Biomedical subjects

R Timpl

Publications and source records attributed to R Timpl.

At least 19 recordsLinked to original sources

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↗

PubMed1992

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.

Amino Acid Sequence↗

PubMed1992

Human alpha 2(VI) collagen gene. Heterogeneity at the 5'-untranslated region generated by an alternate exon.

Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.

Amino Acid Sequence↗

PubMed1992

Binding of the proteoglycan decorin to collagen type VI.

We have examined the interactions between the small dermatan sulfate proteoglycan decorin and collagen types I-VI using solid phase binding assays. The results of these studies showed that 125I-decorin bound most efficiently to collagen type VI in a time- and concentration-dependent manner. Furthermore, this interaction was specific and of moderately high affinity (Kd approximately 3 x 10(-7) M). Binding of decorin to collagen type VI appears to involve the decorin core protein rather than the glycosaminoglycan side chains, since the isolated core protein as well as a recombinant fusion protein containing a major segment (65%) of the human decorin core protein inhibited binding of 125I-decorin to collagen type VI. Other related proteoglycans and their respective core proteins also inhibited the binding of 125I-decorin to collagen type VI, whereas unrelated proteins and isolated glycosaminoglycan chains were without effect. In addition to decorin, collagen type II was also shown to bind to immobilized collagen type VI. Both interactions were effectively inhibited by preincubation of the immobilized collagen VI with decorin or collagen type II. These results suggested that the collagen type VI molecule has binding sites for collagen type II and decorin which are located in close proximity on the collagen type VI molecule. Possible functional roles of these interactions are discussed.

Animals↗

PubMed1992

Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan.

A large portion of the N-terminal globule of human collagen VI was prepared from the culture medium of stably transfected human embryonic kidney cell clones. The recombinant product corresponds to sequence positions 1-1586 of the alpha 3 (VI) chain that consists of eight homologous approximately 200 residue motifs (N9 to N2) being similar to the A domain motif of von Willebrand factor. By ultracentrifugation fragment N9-N2 showed a molecular mass of 180 kDa and an asymmetric shape. Elongated structures that consist of eight small globes (diameter approximately 5 nm) were demonstrated by electron microscopy. The data indicate that each A domain motif represents a separate folding unit which are connected to each other by short protease-sensitive peptide segments. Circular dichroism studies demonstrated about 38% alpha helix, 14% beta sheets and 17% beta turns. Fragment N9-N2 showed binding to heparin which could be abolished by moderate salt concentrations. Heparin binding was assigned to domains N9, N6 and N3 which were obtained after partial proteolysis. Domains N7, N5 and N4 lacked affinity for heparin. In addition, N9-N2 showed strong binding to hyaluronan that required exposure to 6 M urea for full dissociation. Ligand binding studies indicated some affinity of N9-N2 for the triple helical region of collagen VI suggesting a role of the N-terminal globule in the self-assembly of microfibrils. No or only little binding was, however, observed to fibril-forming collagens I and III, several basement membrane proteins and other extracellular proteins. Fragment N9-N2 was also an inactive substrate for cell adhesion.

Amino Acid Sequence↗

PubMed1992

Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development.

Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC50 approximately 1 microgram/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to alpha 6 beta 1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.

Animals↗

PubMed1992

Immunochemical analysis of human kidney reticulin.

This study characterized the nature of reticulin fibrils from human kidney cortex by immunochemical analysis. Controls consisted of type I collagen fibrils derived from the kidney parietal capsule. Most of the fibrils in the capsule ranged in diameter from 60 to 80 nm whereas reticulin fibrils from the cortex ranged from 30-45 nm. Immunochemistry by light and electron microscopic examinations was carried out with antibodies directed against type I and type III collagens, their corresponding aminopropeptides, and decorin (PG-II). The ratio of type I to type III collagen was determined by cyanogen bromide peptide digests. This study showed that reticulin fibrils are hybrids of type I and type III collagens. Double immunoelectron microscopic examination showed that fibrils 20-25 nm consisted mainly of type I collagen some of which retained their aminopropeptide. Larger fibrils 30-35 nm labeled simultaneously for type I and type III collagens. However, most fibrils with diameters between 40-55 nm labeled for type III collagen and its corresponding aminopropeptide. No decorin was detected at the surface of reticulin fibrils. Purified reticulin consisted of 82% type III and 18% type I collagen whereas collagen derived from the capsule revealed 76% type I and 24% type III. The presence of the aminopropeptide of type III procollagen in reticulin fibrils is a striking feature and may play a role in regulating their diameter.

