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• W2024662362 abstract "Full-length and truncated forms of rat thrombospondin-4 (TSP-4) were expressed recombinantly in a mammalian cell line and purified to homogeneity. Biochemical analysis revealed a limited proteolytic processing, which detaches the N-terminal heparin-binding domain from the rest of the molecule and confirmed the importance of the heptad-repeat domain for pentamerization. In electron microscopy the uncleaved TSP-4 was seen as a large central particle to which five smaller globules are attached by elongated linker regions. Binding of TSP-4 to collagens and to non-collagenous proteins could be detected in enzyme-linked immunosorbent assay-style ligand binding assays, by surface plasmon resonance spectroscopy, and in rotary shadowing electron microscopy. Although the binding of TSP-4 to solid-phase collagens was enhanced by Zn2+, that to non-collagenous proteins was not. The interactions of TSP-4 with both classes of proteins are mediated by C-terminal domains of the TSP-4 subunits but do not require an oligomeric structure. Major binding sites for TSP-4 are located in or close to the N- and C-terminal telopeptides in collagen I, but additional sites are detected in more central regions of the molecule. Full-length and truncated forms of rat thrombospondin-4 (TSP-4) were expressed recombinantly in a mammalian cell line and purified to homogeneity. Biochemical analysis revealed a limited proteolytic processing, which detaches the N-terminal heparin-binding domain from the rest of the molecule and confirmed the importance of the heptad-repeat domain for pentamerization. In electron microscopy the uncleaved TSP-4 was seen as a large central particle to which five smaller globules are attached by elongated linker regions. Binding of TSP-4 to collagens and to non-collagenous proteins could be detected in enzyme-linked immunosorbent assay-style ligand binding assays, by surface plasmon resonance spectroscopy, and in rotary shadowing electron microscopy. Although the binding of TSP-4 to solid-phase collagens was enhanced by Zn2+, that to non-collagenous proteins was not. The interactions of TSP-4 with both classes of proteins are mediated by C-terminal domains of the TSP-4 subunits but do not require an oligomeric structure. Major binding sites for TSP-4 are located in or close to the N- and C-terminal telopeptides in collagen I, but additional sites are detected in more central regions of the molecule. thrombospondin cartilage oligomeric matrix protein polyacrylamide gel electrophoresis Tris-buffered saline enzyme-linked immunosorbent assay The thrombospondins (TSPs)1 are a family of multidomain extracellular matrix proteins, characterized by containing epidermal growth factor-like TSP type II domains, calcium-binding TSP type III repeats, a highly conserved C-terminal domain, and an assembly domain that allows oligomerization through the formation of a coiled-coil α-helix. At present, five family members fulfill these criteria, i.e. TSP-1 (1Baenzinger N.L. Brodic G.N. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 240-243Crossref PubMed Scopus (261) Google Scholar, 2Lawler J. Slayter H.S. Coligan J.E. J. Biol. Chem. 1978; 253: 8609-8616Abstract Full Text PDF PubMed Google Scholar, 3Hennessy S.W. Frazier B.A. Kim D.D. Deckwerth T.L. Baumgartel D.M. Rotwein P. Frazier W.A. J. Cell Biol. 1989; 108: 729-736Crossref PubMed Scopus (54) Google Scholar), TSP-2 (4Bornstein P. O'Rourke K. Wikstrom K. Wolf F.W. Katz R. Li P. Dixit V.M. J. Biol. Chem. 1991; 266: 12821-12824Abstract Full Text PDF PubMed Google Scholar), TSP-3 (5Vos H.L. Devarayalu S. De Vries Y. Bornstein P. J. Biol. Chem. 1992; 267: 12192-12196Abstract Full Text PDF PubMed Google Scholar), TSP-4 (6Lawler J. Duquette M. Whittaker C.A. Adams J.C. McHenry K. DeSimone D.W. J. Cell Biol. 1993; 120: 1059-1067Crossref PubMed Scopus (118) Google Scholar), and COMP (cartilage oligomeric matrix protein) (7Hedbom E. Antonsson P. Hjerpe A. Aeschlimann D. Paulsson M. Rosa-Pimentel E. Sommarin Y. Wendel M. Oldberg Å. Heinegård D. J. Biol. Chem. 1992; 267: 6132-6136Abstract Full Text PDF PubMed Google Scholar, 8Mörgelin M. Heinegård D. Engel J. Paulsson