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• W1978202313 startingPage "13586" @default.
• W1978202313 abstract "One of the primary points of regulation of transforming growth factor-β (TGF-β) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-β activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-β complex. Platelet thrombospon-din-1 has TGF-β activity and immunoreactive mature TGF-β associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-β complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin·LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-β1–5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-β since LSKL peptides can competitively inhibit latent TGF-β activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP·TGF-β complex since thrombospondin-bound LAP no longer confers latency on active TGF-β. The mechanism of TGF-β activation by thrombospondin-1 appears to be conserved among TGF-β isoforms as latent TGF-β2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides. One of the primary points of regulation of transforming growth factor-β (TGF-β) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-β activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-β complex. Platelet thrombospon-din-1 has TGF-β activity and immunoreactive mature TGF-β associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-β complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin·LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-β1–5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-β since LSKL peptides can competitively inhibit latent TGF-β activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP·TGF-β complex since thrombospondin-bound LAP no longer confers latency on active TGF-β. The mechanism of TGF-β activation by thrombospondin-1 appears to be conserved among TGF-β isoforms as latent TGF-β2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides. Transforming growth factors-β are a family of small peptide growth factors (25 kDa) involved in the regulation of a variety of cellular functions (1Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3004) Google Scholar, 2Roberts A.B. Wound Rep. Reg. 1995; 3: 408-418Crossref PubMed Scopus (142) Google Scholar, 3Lawrence D.A. Eur. Cytokine Netw. 1996; 7: 363-374PubMed Google Scholar). Processes regulated by TGF-β 1The abbreviation used is: TGF-β, transforming growth factor-β; LAP, latency-associated peptide; NRK, normal rat kidney; BAE, bovine aortic endothelial; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TSP, thrombospondin; ACTH, adrenocorticotropic hormone. include angiogenesis, embryogenesis, wound healing, and inflammation. There are five isoforms of TGF-β, three of which are expressed in mammals. TGF-β is synthesized by virtually all cell types in a latent form that must be activated in order to be recognized by cell-surface receptors and to trigger biological responses (1Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3004) Google Scholar, 2Roberts A.B. Wound Rep. Reg. 1995; 3: 408-418Crossref PubMed Scopus (142) Google Scholar, 3Lawrence D.A. Eur. Cytokine Netw. 1996; 7: 363-374PubMed Google Scholar, 4Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (442) Google Scholar). Mechanisms controlling conversion of the latent complex to the active state are key regulators of TGF-β activity (1Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3004) Google Scholar, 2Roberts A.B. Wound Rep. Reg. 1995; 3: 408-418Crossref PubMed Scopus (142) Google Scholar, 3Lawrence D.A. Eur. Cytokine Netw. 1996; 7: 363-374PubMed Google Scholar, 4Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (442) Google Scholar). Physiological mechanisms of activation are not well understood, although proteolytic processing by plasmin, exposure to reactive oxygen species, and binding to integrins may participate in this process (4Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 47Munger J.S. Huang X. Kawakatsu H. Griffiths M.J.D. Dalton S.L. Wu J. Pittet J.-F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1647) Google Scholar). Our laboratory has shown that interaction of latent TGF-β with the multifunctional platelet and matrix protein thrombospondin-1 (5Adams J. Lawler J. Curr. Biol. 1993; 3: 188-190Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 6Bornstein P. Sage E.H. Methods Enzymol. 1994; 245: 62-84Crossref PubMed Scopus (155) Google Scholar, 7Mosher D.F. Annu. Rev. Med. 1990; 41: 85-97Crossref PubMed Scopus (146) Google Scholar, 8Frazier W.A. Curr. Opin. Cell Biol. 1991; 3: 792-799Crossref PubMed Scopus (162) Google Scholar, 9Bornstein P. FASEB J. 1992; 6: 3290-3299Crossref PubMed Scopus (307) Google Scholar, 10Lahav J. Biochim. Biophys. Acta. 1993; 1182: 1-14Crossref PubMed Scopus (111) Google Scholar) results in activation of latent TGF-β (12Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar, 13Schultz-Cherry S. Ribeiro S.M.F. