Biomedical subjects

R Timpl

Publications and source records attributed to R Timpl.

At least 37 records · Page 2Linked to original sources

PubMed1994

Binding of purified collagen receptors (alpha 1 beta 1, alpha 2 beta 1) and RGD-dependent integrins to laminins and laminin fragments.

Integrins alpha 1 beta 1 and alpha 2 beta 1 when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin-1, which has the chain composition alpha 1 beta 1 gamma 1. The binding was, however, about 10-fold lower than to collagen IV. Only little (alpha 1 beta 1) or no binding (alpha 2 beta 1) was observed to two different laminin isoforms (alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin alpha subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(beta 1 integrin). No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (E1XNd), which lacks the N-terminal beta 1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for alpha 1 beta 1 and alpha 2 beta 1 to the N-terminal region of the laminin alpha 1 chain. Fragment P1 but not intact laminin-1 bound to alpha V beta 3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD = 50 nM for alpha 2 beta 1/laminin-1 binding and a KD = 450 nM for alpha V beta 3/fragment P1 binding and confirmed the anti-beta 1-mediated increase in affinity for alpha 2 beta 1.

Amino Acid Sequence↗

PubMed1994

Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain.

The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.

Amino Acid Sequence↗

PubMed1994

Fibulin-2 (FBLN2): human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes.

Fibulin-2 is a new extracellular matrix protein that we recently identified by characterizing mouse cDNA clones. Fibulin-2 mRNA is prominently expressed in mouse heart tissue and is present in low amounts in other tissues. In this study, we isolated and sequenced a 4.1-kb human fibulin-2 cDNA, which encoded a mature protein of 1157 amino acids preceded by a 27-residue signal sequence. The predicted polypeptide contains three consecutive anaphylatoxin-related segments (domain I) in its central region followed by 10 EGF-like repeats (domain II), 9 of which have a consensus sequence for calcium binding. The 408-residue N-terminal region consists of two separate subdomains, a cysteine-rich segment of 150 residues (Na subdomain) and a cysteine-free segment with a stretch of acidic amino acids (Nb subdomain). The 115-residue C-terminal segment (domain III) is similar to the C variant of fibulin-1. The amino acid sequences of the human and mouse fibulin-2 share approximately 90% identity in domains Na, I, II, and III but only 62% identity in domain Nb. The human cDNA lacks an EGF-like repeat, which is alternatively spliced in the mouse cDNA clones, and a potential cell-binding Arg-Gly-Asp sequence found in the Nb domain of the mouse counterpart. Northern blot analysis of mRNA from various human tissues reveals an abundant 4.5-kb transcript in heart, placenta, and ovary tissue. The expression pattern differs from that of fibulin-1. The fibulin-2 gene was localized by in situ hybridization to the p24-p25 region of human chromosome 3 and to the band D-E of mouse chromosome 6.

Amino Acid Sequence↗

PubMed1994

The fibulin-1 gene (FBLN1) is located on human chromosome 22 and on mouse chromosome 15.

Fibulin-1 is a calcium-binding glycoprotein present in the extracellular matrix and in the serum. The gene coding for fibulin-1 (FBLN1) was located by in situ hybridization of 3H-labeled cDNA probes to human and mouse metaphase chromosomes. The gene was assigned to the q13.2-q13.3 region of human chromosome 22 and to the E-F band of mouse chromosome 15. This finding extends the evolutionary conservation between human chromosome 22 and mouse chromosome 15.

Animals↗

PubMed1994

The laminins.

Laminins are extracellular matrix proteins which consist of alpha, beta and gamma chains with molecular masses of 140-400 kDa. Chain association occurs through a large triple alpha-helical coiled-coil domain towards the C-terminus of each chain. Eight genetically distinct laminin chains (alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 1, gamma 2) and seven different assembly forms (laminins-1 to -7) are known so far. The most extensively characterized laminin-1 (alpha 1 beta 1 gamma 1) shows calcium-dependent self assembly and heterotypic binding to perlecan, nidogen, fibulin-1 and other matrix components. This binding indicates a crucial role in the supramolecular organization of basement membranes. Laminins also possess binding sites for at least six different integrin receptors and are thus involved in many cell-matrix interactions. Such interactions have been shown to be important during embryonic development and for tissue homeostasis and remodelling.