M. J. Biol. Chem. 1992; 267: 6137-6141Abstract Full Text PDF PubMed Google Scholar, 9Oldberg Å. Antonsson P. Lindblom K. Heinegård D. J. Biol. Chem. 1992; 267: 22346-22350Abstract Full Text PDF PubMed Google Scholar). All TSPs except COMP also contain an N-terminal heparin-binding domain, and TSP-1 and TSP-2, in addition, contain procollagen-like domains and properdin-like TSP type I domains. Although TSP-1 and -2 form trimers via their coiled-coil domains, TSP-3, TSP-4, and COMP form pentameric coiled coils. TSP-1 is the best characterized family member. It is found in blood platelets but also in the extracellular matrix formed by many cell types. It has been proposed to bind other matrix proteins, proteolytic enzymes, growth factors, and cellular receptors (for review see Ref.10Adams J.C. Int. J. Biochem. Cell Biol. 1997; 29: 861-865Crossref PubMed Scopus (100) Google Scholar). Mice lacking TSP-1 show a mild skeletal and hematological phenotype together with lung abnormalities and develop pneumonia (11Lawler J. Sunday M. Thibert V. Duquette M. George E.L. Rayburn H. Hynes R.O. J. Clin. Invest. 1998; 101: 982-992Crossref PubMed Scopus (388) Google Scholar). TSP-2 is present in many kinds of connective tissue, including the walls of blood vessels (12Iruela-Arispe L.K. Liska D.J. Sage H. Bornstein P. Dev. Dyn. 1993; 197: 40-56Crossref PubMed Scopus (184) Google Scholar, 13Kyriakides T.R. Zhu Y.-H. Yang Z. Bornstein P. J. Histochem. Cytochem. 1998; 46: 1007-1015Crossref PubMed Scopus (83) Google Scholar), and mice deficient in this protein show connective tissue abnormalities, increased vascular density, and a bleeding diathesis (14Kyriakides T.R. Zhu Y.-H. Smith L.T. Bain S.D. Yang Z. Lin M.T. Danielson K.G. Iozzo R.V. LaMarca M. McKinney C.E. Ginns E.I. Bornstein P. J. Cell Biol. 1998; 140: 419-430Crossref PubMed Scopus (410) Google Scholar). TSP-3 is mainly expressed in lung, cartilage, and brain (12Iruela-Arispe L.K. Liska D.J. Sage H. Bornstein P. Dev. Dyn. 1993; 197: 40-56Crossref PubMed Scopus (184) Google Scholar, 15Qabar A.N. Lin Z. Wolf F.W. O'Shea K.S. Lawler J. Dixit V.M. J. Biol. Chem. 1994; 269: 1262-1269Abstract Full Text PDF PubMed Google Scholar), but little is known about its functions in those tissues. COMP is strongly expressed in all types of cartilage (7Hedbom E. Antonsson P. Hjerpe A. Aeschlimann D. Paulsson M. Rosa-Pimentel E. Sommarin Y. Wendel M. Oldberg Å. Heinegård D. J. Biol. Chem. 1992; 267: 6132-6136Abstract Full Text PDF PubMed Google Scholar) but is also present in tendon (16DiCesare P. Hauser N. Lehman D. Pasumarti S. Paulsson M. FEBS Lett. 1994; 354: 237-240Crossref PubMed Scopus (220) Google Scholar). It has been shown to bind specifically to collagen II (17Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) and might regulate fibril formation or mediate interactions of collagen with other matrix components. TSP-4 was initially identified and cloned from Xenopus laevis (6Lawler J. Duquette M. Whittaker C.A. Adams J.C. McHenry K. DeSimone D.W. J. Cell Biol. 1993; 120: 1059-1067Crossref PubMed Scopus (118) Google Scholar). It is first expressed in late gastrulation, with its mRNA being restricted to somitic mesoderm and skeletal muscle (18Urry L.A. Whittaker C.A. Duquette M. Lawler J. DeSimone D.W. Dev. Dyn. 1998; 211: 390-407Crossref PubMed Scopus (27) Google Scholar). Its complete cDNA sequence has been determined also from humans (19Lawler J. McHenry K. Duquette M. Derick L. J. Biol. Chem. 1995; 270: 2809-2814Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and rat (20Arber S. Caroni P. J. Cell Biol. 1995; 131: 1083-1094Crossref PubMed Scopus (130) Google Scholar). A structural model showing a pentameric structure was proposed based on the characterization of the human recombinant protein (19Lawler J. McHenry K. Duquette M. Derick L. J. Biol. Chem. 1995; 270: 2809-2814Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and confirmed by analysis of bovine TSP-4 isolated from tendon tissue (21Hauser N. Paulsson M. Kale A.A. DiCesare P. FEBS Lett. 1995; 368: 307-310Crossref PubMed Scopus (50) Google Scholar). Some results indicate that in tendon TSP-4 subunits may occur in hybrid pentamers together with COMP subunits (22Hecht J.T. Deere