Collagen↗

PubMed1991

Localization of a major nidogen-binding site to domain III of laminin B2 chain.

The large pepsin fragments P1 and P1X, which comprise most of the rod-like domains III of the three short arms of laminin from the mouse Engelbreth-Holm-Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70-80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine-rich repeats with similarity to epidermal growth factor showed substantial nidogen-binding activity. Further degradation of this component to an active 28-kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine-rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.

Amino Acid Sequence↗

PubMed1991

Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1.

Cyclic Arg-Gly-Asp-Phe-Val peptides with either D-Phe or D-Val residues were 20- to more than 100-fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly-Arg-Gly-Asp-Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding of platelet receptor alpha IIb beta 3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all-trans conformation with a beta II' and gamma turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity of inhibition.

Amino Acid Sequence↗

PubMed1991

Molecular characterization of cuticle and interstitial collagens from worms collected at deep sea hydrothermal vents.

Two different collagens were isolated and characterized from the body walls of the vestimentiferan tube worm Riftia pachyptila and the annelid Alvinella pompejana, both living around hydrothermal vents at a depth of 2600 m. The acid-soluble cuticle collagens consisted of a long triple helix (2.4 microns for Alvinella, 1.5 microns for Riftia) terminating into a globular domain. Molecular masses of 2600 and 1700 kDa, respectively, were estimated from their dimensions. The two cuticle collagens were also quite different in amino acid composition, in agreement with their different supramolecular organizations within tissues. Interstitial collagens corresponding to cross-striated fibrils underneath the epidermal cells could be solubilized by digestion with pepsin and consisted of a single alpha-chain. They were similar in molecular mass (340 kDa) and length (280 nm) but differed in composition and banding patterns of segment-long-spacing fibrils. This implicates significant sequence differences also in comparison to fibril-forming vertebrate collagens, although all form typical quarter-staggered fibrils. The thermal stability of the worm collagens was, with one exception (interstitial collagen of Riftia), in the range of mammalian and bird collagens (37 to 46 degrees C), and thus distinctly above that of shallow sea water annelids. Yet, their 4-hydroxyproline contents were not directly correlated to this stability. About 20% of Riftia collagen alpha-chain sequence was elucidated by Edman degradation and showed typical Gly-X-Y repeats but only a limited homology (45 to 58% identity) to fibril-forming vertebrate collagens. A single triplet imperfection and the variable hydroxylation of proline in the X position were additional unique features. It suggests that this collagen represents an ancestral form of fibril-forming collagens not directly corresponding to an individual fibril-forming collagen type of vertebrates.

Amino Acid Sequence↗

PubMed1991

Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.

A cDNA construct (approximately 1 kb) of human BM-40 in a plasmid with the cytomegalovirus promoter and enhancer was used to produce several stable clones by transfecting two human cell lines (293, HT 1080). These clones showed a high expression of exogenous 1-kb BM-40 mRNA and no or only little endogenous 2.2-kb mRNA. These clones also secreted BM-40 at high rates (5-50 micrograms ml-1 day-1) into serum-free culture medium as shown by electrophoresis, radioimmunoassay and metabolic labelling. Transfection with the plasmid and overexpression of BM-40 had no effect on cell spreading, proliferation rate and adhesion patterns to extracellular matrix substrates. Recombinant human BM-40 was purified by anion-exchange chromatography and showed the expected N-terminal sequence and amino acid composition. The protein was also identical or similar to authentic BM-40 purified from the mouse Engelbreth-Holm-Swarm tumor in hexosamine content, electrophoretic mobility, circular dichroism and binding activity for calcium and collagen IV. Reduction of both authentic and recombinant BM-40 decreased binding activity which indicates correct formation of disulfide bonds in the recombinant protein. A specific and sensitive radioimmunoassay for human BM-40 was shown to be useful for detecting small quantities of the protein in human cell culture medium and blood. No significant cross-reaction was, however, detected between human and mouse BM-40.

Amino Acid Sequence↗

PubMed1991

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV.

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.

Amino Acid Sequence↗

PubMed1991

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells.

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.

Adult↗