Animals↗

PubMed1994

Structure and stability of the triple-helical domains of human collagen XIV.

Two triple-helical domains, Col 1 and Col 2, were obtained from a pepsin digest of human placental collagen XIV and separated from each other under nondenaturing conditions. Edman degradation demonstrated 106 amino acids residues in the Col 1 and 149 residues in the Col 2 domain. All except one of the 37 prolines in the Yaa position of the Gly-Xaa-Yaa triplets were completely hydroxylated to 4-hydroxyproline, and there were three imperfections in the triplet repeat. Partial or complete hydroxylation and glycosylation were found for all seven lysines in the Yaa position. Domain Col 1 was joined by disulfide bonds into a trimer, while Col 2 appeared as a mixture of monomers and disulfide-linked dimers. Circular dichroic spectra were typical for the collagen triple helix and revealed relatively high melting temperatures for Col 1 (38 degrees C) and Col 2 (43 degrees C). An almost perfect refolding of the triple helix was observed for Col 1 but not for Col 2, emphasizing the importance of disulfide bonds for the folding kinetics and in part the stability of the triple helix. Circular dichroic spectra of the large nontriple helical domain, NC3, of collagen XIV indicated 11% alpha helix and 63% beta structure. Comparative melting profiles of NC3 and intact collagen XIV indicated that the triple helices in intact collagen XIV have a melting temperature of 44 degrees C.

Amino Acid Sequence↗

PubMed1994

Adhesion of human epidermal keratinocytes to laminin.

We have examined the mechanism by which human epidermal keratinocytes adhere to the A/B1/B2 (alpha 1 beta 1 gamma 1) form of laminin. Adhesion could be completely inhibited with an antibody to the beta 1 integrin subunit or a combination of antibodies recognising the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins. Keratinocytes adhered in the presence of magnesium and manganese ions, but calcium ions did not support adhesion and inhibited adhesion when combined with magnesium and manganese. The effects of anti-integrin antibodies (including a stimulatory antibody to the beta 1 subunit) were not influenced by specific cations, with the exception that inhibition by an antibody to alpha 2 beta 1 was abrogated by the presence of manganese ions. The E3 and E8 proteolytic fragments of laminin did not support keratinocyte adhesion and heat inactivation of the E8 site in intact laminin did not reduce adhesion. Three laminin fragments that did support adhesion were P1, E4 and E1X-Nd, P1 activity being attributable at least in part to the RGD site; antibody blocking experiments suggested that adhesion to these fragments was primarily via alpha 3 beta 1. The synthetic peptide GD-6, derived from the carboxy terminus of the laminin A chain (included within E3) did support adhesion, but the significance of this observation is unclear, since a scrambled control peptide could also support adhesion. In conclusion, keratinocyte adhesion to A/B1/B2 laminin involves three integrins and multiple binding sites that are different from those defined previously.

Amino Acid Sequence↗

PubMed1994

Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition.

The inhibitory capacities of six different disintegrins and one related neurotoxin analogue for the binding of RGD-dependent integrins to either fibrinogen, vitronectin or fibronectin were compared in solid phase assays. Echistatin and flavoridin were the most active inhibitors for alpha V beta 3 and alpha 5 beta 1 integrins and moderately exceeded the activity of the natural protein ligands. The same disintegrins together with eristostatin, bitistatin and barbourin were also very potent inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. For all three integrins, albolabrin showed the lowest affinity, but it still clearly exceeded that of synthetic GRGDS. However, assay conditions may determine these relative affinities, as shown for the alpha IIb beta 3 and alpha V beta 3 integrins when used either in immobilized or soluble form. For alpha IIb beta 3, however, a close correlation was found between KD values determined in platelet binding assays and the concentrations required for half maximal inhibition of three disintegrins. The inhibiting capacity of disintegrins in assays with purified integrins also correlated reasonably well with their inhibition of cell attachment to RGD-dependent protein substrates. However, sequence differences in the RGD loops of the various disintegrins may not fully account for the 20-100-fold difference in their binding capacities. This was particularly evident for echistatin and albolabrin, which differ in this region only by two conservative substitutions but have considerably different inhibitory activities. More remote regions of the disintegrins and alignment of disulfide bridges are therefore likely to contribute to their affinity and selectivity.