M. Putnam E. Cole W. Vertel B. Chen H. Lawler J. Matrix Biol. 1998; 17: 269-278Crossref PubMed Scopus (98) Google Scholar). In chick its mRNA is found in osteogenic tissues and corneal fibroblasts, but not in somitic mesoderm or skeletal muscle (23Tucker R.P. Adams J.C. Lawler J. Dev. Dyn. 1995; 203: 477-490Crossref PubMed Scopus (57) Google Scholar) as in frog and humans, showing that the pattern of tissue-specific expression may vary between species. Arber and Caroni (20Arber S. Caroni P. J. Cell Biol. 1995; 131: 1083-1094Crossref PubMed Scopus (130) Google Scholar) isolated rat TSP-4 cDNA from a subtractive cDNA library prepared from denervated skeletal muscle. Using a polyclonal antibody raised against a synthetic peptide derived from the C-terminal domain, they could detect TSP-4 in the interstitial cells in both skeletal and heart muscle. The expression of TSP-4 was elevated after denervation of skeletal muscle, with the signal intensity increasing specifically in the interstitial cells. Using in situ hybridization as well as immunofluorescence localization of TSP-4 combined with α-bungarotoxin labeling, they could show that TSP-4-expressing cells were in the immediate vicinity of the neuromuscular junction. In addition, the expression of TSP-4 by a variety of neuronal cells was detected, andin vitro assays showed that TSP-4 possesses a neurite outgrowth-promoting activity (20Arber S. Caroni P. J. Cell Biol. 1995; 131: 1083-1094Crossref PubMed Scopus (130) Google Scholar). Our identification of TSP-4 as an abundant non-collagenous protein in tendon extracellular matrix (21Hauser N. Paulsson M. Kale A.A. DiCesare P. FEBS Lett. 1995; 368: 307-310Crossref PubMed Scopus (50) Google Scholar) prompted us to investigate whether TSP-4 plays a role in extracellular matrix structure and assembly. In the present work we have recombinantly expressed full-length and truncated forms of TSP-4 in mammalian cells, characterized their structure, and used them in interaction studies to demonstrate that TSP-4 binds with high affinity to both collagenous and non-collagenous extracellular matrix proteins via its C-terminal region and that the former interaction is enhanced by Zn2+ ions. Total RNA was isolated from the skin of 4-day-old rats by the guanidinium isothiocyanate method. Reverse transcription-polymerase chain reaction was performed with Expand-Polymerase using the following primers: primer 1, 5′-TTAAACTAGTAGCGGGCGCCCAGGCCAC; primer 2, 5′-GTAAAGCTTCCAACACCGCACACACACG; primer 3, 5′-GCATGCGCAGTGCATTGAGG; primer 4, 5′-GCCAAGCTTAGAAGGCAGTTGTGAGATTGC; primer 5, 5′-GGACTAGTAAGTGAGCCGCTGGC; primer 6, 5′-TCAACATCAGTGCACACCTGC; primer 7, 5′-GGACTAGTACCTCTCAGCTTCCAG; primer 8, 5′-AAGTCAGTCGACTCATTAGCCACAAGCCTGGCACTCAGC; primer 9, 5′-TTGCGGCCGCTCATTACTGCTGCAGGAAACAGTCTTGTAGG. cDNA encoding the recombinant full-length TSP-4 was obtained by ligation of two shorter cDNAs amplified with the primer pairs 1/2 and 3/4, respectively, over a common FspI site in pCRII (Invitrogen). Fragment NT+CC was obtained with the primer pair 1/8 and NT with the pair 1/9. The cDNAs for CT and CT+CC were based on that for the full-length protein. For both fragments only the 5′-end was changed by using the primer pair 5/6 for CT+CC and 7/6 for CT. The primer combinations introduced a novel SpeI site at the 5′-end and a stop codon, a NotI or a BamHI site, respectively, at the 3′-end. The products were restriction-digested with NheI and NotI/BamHI and, after purification by agarose gel electrophoresis, were inserted in-frame with the BM40 signal peptide in the expression vector pCEP-Pu (24Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar) digested with the same enzymes. DNA sequencing was performed on the ALF- express DNA sequencer (Amersham Pharmacia Biotech) using the Cy5TM Auto Read sequencing kit (Amersham Pharmacia Biotech). The recombinant plasmids were introduced by electroporation into the human embryonic kidney cell line 293-EBNA (Invitrogen). The transfected cells were selected with 0.5 μg/ml puromycin and grown to confluency. Secretion of full-length or truncated forms of TSP-4 into the medium was confirmed by comparison in SDS-PAGE of medium samples from transfected and wild-type cells. For