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R Timpl

Publications and source records attributed to R Timpl.

At least 289 records · Page 16Linked to original sources
PubMed1985

Structure and biology of the globular domain of basement membrane type IV collagen.

A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpasture's syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.

Animals↗
PubMed1985

Extracellular matrix and epithelial differentiation of Wilms' tumor.

Different histologic types of 82 Wilms' tumors were graded on the basis of the histologic pattern. Representative tumors of each group were analyzed by the immunoperoxidase method for evaluation of the histogenesis of Wilms' tumor and the value of antibodies against extracellular matrix (ECM) components (collagens I and III, laminin, fibronectin) in differential diagnosis of different types of Wilms' tumors. The tubules of classic Wilms' tumor expressed laminin, which could be seen also in and around some blastemal cells. Blastema and tubules were negative for interstitial collagens, but Type I and III collagen were prominent in the fibrovascular stroma. The monomorphous tubular, psammomatous and rosetting tumors expressed laminin, but no interstitial collagens. In sarcomas, only the blastemal variant of spindle-cell sarcomas was negative for interstitial collagens, which were abundantly seen in all other sarcomas. While spindle-cell sarcomas were devoid of laminin, the highly malignant rhabdoid and clear-cell sarcomas expressed laminin in a characteristic dotted fashion. Staining for fibronectin gave varying results and had therefore only a limited value in distinguishing different types of Wilms' tumors. However, the antibodies against interstitial collagens and against the basement membrane glycoprotein laminin turned out to be a useful adjunct in differential diagnosis and classification, especially of sarcomatoid Wilms' tumors. The basement membrane of normal nephrons is similar to that in tubules of triphasic Wilms' tumor, but the ECM of blastemas is different. This transformed phenotype might represent a maturation arrest of the blastemal cell when compared with the expression of proteins during normal nephrogenesis.

Adenocarcinoma↗
PubMed1985

Tumor growth and metastasis increases the serum concentration of basement membrane proteins.

Serum concentrations of two basement membrane proteins, laminin and collagen type IV, were studied in three spontaneously metastasising experimental tumors: 3LL Lewis Lung and B16 melanoma of the mouse and R3230 adenocarcinoma of the rat. In the Lewis Lung tumor the mass of the primary tumor or the degree of metastasis correlated well with the concentration of serum laminin. In B16 melanoma the level of serum laminin was only significantly raised when the tumor had metastasised to the lung. No correlation between tumor mass and serum laminin was found in the R3230 adenocarcinoma. The serum concentrations of collagen IV, however, were raised to a certain extent during primary tumor growth and metastasis. Immunofluorescent staining for laminin in the Lewis Lung tumor demonstrated positive reactions in the primary tumor and in metastases of the lung. The data indicate the possibility of using similar tests for clinical tumor diagnosis.

Adenocarcinoma↗
PubMed1984

Domain structure of cartilage proteoglycans revealed by rotary shadowing of intact and fragmented molecules.

The rotary-shadowing technique for molecular electron microscopy was used to study cartilage proteoglycan structure. The high resolution of the method allowed demonstration of two distinct globular domains as well as a more strand-like portion in the core protein of large aggregating proteoglycans. Studies of proteoglycan aggregates and fragments showed that the globular domains represent the part of the proteoglycans that binds to the hyaluronic acid, i.e. the hyaluronic acid-binding region juxtapositioned to the keratan sulphate-attachment region. The strand-like portion represents the chondroitin sulphate-attachment region. Low-Mr proteoglycans from cartilage could be seen as a globule connected to one or two side-chain filaments of chondroitin sulphate.

Animals↗
PubMed1984

Structure and interactions of heparan sulfate proteoglycans from a mouse tumor basement membrane.