Amino Acid Sequence↗

PubMed1994

Immunological properties and tissue localization of two different collagen types in annelid and vestimentifera species.

Rabbit antisera against cuticle and interstitial collagens from shallow sea water and hydrothermal vent annelids (Arenicola marina, and the pompeii worm Alvinella pompejana) and the vestimentiferan tube worm Riftia pachyptila showed a clear distinction between the two types of collagens, a broad cross-reactivity among the worm collagens and no reactions with various mammalian collagens. The antibodies reacted with various epitopes found on both triple helical and unfolded collagens. The cuticle collagens were localized by immunofluorescence to the outer surface of the epidermis and in annelids additionally to the anterior part of the digestive tract. The interstitial collagen was detected underneath the epidermis and between distinct muscle layers. Both collagens were also detected in the anterior obturaculum, a tissue unique to vestimentifera. They were located either in the periphery of the tissue (cuticle collagen) or in the central part (interstitial collagen), which appeared to be a large extracellular matrix. Both collagens, however, showed a different supramolecular organization in the obturaculum when compared to the posterior body wall collagens. The identity of the interstitial collagens from the two locations was verified by biochemical analysis. These data demonstrate a very special and rigid matrix structure in the obturaculum, which may adapt it to specific physiological functions.

Animals↗

PubMed1993

Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein.

The adhesive glycoprotein vitronectin (VN) shows a high degree of conformational flexibility implicating that different molecular forms of the molecular may exist. Conformation-dependent monoclonal antibodies 13H1 or 16A7 that, per se, did not react with plasma VN bound to VN treated with heparin, chaotropes, detergents, pH below 6, or by heating at 56 degrees C. Dependent on the stimulus, recognition of VN by these antibodies varied and preceded heparin binding and self-association of VN resulting in the formation of noncovalently linked multimeric species of the protein. Both monoclonal antibodies also reacted with VN in serum or in platelet releasates as well as with VN in extracellular matrices of endothelial cells and inhibited cell adhesion on immobilized VN. Critical VN levels were needed for concentration-dependent multimerization indicating a nonlinear type of polymerization process. The nature of VN multimers was judged by nondenaturing gel electrophoresis, gel filtration, and sucrose gradient ultracentrifugation and revealed the formation of 3- to 16-mer multimeric species within an M(r) range of 200-1200 kDa representing a mean sedimentation coefficient of 9.6 S. In electron microscopy, multimeric VN occurred as globular specimens with an average diameter of 15-28 nm (monomeric plasma VN, 6-8 nm). In contrast to plasma VN, VN multimers were efficiently stabilized by covalent inter-molecular bonds following chemical or transglutaminase-induced cross-linking. A synthetic peptide comprising the central heparin binding region of VN (residues 348-361) not only bound to plasma VN but induced its multimerization also in plasma. During plasmin proteolysis of VN, fragments were generated that lacked the heparin binding region and that lost the ability to multimerize following urea or detergent treatment, implicating that the highly basic region is essential for multimer formation. These data suggest that non-plasma forms of VN, which are abundant in platelets and subendothelium, represent the prototype conformer of the reactive heparin binding form of VN. Our findings implicate that conformationally altered forms of VN enable the adhesive protein to multimerize in a characteristic fashion and thereby endow extracellular matrix sites with unique multivalent properties.

Amino Acid Sequence↗

PubMed1993

The extracellular matrix glycoproteins BM-90 and tenascin are expressed in the mesenchyme at sites of endothelial-mesenchymal conversion in the embryonic mouse heart.