purification of the full-length protein, serum-free culture medium was dialyzed against 2 murea, 75 mm NaCl, 15 mm Tris-HCl, pH 7.4 and applied to a column of DEAE-Sepharose FF (Amersham Pharmacia Biotech), equilibrated in the same buffer. The column was eluted with a gradient of 75–500 mm NaCl. TSP-4-containing fractions were pooled, applied to a molecular sieve column of Sepharose CL4B (Amersham Pharmacia Biotech) equilibrated in 75 mm NaCl, 15 mm Tris-HCl, pH 7.4. Final purification and concentration was achieved through binding of TSP-4 to a column of heparin-Sepharose CL6B (Amersham Pharmacia Biotech) in the same buffer and stepwise elution with 0.3 m NaCl. The TSP-4 fragments CT and CT+CC were purified in an analogous manner, but because they lack the heparin-binding domain, final concentration was achieved by stepwise elution from a small column of DEAE-Sepharose FF. For the analogous purification of the smaller and more basic fragments NT and NT+CC, an ion exchanger of SP-Sepharose FF (Amersham Pharmacia Biotech) and a molecular sieve of Sephadex G-75 (Amersham Pharmacia Biotech) were used. The purity and size of all purified proteins were assessed by SDS-PAGE performed according to the protocol of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) with or without prior reduction of disulfide bonds. Alternatively, full-length TSP-4 was isolated from cell culture medium in the absence of 2 m urea but with 1.8 mmcalcium chloride added to all buffers. Anion exchange and heparin affinity chromatographies were otherwise performed as described above. Final pools were dialyzed against TBS containing 1.8 mmcalcium (TBS-Ca). To analyze the effect of urea, TSP-4 purified in the presence of calcium was dialyzed extensively against 2 murea in the absence of calcium. The urea was then removed by dialysis against TBS-Ca. For visualization of complexes by rotary shadowing electron microscopy, full-length TSP-4, COMP, and collagen I were dialyzed overnight at 4 °C against 0.2 mammonium formate, containing 1 mm ZnCl2. When desired Na2EDTA was added to a final concentration of 5 mm followed by incubation for 1 h at room temperature. After addition of a 4-fold molar surplus of collagen I to the TSP-4 or COMP samples, the mixtures were incubated for 2 h at room temperature. The samples were diluted 1:1 with 80% glycerol, sprayed onto freshly cleaved mica, dried in vacuum, and rotary-shadowed with platinum/carbon (26Engel J. Furthmayr H. Methods Enzymol. 1987; 145: 3-78Crossref PubMed Scopus (137) Google Scholar). The replicas were floated off onto distilled water, picked up on 400-mesh copper grids, and examined in a Zeiss EM 902A electron microscope. Rat COMP was prepared recombinantly using EBNA 293 cells in a manner analogous to the preparation of TSP-4. 2J. Thur and P. Maurer, unpublished results. TSP-4 was also subjected to negative staining using 0.75% uranyl formate. All collagens used in ligand binding assays were purchased from Sigma. They had been isolated after pepsin digestion from the following tissues: collagen I from calf skin, collagen II from bovine tracheal cartilage, and collagens III, IV, and V from human placenta. Each collagen was dissolved in 0.1 macetic acid, pH 2.5. Murine laminin-1 (27Paulsson M. J. Biol. Chem. 1988; 263: 5425-5430Abstract Full Text PDF PubMed Google Scholar), bovine fibronectin (28Miekka S.I. Ingham K.C. Menache D. Thromb. Res. 1982; 27: 1-14Abstract Full Text PDF PubMed Scopus (246) Google Scholar), human BM40, 3B. Kaufmann and P. Maurer, unpublished results. and murine matrilin-2 (29Piecha D. Muratoglu S. Mörgelin M. Hauser N. Studer D. Kiss I. Paulsson M. Deák F. J. Biol. Chem. 1999; 274: 13353-13361Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) were isolated in our laboratory. Polyclonal antibodies specific for TSP-4 were generated at Eurogentec (Belgium) by immunizing rabbits with full-length TSP-4 and the TSP-4 fragment NT, respectively. The antiserum against TSP-4 was affinity-purified on a column of TSP-4 Sepharose CL4B prepared by CNBr coupling. Additionally, antisera against murine laminin-1, bovine fibronectin, human BM40, and murine matrilin-2 were raised in rabbits. Collagens were biotinylated for detection (30Meier T. Arni S. Malarkanna S. Poincelet M. Hoessli D. Anal. Biochem. 