Various forms of heparan sulfate proteoglycan were solubilized from the mouse Engelbreth-Holm-Swarm (EHS) sarcoma by extraction with 0.5 M NaCl, collagenase digestion and extraction with 4 M guanidine. They could be separated into high (greater than or equal to 1.65 g/ml) and low (1.38 g/ml) buoyant density variants. The high-density form from the NaCl extract and collagenase digest had Mr = 130000 and So20,W = 4.5 S and contained 4-10% protein, indicating Mr = 5 000-12 000 for the protein core. This proteoglycan exhibited polydispersity as shown by rotary shadowing electron microscopy and ultracentrifugation. An average molecule consisted of four heparan sulfate chains (Mr = 29 000) each with a length of 32 +/- 10 nm. The low-density form (Mr about 400 000) could not be completely purified and contained about 50% protein. As shown by radioimmunoassay, the various proteoglycans shared similar protein cores. Labeling of the tumor in vivo or in vitro demonstrated preferential incorporation of radioactive sulfate in the high-density form. The high-density proteoglycan interacted in affinity chromatography by virtue of its heparan sulfate chains with laminin, fibronectin, the globular domain NC1 and the triple helix of collagen IV. These interactions were abolished at moderate concentrations of NaCl (0.1-0.2 M) and in the presence of heparin, chondroitin sulfate or dextran sulfate. Interactions with the globule NC1 could also be demonstrated by velocity band centrifugation in sucrose gradients and a binding constant of about 10(6) M-1 was derived.

Animals↗
PubMed1984

Immunochemistry, genuine size and tissue localization of collagen VI.

Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple-helical and globular structures. Antisera prepared against a constituent-chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross-react with other collagen types I-V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide-bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers. Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross-striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.

Cells, Cultured↗
PubMed1984

Sequence comparison of pepsin-resistant segments of basement-membrane collagen alpha 1(IV) chains from bovine lens capsule and mouse tumour.

The C-terminal peptic fragment P1 (about 518 amino acid residues) of bovine lens-capsule collagen alpha 1(IV) chain was cleaved with CNBr and trypsin. The peptides were purified and characterized, allowing their ordering within the P1 fragment by comparison with a corresponding section of mouse collagen alpha 1(IV) chain [Schuppan, Glanville & Timpl (1982) Eur. J. Biochem. 123, 505-512]. About 67% of the sequence of bovine collagen fragment P1 was determined by Edman degradation. Comparison with the sequence of the corresponding mouse collagen fragment P1 showed 76% identity for positions Xaa and Yaa of the triplet structures Gly-Xaa-Yaa. Invariance was found for the positions of two non-triplet interruptions and of 3-hydroxyproline residues, pointing to the functional importance of these structures.

Amino Acid Sequence↗
PubMed1984

Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.

Amino Acid Sequence↗
PubMed1984

Binding of Streptococcus pyogenes to laminin.

Some strains of Streptococcus pyogenes isolated from infected human tissues were shown to bind laminin, a major component of basement membranes. Binding of 125I-laminin to bacteria was time dependent and functionally irreversible. Of several unlabeled proteins tested in competition experiments, laminin and fibrinogen inhibited binding of the radiolabeled protein. The inhibitory effect exerted by fibrinogen was apparently not caused by a binding to the laminin receptors. The number of receptors available for laminin on cells of the strain examined ranged from 0 to 10(3) depending on the media used to grow the bacteria and an apparent KD of 4 X 10(-8)M was calculated for the reaction. Bacterial cells incubated with proteolytic enzymes lose the ability to bind laminin, and a trypsin digest contained active receptors capable of competing with intact cells for 125I-laminin. Active receptors may be adsorbed on a column of laminin-Sepharose but not on Sepharose gels substituted with fibrinogen or fibronectin. After radiolabeling the proteins in the trypsin digest a laminin-binding 125I-labeled protein (Mr greater than 10(6] was isolated by affinity chromatography from a receptor positive strain. Similar components could not be isolated from a strain apparently lacking laminin receptors. Therefore, this protein was tentatively identified as a laminin receptor of streptococci.

Binding, Competitive↗
PubMed1984

Subunit structure and assembly of the globular domain of basement-membrane collagen type IV.