BM-90 is a novel glycoprotein initially isolated from the extracellular matrix of a mouse tumor. We here studied the expression of BM-90 during embryonic development of the mouse heart and compared its expression pattern with that of tenascin and laminin. Distribution was studied by immunofluorescence using antibodies specifically raised against mouse BM-90, laminin and tenascin. Some expression of BM-90 was seen in myocardial basement membranes at early developmental stages, but expression abruptly decreased from these sites at day 12 of embryogenesis. Laminin B chains were also found in the muscle basement membranes early but did not decrease with advancing development. The most striking observation was the markedly enriched expression of BM-90 in the endocardial cushion tissue (ECT). The ECT is derived from mesenchymal cells converted from endothelium and they will form the cardiac valves and septa. In the ECT, BM-90 showed considerable co-distribution with tenascin, but tenascin expression was more focal and did not mark all areas of the ECT. Northern blot data show that BM-90 and tenascin were produced by the developing heart. With antibodies detecting A, B1 and B2 chains of mouse laminin, no immunoreactivity was seen in the ECT. Our data thus show clear-cut differences in the molecular composition of the ECT and muscle basement membranes in the developing heart. The focal expression of BM-90 in the ECT suggests that BM-90 could be involved in epithelial-mesenchymal transitions.

Animals↗

PubMed1993

Structural basis of beta 1 integrin-mediated cell adhesion to a large heparan sulfate proteoglycan from basement membranes.

In a cell attachment assay, several cell lines were found to adhere and spread on heparan sulfate proteoglycan purified from a basement membrane-producing tumor. This adhesion was clearly distinct from that on laminin. Cell adhesion to the proteoglycan was completely inhibited by three different antibodies against integrin beta 1 subunit, while inhibitory antibodies against beta 3 and alpha 2 to alpha 6 subunits were without strong effects. Removal of heparan sulfate from the proteoglycan diminished cell attachment, but addition of heparin to the cells did not affect adhesion to the proteoglycan. This suggests that both the heparan sulfate side chains and core protein structures are required for efficient cell adhesion. Studies with proteolytic fragments and synthetic RGD peptides showed that the single RGD sequence of mouse proteoglycan is not involved in cellular recognition. Characterization of fragments also allowed to localize cell adhesion and heparan sulfate attachment sites to the same 160 kDa core protein structure but not to fragments derived from the N-terminal portion of the proteoglycan.

Amino Acid Sequence↗

PubMed1992

Amino-acid sequence and cell-adhesion activity of a fibril-forming collagen from the tube worm Riftia pachyptila living at deep sea hydrothermal vents.

We have determined the amino acid sequence of the alpha chain of a fibril-forming collagen from the body wall of the marine invertebrate Riftia pachyptila (vestimentifera) by Edman degradation. The pepsin-solubilized collagen chain consists of a 1011-residue triple-helical domain and short remnants of N- and C-telopeptides. The triple-helical sequence showed one imperfection of the collagen Gly-Xaa-Yaa triplet repeat structure due to a Gly-->Ala substitution. This imperfection is correlated to a prominent kink in the molecule observed by electron microscopy. No strong sequence similarity was found with the fibril-forming vertebrate collagen types I-III, V and XI except for the invariant Gly residues. However, one of the two consensus cross-linking sequences was well conserved. The Riftia collagen shared with the vertebrate collagens many post-translational modifications. About 50% of the Pro and Lys residues are found in the Yaa position and were extensively hydroxylated to 4-hydroxyproline (4Hyp) and hydroxylysine (Hyl). A few proline residues in Xaa position were partially hydroxylated to either 4Hyp or 3Hyp. Despite the low sequence similarity, Riftia collagen was a potent adhesion substrate for two human cell lines. Cell adhesion could be inhibited by antibodies against the integrin beta 1 subunit but not by RGD peptides. This biological activity is apparently conserved in fibril-forming collagens of distantly related species but does not require the two RGD sequences present in Riftia collagen.

Amino Acid Sequence↗

PubMed1992

Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides.