1992; 204: 220-226Crossref PubMed Scopus (95) Google Scholar). Inital ligand binding studies were performed in solid phase assays either with TSP-4 proteins as soluble ligands or coated to plastic. 96-well plates (Maxisorb, NUNC) were coated overnight at 4 °C with 10 μg/ml collagens I, II, III, IV, and V, laminin-1, fibronectin, BM40, matrilin-2, or TSP-4 proteins. The collagens were coated from 0.5m acetic acid and the non-collagenous proteins from 0.15m NaCl, 50 mm Tris, pH 7.4 (TBS). After rinsing with 0.04% Tween 20 in TBS for 1 h, the wells were blocked with 200 μl of 1 mg/ml ovalbumin in TBS at room temperature. After rinsing as above, the coated wells were incubated for 1 h at room temperature with 10 μg/ml TSP-4 diluted in TBS containing either 1.25 mm CaCl2, 0.22 mmZnCl2, 0.9 mm MgCl2, 0,02 mm MnCl2, or 10 mm EDTA. In separate experiments the zinc concentration was varied between 0 and 1 mm. For dose-response curves full-length TSP-4 was added at different concentrations between 0 and 4 μg/ml. Bound full-length TSP-4 and the recombinant fragments CT and CT+CC were detected using the polyclonal antibodies against full-length TSP-4. For the detection of the N-terminal TSP-4 fragments, the polyclonal antibodies against the fragment NT were used. The BIAcore 2000 biosensor system (BIAcore, Sweden) was used to further characterize the interactions between TSP-4 and other extracellular matrix proteins. Immobilization of proteins to the CM5 sensor chip was achieved byN-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccimide coupling procedure at a flow of 5 μl/min. The surface of the carboxymethylated sensor chip was activated by addition of 40 μl of 0.05 m N-hydroxysuccimide, 0.2 m N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride solution. TSP-4 (100 μg/ml) was coupled to the activated chip in 0.15 m sodium chloride, 10 mmsodium citrate, pH 3.2, for 8 min. The residual activated esters of the surface were deactivated by blocking with 1 m ethanolamine in 0.1 m sodium hydrogen carbonate, pH 5.3. One surface on each chip was activated and deactivated under identical conditions without coupling a protein and was used as a blank. Collagens or laminin-1 (10 μg/ml in TBS containing 0.5 mmZnCl2) were pumped over the TSP-4 sensor chip at a flow rate of 80 μl/min for 2 min. In separate experiments the concentration of collagen I was varied between 2.5 and 10 μg/ml. For other experiments collagens (100 μg/ml) were coupled on an activated sensor chip from 0.5 m sodium acetate, pH 4.0. Similarly, laminin-1 (100 μg/ml) was coupled from 10 mm sodium citrate, pH 5.0. Deactivation was performed as above. Binding of full-length TSP-4 (50 μg/ml) and fragment CT (25 μg/ml) as soluble ligands to surfaces with immobilized collagens or laminin-1 were performed at a flow of 50 μl/min. The BIAevaluation version 2.1 software was used to calculate dissociation constants. RNA was isolated from the skin of 4-day-old rats, and cDNA coding for TSP-4 was synthesized by reverse transcription. The primers used were based on the sequence derived from rat brain (20Arber S. Caroni P. J. Cell Biol. 1995; 131: 1083-1094Crossref PubMed Scopus (130) Google Scholar), which lacks the 5′-region coding for the signal peptide. For this reason the amplified sequence started with the N-terminal, heparin-binding domain of TSP-4. Two overlapping cDNAs with lengths of 1305 and 1617 base pairs were amplified and ligated to obtain the full-length cDNA for TSP-4. Sequencing revealed four discrepancies to the published sequence of rat TSP-4 (20Arber S. Caroni P. J. Cell Biol. 1995; 131: 1083-1094Crossref PubMed Scopus (130) Google Scholar). A deletion of three nucleotides1308GGC1310 and two single nucleotide alterations G1961 → C and C1978 → T occurred in our sequence. These changes were confirmed by repeated cDNA synthesis and sequencing. The deletion is also found in sequences for TSP-4 previously determined for X. laevis and humans (6Lawler J. Duquette M. Whittaker C.A. Adams J.C. McHenry K. DeSimone D.W. J. Cell Biol. 1993; 120: 1059-1067Crossref PubMed Scopus (118) Google Scholar, 19Lawler J. McHenry K. Duquette M. Derick L. J. Biol. Chem. 