These antibodies failed to react with interstitial connective tissue. Absorption of antibodies against type IV tumor collagen with mouse or human kidney homogenate abolished or significantly decreased their reaction with tumor tissue. The findings indicate that various basement membranes may contain related or identical collagenous proteins which show a high degree of interspecies homology.
Absorption↗
Induction of a basement membrane glycoprotein in embryonic kidney: possible role of laminin in morphogenesis.
The glycoprotein laminin is found exclusively in the basement membranes of adult tissues, not in the mesenchymal stroma. We studied the appearance and distribution of laminin during the early formation of kidney tubules in mouse embryos and in an in vitro transfilter model system. In immunofluorescence using affinity-purified antibodies, the distribution of laminin showed a clear correlation, both spatially and temporally, to the early stages of tubule formation. In vivo, laminin was first detected in a punctate pattern in areas where the pretubular aggregates form; later, it became confined to the basement membranes of the tubules. In experiments in vitro, the nephrogenic mesenchyme was found to form tubules after 12-24 hr of transfilter contact with the inductor. The first laminin spots were found after 12 hr of culture, 24 hr before overt morphogenesis. As the mesenchymal cells began to aggregate and elongate (at 36 hr), laminin was detected in those cells destined to become epithelial, and at 48 hr it was not found in cells remaining in the stroma. In more mature tubules (at 72 hr), laminin was seen as a sharp band in the basement membranes. It is suggested that laminin is involved in the increased cell adhesiveness during the early aggregation of the nephrogenic mesenchyme.
Animals↗
Immunofluorescence analysis of collagen, fibronectin, and basement membrane protein in scleroderma skin.
Scleroderma skin and the subcutaneous tissue was studied by indirect immunofluorescence with specific antibodies against interstitial collagens and procollagens, against fibronectin and against the basement membrane proteins Type IV collagen and laminin. Staining for Type I procollagen and fibronectin was distinctly increased in the lower dermis and subcutaneous tissue. When compared with normal skin the data suggests that fibrosis may start around capillaries and in close proximity to adipose cells. Additional changes in the distribution to Type IV collagen and laminin were found in some patients and probably reflect the alterations in small blood vessels.
Adult↗
Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization.
The kinetics of triple-helix formation in type III pN-collagen, type III collagen and a quarter fragment of type III collagen was followed by optical rotation and circular dichroism. Kinetic intermediates were detected by trypsin digestion and polyacrylamide gel electrophoresis. The end products of refolding at 25 degrees C were identical to the native molecules according to their melting profiles, molecular weights and sedimentation behavior. Only at low temperatures (4-15 degrees C) were mismatched structures of lower stability formed. At 25 degrees C helix formation started exclusively at the set of three disulfide bridges which link the three chains at the carboxy-terminal end. The growth of the triple helix proceeds from this single nucleus at a rather uniform rate in a zipper-like fashion. This gives rise to zero-order kinetics over a large fraction of the conversion. Consequently the time of half conversion is proportional to the length of the molecule. From the appearance and disappearance of intermediates the growth of the triple helix could be observed directly. The rate of helix propagation is determined by the rate of cis leads to trans isomerization of peptide bonds. A model mechanism was devloped which quantitatively described the overall kinetics as well as the time course of the intermediates with a single set of parameters: the rate constant of cis leads to trans isomerization k = 0.015 s-1 and an average number of 30 tripeptide units in uninterrupted stretches of residues with all peptide bonds in trans configuration.
Animals↗
7-S collagen: characterization of an unusual basement membrane structure.
A new type of collageneous structure, tentatively named 7-S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistance towards further degradation by bacterial collagenase at 20 degrees C. 7-S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl-galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360,000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm = 48 degrees C and 70 degrees C. The more stable domain could be isolated as an homogeneous fragment (Mr = 225,000) after a second digestion with collagenase at 37 degrees C. This fragment contained all the disulfide bonds (42 Cys/1,000 residues) of the original molecule. Electron microscopy showed a rod-like structure in agreement with the hydrodynamic properties of 7-S collagen. The dimensions of these peptides were 3 X 95 nm (long form) and 2.4 X 40-50 nm (short form). Complete reduction of 7-S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27,000-153,000 which differ from each other by Mr increments 25,000-27,000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross-linked or represent fragments produced during proteolytic treatments. Complete reduction of 7-S collagen under non-denaturing conditions lowered the thermal transiton of the triple helix to 48 degrees C but did not change its molecular weight except when exposed to dissociating solvents. 7-S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37 degrees C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross-reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7-S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.
Amino Acids↗
Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line.
The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.
Amino Acids↗
Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin, and fibronectin.
Specific antibodies to collagen type IV, laminin, and fibronectin were used to localise these proteins by indirect immunofluorescence in frozen sections of normal and fibrotic liver. In normal livers distinct staining was found in basement membranes of blood and lymph vessels, of bile ducts and ductules and around nerve axons. Positive reactions for type IV collagen and fibronectin were also observed in the perisinusoidal space, while hepatocytes and most of the interstitial matrix of portal fields remained unstained. Liver specimens obtained from patients with alcoholic liver disease (fatty liver, hepatitis or cirrhosis) and chronic active hepatitis showed a more intense reaction with the antibodies in the perisnusoidal space including now distinct staining for laminin. These patterns were particularly prominent at borders between fibrotic septa and remnants of parenchyma or pseudolobules. Strong reactions were also found for type IV collagen and fibronectin in the periportal interstitium and in large fibrotic areas. The findings support previous electron-microscopical and chemical evidence for increased basement membrane production in human liver fibrosis and demonstrate that this may involve different proteins and occur at different anatomical sites.