The Arg-Gly-Asp (RGD) sequence is a universal cell-recognition site of various extracellular proteins that interact with integrin cell-surface receptors. In order to design low-molecular-mass RGD protein antagonists, the determination of the biologically active conformation is a prerequisite. We present a method that yields detailed insight into the steric factors which govern the binding of the ligands to their receptors by systematically scanning the conformational space accessible for the tripeptide sequence RGD. The investigation is based on the conformationally controlled design of homodetic cyclic oligopeptides and their structural determination, coupled with biological assays. For this purpose, a whole set of cyclic pentapeptides and hexapeptides has been synthesized and their three-dimensional structures in solution analyzed by modern two-dimensional NMR techniques in combination with restrained and free molecular dynamics simulations. Their biological activity was compared with that of linear GRGDS in inhibition assays of tumor cell adhesion to laminin P1 and vitronectin substrates. An up to 100-fold, and in part selective, increase in activity was observed for two cyclic pentapeptides. Most other peptides showed a decreased activity which, however, was useful to correlate activity with rather small variations in conformation. Detailed comparative studies of the systematically designed conformations and the corresponding anti-adhesive activities offer an access to lead structures for a rational indirect drug design of peptide and peptidomimetic pharmaceuticals with strong interfering activity for integrin-mediated cell-cell and cell-matrix interactions.

Amino Acid Sequence↗

PubMed1992

Effect of transforming growth factor-beta on collagen type VI expression in human dermal fibroblasts.

Steady-state mRNA levels and protein synthesis of collagen type VI were determined after stimulation of human dermal fibroblasts with transforming growth factor-beta (TGF beta). While there was a 227% increase in the alpha 3(VI) subunit mRNA at maximal TGF-beta concentration, alpha 1(VI) and alpha 2(VI) subunit mRNA levels remained unchanged. Concomitantly collagen type VI immuno-reactive material increased up to 172% of controls in cell culture medium and cell layer extracts. Regulation of alpha 3(VI) gene expression is therefore critical for the control of collagen type VI synthesis and determines the deposition of collagen type VI heterotrimeric molecules.

Blotting, Northern↗

PubMed1992

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.

Basement Membrane↗

PubMed1992

Urokinase binding to laminin-nidogen. Structural requirements and interactions with heparin.

Recently we have shown that heparin and related sulfated polyanions are low-affinity ligands of the kringle domain in the amino-terminal region (ATF) of human urokinase (u-PA), and proposed that this may facilitate loading of u-PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin-nidogen) for u-PA binding, and found that laminin-nidogen is also a ligand of the u-PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin-nidogen showed that there are u-PA binding sites in fragment E4 of laminin as well as in nidogen. The long-arm terminal domain of laminin (fragment E3), which contains a heparin-binding site, competed for binding of u-PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u-PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy-terminal domain. Direct binding assays confirmed that u-PA binds to nidogen through a site in the u-PA ATF. We conclude that u-PA binds to laminin-nidogen by interactions involving the ATF region of u-PA, the E4 domain of laminin and the rod or amino-terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u-PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.

Binding, Competitive↗

PubMed1992

Cloning and chromosomal location of human alpha 1(XVI) collagen.

We have characterized cDNA clones that encode a newly discovered collagenous polypeptide. A 4-kilobase (kb) cDNA clone was initially isolated by screening a human fibroblast cDNA library with a probe encoding the collagenous domain of the human alpha 3(VI) collagen. Subsequent screening of another fibroblast cDNA library yielded overlapping clones having a total length of 5.4 kb, which contained an open reading frame of 1603 amino acids including a 21-amino acid signal peptide. The predicted polypeptide consists of 10 collagenous domains 15-422 amino acids long, which were interspersed with 11 noncollagenous (NC) domains. Except for a large N-terminal NC11 domain of 312 residues, most of the NC domains were short (11-39 residues) and cysteine-rich. The overall structural arrangement differed significantly from other known collagen chains. Further analysis indicated that the deduced polypeptide exhibited several structural features characteristically seen in members of the fibril-associated collagen, types IX, XII, and XIV. In addition, the cysteine-rich motifs in the NC domains resembled those found in the cuticle collagen of Caenorhabditis elegans. Northern blot analyses showed hybridization of the cDNA to a 5.5-kb mRNA in human fibroblasts and keratinocytes. The gene was localized by in situ hybridization to band p34-35 of human chromosome 1. The data clearly support the conclusion that the cDNA encodes a collagen chain that has not been previously described. We suggest that the cDNA clones encode the alpha 1 chain of type XVI collagen.

Amino Acid Sequence↗