1995; 270: 2809-2814Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The single base changes produced nucleotide triplets encoding amino acid residues conserved at these sites in other species. Similarly, the sequence 1311GCG1313 instead of GAC was found repeatedly and appeared plausible, because it is identical with that in X. laevis and conserves the acidic nature of the amino acid residue coded for in the human sequence. A silent mutation G1115 → T was not further investigated. The full-length and four truncated cDNAs were inserted into the pCEP-Pu vector, utilizing the signal sequence of BM40 (24Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar). The constructs represented the full-length TSP-4 (TSP-4), the C-terminal domains together with the α-helical coiled-coil region (CT+CC), the C-terminal domains alone (CT), the N-terminal heparin-binding domain together with the coiled-coil region (NT+CC), and the N-terminal heparin-binding domain (NT) (Fig. 1). The plasmids were introduced into the human embryonic kidney 293-EBNA cell line, where they were stably maintained in episomal form. The secreted recombinant proteins were purified chromatographically from serum-free culture media. All proteins were purified in a buffer containing 2m urea to increase solubility and purification yields. To demonstrate that the use of 2 m urea does not cause irreversible structural or functional changes, smaller amounts of the full-length TSP-4 were purified by a procedure avoiding all potentially denaturing agents and retaining divalent cations. All recombinant proteins were analyzed by automated amino acid sequencing, and in each case yielded a major sequence confirming the expected cleavage of the BM40 signal peptide. The TSP-4 proteins were subjected to SDS-PAGE under both reducing and non-reducing conditions (Fig. 2). In each case the reduced protein product, when compared with reference proteins of known mass, yielded a molecular mass equal to or somewhat higher than that predicted from the amino acid sequence (Fig. 2 B). The full-length TSP-4 gave a major band at 135 kDa and a minor one at 120 kDa. When not reduced, those proteins containing the coiled-coil domain migrated to positions compatible with the expected pentameric structure (Fig. 2 A).Figure 1Domain structure of the full-length TSP-4 and of the recombinant fragments produced. TSP-4, full-length TSP-4 subunit; CT+CC, the C-terminal part with, and CT without, the coiled-coil domain; NT+CC, the N-terminal, heparin-binding domain with, and NT without, the coiled-coil domain. TN, the N-terminal, heparin-binding domain; CC, the coiled-coil domain; EG, epidermal-growth factor like domains with the asterisksdenoting calcium-binding EG domains; and TC, the C-terminal globular domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2SDS-PAGE of the purified recombinant TSP-4 proteins under non-reducing (A) and reducing (B) conditions. For explanation of the abbreviations see the legend to Fig. 1. Fn designates the migration position of non-reduced fibronectin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The purified, full-length TSP-4 was also analyzed by both negative staining and rotary shadowing electron microscopy (Fig.3). The particles seen were comparatively homogenous with most showing up to five small globules extending via a thinner segment from a central accumulation of mass. Based on the proposed structure of TSP-4 (19Lawler J. McHenry K. Duquette M. Derick L. J. Biol. Chem. 1995; 270: 2809-2814Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), each of the small external globules corresponds to the C-terminal part of one subunit and the central mass is made up by the five N-terminal heparin-binding domains, which are held at close distance from each other by the coiled coil and are not individually resolved. The accumulation of stain at the center of the particles was more extensive than in earlier images of bovine TSP-4 (21Hauser N. Paulsson M. Kale A.A. DiCesare P. FEBS Lett. 1995; 368: 307-310Crossref PubMed Scopus (50) Google Scholar). A variety of purified extracellular matrix proteins were coated to plastic and after saturation of free binding sites, a solution of 10 μg/ml of full-length recombinant TSP-4 was incubated in the wells. Bound TSP-4 was detected by use of a specific antiserum raised against the full-length protein. The panel of potential ligands was chosen to include collagens I–V as well as laminin-1, fibronectin, matrilin-2, and BM40 as examples for non-collagenous proteins. All these proteins show an at least partial codistribution with TSP-4 as determined by immunohistochemistry on sections of mouse tissues. 