Adolescent↗
Characterization of pepsin fragments of laminin in a tumor basement membrane. Evidence for the existence of related proteins.
Laminin was extracted with neutral buffer from a tumor basement membrane and subjected to extensive degradation by pepsin. The treatment released two homogenous fragments P1 (Mr = 290,000) and P2 (Mr = 45,000) in addition to a mixture of smaller peptides. Fragments P1 and P2 together contained more than 90% of the disulfide bonds and accounted for about one third of the mass of laminin. Both peptides differed in amino acid composition, immunological properties and a complex chain structure demonstrating the existence of two disulfide-bonded domains in the molecule. Part of the laminin in the tumor matrix could only be solubilized by pepsin treatment and several fragments were purified. The major fragment P1i closely resembled P1 of soluble laminin in its chemical and immunological properties. Minor fragments Pa, Pb, Pc and Pd (Mr = 44,000-74,000) were only related to P1 or P2 in amino acid composition, chain pattern and antigenicity. The data were interpreted as indicating that proteins similar but not identical to laminin exist in the basement membrane and account for the minor peptide variants.
Amino Acids↗
Mummies from Peru. Demonstration of antigenic determinants of collagen in the skin.
This paper describes immunofluorescence studies on the possible preservation of antigenic determinants of different types of collagen in sections of the skin of human mummies from Peru. The age of the mummies was dated from the 4th to the 14th century AD. Using specific antibodies, it was possible to demonstrate type I and type III collagen. The antigenic determinants specific for procollagen type I, procollagen type III and the type IV collagen component of basement membranes were not preserved.
Adolescent↗
Study on the nature of the Goodpasture antigen using a basement membrane-producing mouse tumour.
Autoantibodies in the sera of patients with Goodpasture's syndrome showed a strong reaction in indirect immunofluorescence tests on unfixed, frozen sections of a mouse tumour (EHS sarcoma), previously shown to produce extracellular basement membrane. Anti-basement membrane antibodies from patients with bullous pemphigoid failed to react with the mouse tumour, but showed a distinct reaction with cylindroma tissue. Absorption of Goodpasture sera with tumour homogenate completely abolished their reaction on sections of human and murine kidney. Basement membrane (type IV) collagen and a high molecular weight, non-collagenous glycoprotein were isolated from the tumour matrix and studied in absorption experiments and radioimmunoassays. Little or not reaction was observed with Goodpasture patients' sera indicating that neither of these two proteins is the major antigen involved in the disease. Antigenic material reacting with Goodpasture sera was extracted from the tumour in neutral salt solutions, suggesting that it is a non-collagenous protein.
Animals↗
Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.
Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen.
Antibodies↗
Laminin--a glycoprotein from basement membranes.
We have isolated a large noncollagenous glycoprotein, laminin, from a mouse tumor that produces basement membrane. The protein consists of at least two polypeptide chains (Mr = 220,000 and Mr = 440,000) joined to each other by disulfide bonds. Laminin and type IV collagen are major constituents of the tumor. Laminin is distinctly different from fibronectin, another component of basement membranes, in amino acid composition and immunological reactivity. Pepsin digestion of laminin releases a large, cystine-rich fragment which retains most of the antigenicity of the original protein. Immunological studies using purified antibody against laminin show that it is produced by a variety of cultured cells. In addition, these antibodies react with the basement membranes of normal tissues, suggesting that this protein or an immunologically related protein is a constituent of the basement membranes of these tissues.
Amino Acids↗
Type III procollagen is the major collageneous component produced by a continuous rhabdomyosarcoma cell line.
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Cell Line↗
Immunological approaches to the structure of fibrinogen.
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Amino Acid Sequence↗
Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.
The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.
Amino Acid Sequence↗
Structure of antigenic determinants in the N-terminal region of dermatosparactic sheep procollagen type I.
About half of the rabbit antisera raised against type-I procollagen, p alpha 1(I) chain or nonreduced procollagen peptides reacted in a radioimmunoassay with the reduced form of peptide Col 1, which comprises the whole non-collagenous region at the N-terminus of procollagen. Proteolytic fragments prepared from reduced peptide Col 1 were still effective inhibitors of the antibodies and allowed the localization of two antigenic determinants. The antigenically active regions have the sequences less than Glu-Glu-Glu-Gly-Gln-Gln-Glu and Gly-Asp-Thr-Gly-Pro-Arg, and are located at the N- and C-termini of the peptide respectively. Antibodies raised against reduced peptide Col 1 bind to a determinant localized in a different region of the peptide.
Amino Acid Sequence↗
Identification of a new basement membrane collagen by the aid of a large fragment resistant to bacterial collagenase.
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Amino Acids↗