The globular domain of collagen IV was solubilized by collagenase digestion from a mouse tumor, human placenta and bovine aorta and was purified by chromatographic methods. The materials show a unique, mainly non-collagenous amino acid composition and contain small amounts of glucosamine and galactosamine. The globular structures with Mr = 170 000 appear as a hexameric assembly originating from two collagen IV molecules. Subunits of this assembly are two different dimers Da and Db (Mr about 56 000) and monomers (Mr = 28 000). Their N-terminal amino acid sequences start with short triple-helical sequences, which overlap with the C-terminal triple helix of the alpha 1(IV) and alpha 2(IV) chain, demonstrating that the globule originates from the C terminus of collagen IV. Dimers arise from monomers by disulfide cross-linking (form Db) and/or formation of non-reducible cross-links (form Da). Reduction under non-denaturing conditions causes partial dissociation of the globule and of collagen IV dimers, indicating that reducible cross-links are formed between monomers of two different collagen IV molecules. Dissociation of the hexamer into the subunits can be achieved with 8 M urea, sodium dodecyl sulfate or in the pH range 2.5-4. The latter indicates that carboxyl groups are essential for association. Mixtures of the subunits (monomers and dimers) or purified dimers reassemble in neutral buffer into hexamers as shown by ultracentrifugation and electron microscopy. Reconstituted hexamers, however, dissociate in a much broader pH range than the native globules. Circular dichroic spectra indicate that the structure is more completely refolded from acid-treated than from urea-treated material. These data suggest that globules originating from monomers (as existing in single collagen IV molecules) are stabilized by the adjacent triple helix. Covalent cross-link formation stabilizes the globular structure and allows reconstitution in stoichiometric proportions.

Animals↗
PubMed1984

Laminin, proteoglycan, nidogen and collagen IV: structural models and molecular interactions.

Major components of basement membranes, including collagen IV, laminin, heparan sulphate proteoglycan and nidogen, were isolated from the matrix of the EHS sarcoma. The purified components were analysed for their domain structure and for the participation of distinct domains in molecular interactions and cell binding. Collagen IV consists of four domains which have triple helical or non-collagenous structures. Self-assembly of the protein into a network-like organization occurs by specific interactions between N-terminal triple helical segments and between the C-terminal globules. Cell binding requires a central triple helical segment. Laminin has the shape of an asymmetrical cross; different globular domains within this structure mediate binding to proteoglycan and to cells. The proteoglycan consists of four heparan sulphate chains attached to a small protein core. These chains have the potential to bind laminin, fibronectin and collagen IV. Nidogen was isolated in several molecular forms which showed either self-aggregation or binding to laminin.

Animals↗
PubMed1984

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival.

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival-potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity-purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin-binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell-binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod-like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin-binding domain of laminin is therefore responsible for its effects on neurons.

Animals↗
PubMed1984

Glomerular basement membrane synthesis and serum concentration of type IV collagen in streptozotocin-diabetic rats.

Glomerular basement membrane synthesis was measured in vivo in diabetic rats at various times after streptozotocin injection. Simultaneously the type IV collagen concentration in serum was determined by a radioimmunoassay specific for the terminal cross-linking domain, 7S collagen. Basement membrane collagen synthesis and the 7S collagen levels were markedly raised in diabetic animals 18-34 days after streptozotocin injection and showed a significant correlation. The results suggest that the amount of serum 7S collagen reflects increased basement membrane synthesis in a diabetic situation. Both basement membrane collagen synthesis and serum 7S collagen concentration also show a positive correlation with the blood glucose levels of diabetic rats. This indicates that the degree of metabolic dysregulation influences basement membrane synthesis.

Age Factors↗
PubMed1984

Dual origin of glomerular basement membrane.

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowman's capsule).

Allantois↗
PubMed1984

Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death.

The role of collagen I for midgestation development was studied in homozygous Mov 13 embryos, which cannot synthesize alpha 1(1) mRNA as a result of insertional mutagenesis and most of which die between day 12 and 14 of gestation. No type I collagen was detected in mutant embryos, while the distribution of other collagens, laminin, and fibronectin was not affected. Mutant embryos develop normally up to day 12 of gestation, suggesting that collagen I has no essential role in the early phase of morphogenesis. The first pathological events were detected in hemopoietic cells of the liver, followed by necroses of mesenchymal cells in other parts of the embryo. The sudden death is caused by the rupture of a major blood vessel, indicating an important role for collagen I in establishing the mechanical stability of the circulatory system. Our results furthermore suggest that complex cell interactions in embryonic development such as those in early hemopoiesis may depend on the presence of collagen type I.

Animals↗