4L. Narouz-Ott, unpublished results. Interactions were detected with all proteins in the panel with the exception of collagen IV and BM40 for which only low or insignificant binding was seen at the concentrations used (Fig. 4). Because many extracellular matrix proteins, including COMP with its close structural relationship to TSP-4 (17Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), are known to have specific requirements for divalent cations when binding ligands, the assay was performed in the presence of a variety of such ions. Zn2+enhanced binding of TSP-4 to collagens, in agreement with previous observations for COMP (17Rosenberg K. Olsson H. Mörgelin M. Heinegård D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), whereas binding to non-collagenous proteins did not show a strong requirement for Zn2+ (Fig. 4) or other divalent cations that were used at their physiological plasma concentrations. In further ELISA-style assays the binding of TSP-4 to the various collagens was determined in the presence of varying concentrations of Zn2+. A plateau of maximal binding was reached in the range of 0.1–0.5 mm (results not shown). The interaction between TSP-4 and collagen I was investigated in greater detail. Incubation of increasing concentrations of TSP-4 with immobilized collagen I in the presence of Zn2+ showed a saturable binding with an apparent K d of 2 nm (Fig. 5 A). Independent determination of the color yield of TSP-4 coated directly to plastic allowed estimation that, at half-saturation, 18% of all TSP-4 molecules added were bound to collagen I. A sample that had been purified in the absence of urea and in the presence of divalent cations, dialyzed into the buffer with 2 m urea used for most purifications, and then back into a physiological buffer, gave a binding curve identical to that obtained for TSP-4, which had never been exposed to urea (Fig. 5 A). For both kinds of samples, binding occurring in the presence of 1 mm Zn2+was abolished when this cation was removed and replaced by 1 mm Ca2+ or 1 mm EDTA (Fig.5 B). Collagens I–IV were assayed for binding to full-length TSP-4 that had been immobilized to a BIAcore CM5 sensor chip (Fig.6 A). The collagens were used at 10 μg/ml in a buffer containing 0.5 mmZn2+. Collagens I–III showed binding, in agreement with the ELISA-style binding assays, whereas collagen IV again showed no or only weak association. The dissociation was biphasic for all bound collagens. Dissociation rate constants k disswere separately fitted for the fast and slow phases. The resultingk diss values differed between the two phases by a factor of less than seven. This difference was correspondingly also found in the calculated equilibrium dissociation constantsK d. Different binding sites for TSP-4 are present on collagens (see below) and could be the reason for the biphasic dissociation. Nonetheless, we give mean K d values for the binding of TSP-4 to collagens, because other causes of biphasic dissociation cannot be excluded. Thus dissociation constants of 8.5 ± 6.5 nm for collagen I, 4.4 ± 3.0 nm for collagen II, and 38.5 ± 4.0 nm for collagen III were obtained. Experiments in which collagen I was passed over the sensor chip with immobilized TSP-4 at d" @default.
• W2024662362 created "2016-06-24" @default.
• W2024662362 creator A5003611587 @default.
• W2024662362 creator A5035706902 @default.
• W2024662362 creator A5049839584 @default.
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• W2024662362 date "2000-11-01" @default.
• W2024662362 modified "2023-10-16" @default.
• W2024662362 title "Thrombospondin-4 Binds Specifically to Both Collagenous and Non-collagenous Extracellular Matrix Proteins via Its C-terminal Domains" @default.
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