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar, 14Schultz-Cherry S. Lawler J. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26783-26788Abstract Full Text PDF PubMed Google Scholar, 15Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Thrombospondin purified from human platelets (thrombospondin-1) is associated with TGF-β activity (11Murphy-Ullrich J.E. Schultz-Cherry S. Hook M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar). The site in thrombospondin responsible for latent TGF-β activation has been localized to the type 1 repeats (14Schultz-Cherry S. Lawler J. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26783-26788Abstract Full Text PDF PubMed Google Scholar): specifically, the KRFK sequence located between the first and second type 1 repeats of thrombospondin-1 (15Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). To better understand the mechanism of thrombospondin-mediated activation of latent TGF-β, we sought to determine the region of the latent TGF-β complex recognized by the TGF-β-activating sequence KRFK in thrombospondin. Small latent TGF-β (reviewed in Refs. 1Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (3004) Google Scholar, 2Roberts A.B. Wound Rep. Reg. 1995; 3: 408-418Crossref PubMed Scopus (142) Google Scholar, 3Lawrence D.A. Eur. Cytokine Netw. 1996; 7: 363-374PubMed Google Scholar, 4Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (442) Google Scholar) is a dimeric complex of ∼100 kDa, composed of two identical chains in which an amino-terminal 278-amino acid latency-associated peptide (LAP) is noncovalently associated with the carboxyl-terminal 112-amino acid active peptide. This latent complex is the product of a single gene. Prior to secretion, LAP is enzymatically cleaved from the active peptide (16Gentry L.E. Lioubin M.N. Purchio A.F. Marquardt H. Mol. Cell. Biol. 1988; 8: 4162-4168Crossref PubMed Scopus (209) Google Scholar), and the integrity and latency of the secreted complex are presumably maintained via electrostatic interactions (17Brown P.D. Wakefield L.M. Levison A.D. Sporn M.B. Growth Factors. 1990; 3: 35-43Crossref PubMed Scopus (307) Google Scholar). Although latent TGF-β can also exist as a large complex in which small latent TGF-β is associated with a latent TGF-β-binding protein, the presence of the latent TGF-β-binding protein is neither necessary nor sufficient to confer latency on the active peptide (18Miyazono K. Hellman U. Wernstedt C. Heldin C.-H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar, 19Wakefield L.M. Smith D.M. Flanders K.C. Sporn M.B. J. Biol. Chem. 1988; 263: 7646-7654Abstract Full Text PDF PubMed Google Scholar, 20Flaumenhaft R. Abe M. Sato Y. Miyazono K. Harpel J.G. Heldin C.-H. Rifkin D. J. Cell Biol. 1993; 120: 995-1002Crossref PubMed Scopus (226) Google Scholar). On the other hand, latency is dependent on the presence of LAP, and modification of the cysteine residues responsible for LAP dimerization results in altered TGF-β secretion (21Sha X. Yang L. Gentry L.E. J. Cell Biol. 1991; 114: 827-839Crossref PubMed Scopus (59) Google Scholar, 22Miller D.M. Ogawa Y. Iwata K.K. ten Dijke P. Purchio A.F. Soloff M.S. Gentry L.E. Mol. Endocrinol. 1992; 6: 694-702Crossref PubMed Scopus (28) Google Scholar, 23McMahon G.A. Dignam J.D. Gentry L.E. Biochem. J. 1996; 313: 343-351Crossref PubMed Scopus (68) Google Scholar), suggesting that the tertiary structure of LAP is important for the formation of the latent TGF-β complex. Gentry and co-workers (21Sha X. Yang L. Gentry L.E. J. Cell Biol. 1991; 114: 827-839Crossref PubMed Scopus (59) Google Scholar) showed through mutagenesis studies that amino acids 40–80 in LAP are important for maintenance of the latency of the complex. In a previous study, we observed that antibodies raised against a sequence present in the amino terminus of LAP (residues 81–94) inhibited activation of latent TGF-β by thrombospondin (13Schultz-Cherry S. Ribeiro S.M.F. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar). These observations led us to propose that thrombospondin-mediated activation of latent TGF-β involves interactions between the thrombospondin activation sequence (KRFK) and a site present in the amino-terminal region of LAP. We now show that LAP is complexed with thrombospondin-1 in association with biologically active TGF-β and that the thrombospondin-1 sequence KRFK binds LAP through interactions that involve a specific sequence at the amino terminus of β1-LAP (L54SKL57). The KRFK sequence in thrombospondin-1 and the LSKL sequence in LAP are apparently critical for activation of latent TGF-β by thrombospondin-1 since soluble LSKL peptides can competitively block activation of latent TGF-β by either thrombospondin-1 or KRFK-containing peptides. LAP binding to thrombospondin may play a role in preventing re-formation of the latent complex. In addition, the mechanism of thrombospondin-mediated activation of latent TGF-β appears to be conserved in the mammalian isoforms of TGF-β since thrombospondin-1 can also activate latent TGF-β2 in an LSKL-sensitive manner. Thrombospondin-1, native or strip- ped of TGF-β activity, was purified as described (11Murphy-Ullrich J.E. Schultz-Cherry S. Hook M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar) from human platelets obtained from the Birmingham American Red Cross. Thrombospondin purity was assessed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. The depletion of TGF-β activity in preparations of stripped thrombospondin was confirmed in the NRK colony formation assay (11Murphy-Ullrich J.E. Schultz-Cherry S. Hook M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar). The peptides used in this work were synthesized by the University of Alabama at Birmingham Comprehensive Cancer Center/Peptide Synthesis and Analysis shared facility. Initial peptides and peptide 246 used in this study were a gift from Dr. David Roberts (NCI, National Institutes of Health). Recombinant latent TGF-β2 was a generous gift from Dr. Patricia Segarini and Celtrix Corp. (Palo Alto, CA), and was purified as described (24Lioubin M.N. Madisen L. Marquardt H. Roth R. Kovacina K.S. Purchio A.F. J. Cell. Biochem. 1991; 45: 112-121Crossref PubMed Scopus (19) Google Scholar). Recombinant latent TGF-β1 was a gift from Jane Ranchelis (Bristol-Myers Squibb, Seattle, WA). Monoclonal antibody 133 against stripped thrombospondin-1 was developed in a joint effort between our laboratory and the University of Alabama at Birmingham Hybridoma core facility (12Schultz-Cherry S. Murphy-Ullrich J.E. J. Cell Biol. 1993; 122: 923-932Crossref PubMed Scopus (401) Google Scholar). Recombinant human β1-LAP (catalogue no. 246-LP/CF) and mouse monoclonal and goat polyclonal anti-LAP antibodies (catalogue no. AB-246-NA) were purchased from R&D Systems (Minneapolis, MN). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Bovine aortic endothelial (BAE) cells were isolated in our laboratory from aortas obtained at a local abattoir and were characterized by Dil-AcLDL uptake and staining for factor VIII antigen, according to established protocols. Stocks were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter glucose and supplemented with 20% fetal bovine serum. Conditioned media experiments were performed in the presence of reduced serum concentrations as described in the figure legends. NRK-49F cells (CRL-1570) were purchased from the American Type Culture Collection (Rockville, MD) and were kept in DMEM supplemented with 10% calf serum. All cells were routinely tested for mycoplasma. Equimolar concentrations of stripped thrombospondin-1 or peptides (11 nm) were incubated with recombinant latent TGF-β (2 nm) in a final volume of 0.5 ml of PBS for 1 h at 37 °C (13Schultz-Cherry S. Ribeiro S.M.F. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar). Alternatively, stripped thrombospondin-1 or peptides were incubated with BAE cell-conditioned media as described (11Murphy-Ullrich J.E. Schultz-Cherry S. Hook M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar). A positive control for latent TGF-β activation consisted of heat treatment of the latent complex at 80 °C for 5 min. TGF-β activity was assayed based on its ability to stimulate growth of NRK fibroblasts in suspension as described (11Murphy-Ullrich J.E. Schultz-Cherry S. Hook M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar). In brief, 1–3 × 103NRK cells were plated in a 0.3% agar suspension in the presence of epidermal growth factor (2.5 ng/ml; Life Technologies, Inc.) and in the presence or absence of TGF-β and incubated at 37 °C for 7 days. At the end of this incubation period, colonies containing 8–10 cells (i.e. colonies larger than 62 μm) were counted. Active TGF-β (2.5 ng/ml) was used as a positive control. Experiments were performed in triplicate at least twice. Samples were separated by SDS-polyacrylamide gel electrophoresis (% acrylamide indicated in the figure legends) and transferred to nitrocellulose membranes (2 h, 100 V). Nonspecific protein-binding sites present in the membranes were blocked by incubation with 0.5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (Tris-buffered saline/Tween). Membranes were then incubated with primary antibodies diluted in Tris-buffered saline/Tween (antibody 133 used at 0.05 μg/ml, goat polyclonal anti-LAP at 1 μg/ml, and other antibodies and dilutions specified in the figure legends) followed by extensive washes in Tris-buffered saline/Tween with 0.1% Tween 20. After washing, membranes were incubated with peroxidase-conjugated secondary antibodies (peroxidase-conjugated goat anti-mouse IgG used at 0.1 μg/ml for 1 h at room temperature, peroxidase-conjugated rabbit anti-goat IgG at 0.08 μg/ml, and dilutions and incubation times for other antibodies as indicated in the figure legends) and developed by enhanced chemiluminescence (Pierce) according to the manufacturer's instructions. Multiple exposures were obtained to assure linearity of the response. Peptide KRFKQDGGC or TRIRQDGGC (5 mg/1 ml in 50 mm Tris and 5 mm EDTA, pH 8.5) was coupled to Sulfolink (1 ml; Pierce) according to the manufacturer's instructions and equilibrated in PBS. 2.4 μmol of peptide KRFKQDGGC or 3.3 μmol of peptide TRIRQDGGC were coupled to the Sulfolink resin. Recombinant human β1-LAP (10 μg/0.5 ml of PBS, 0.28 nmol) was loaded and incubated with the affinity matrix (bed volume = 1 ml) for 20 min at room temperature and then circulated through the column five times. Prior to elution, the column was washed with 25 ml of PBS. Proteins bound to the affinity matrix were then eluted stepwise, first with 4 ml of peptide SLLK, followed by 10 ml of peptide LSKL and, for the TRIR affinity column, peptide TRIR (all peptides at 86 μm, a 150-fold molar excess to LAP). Fraction size was 0.25 ml, and all LAP protein eluted between fractions 3 and 5 (0.75–1.25 ml). Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with anti-LAP antibodies. Stripped thrombospondin (11 nm) and recombinant human β1-LAP (28 nm) were incubated together in a total volume of 0.5 ml of PBS in the presence or absence of peptide KRFK, TRIR, or KRAK, (11 μm) or peptide LSKL, SLLK, or RGQILSKLRL (28 μm). Peptides were used at a 1000-fold molar excess to either TSP or LAP, respectively. When peptides were present, each protein was preincubated with the appropriate inhibitory peptide for 30 min at 4 °C (TSP preincubated with LSKL and LAP preincubated with KRFK). The second protein was then added to the peptide/protein mixture and incubated together for 1 h at 4 °C. The protein mixture was incubated for 1 h at 4 °C with goat polyclonal antibodies raised against recombinant human LAP (0.5 μg of antibody/0.5 ml of sample), followed by a 30-min incubation with protein G-Sepharose 4B beads (Sigma) in 10 mm Tris, pH 7.4, containing 150 mm NaCl, 1% Triton X-100, and 0.5% Nonidet P-40. Alternatively, the protein mixture was incubated for 1 h at 4 °C with GammaBind G-Sepharose (catalogue no. 17-0885-01, Amersham Pharmacia Biotech) conjugated with monoclonal antibody 133 in wash buffer (PBS containing 1 g/liter ovalbumin and 5 ml/liter Tween 20; 10 μg of antibody/0.5 ml of matrix). Immune complexes were washed with wash buffer, resuspended in reducing Laemmli buffer, and analyzed by Western blotting with monoclonal antibody 133 against thrombospondin or with goat polyclonal anti-LAP antibodies. For dose-response inhibition assays, LAP was preincubated with 0.11–110 μm peptide KRFK, whereas thrombospondin was incubated with 0.28–280 μm peptide LSKL. For immunoprecipitation of proteins from media conditioned by BAE cells, ∼3 × 106 cells were incubated for 24 h in 3 ml of DMEM containing insulin-transferrin-sodium selenite media supplement (Sigma) at the concentration recommended by the manufacturer. Conditioned media were harvested and immediately incubated with goat polyclonal antibodies against recombinant human LAP (10 μg/ml) for 1 h at 4 °C, followed by incubation with protein G-Sepharose 4B beads for 1 h at 4 °C. Immune complexes were washed and analyzed by Western blotting with monoclonal anti-TSP antibodies as described above. To deplete native thrombospondin of LAP, 20 μg of thrombospondin in 25 μl of PBS were incubated three times with 25 μl of goat anti-LAP antibodies coupled to CNBr-activated Sepharose (coupling per manufacturer's instructions) for 20 min each time. Following each incubation, samples were centrifuged, and the supernatant was transferred to another tube containing antibodies coupled to resin. Prior to the first incubation and following the last incubation, protein concentration in the sample was measured by A 280 nm using a molar extinction coefficient of 1.27. Sample volumes to be tested for TGF-β activity and Western-blotted for LAP were adjusted so that the same amount of protein (6.25 μg) was used in all cases. Assay conditions were those previously described as ideal for re-formation of the latent TGF-β complex (22Miller D.M. Ogawa Y. Iwata K.K. ten Dijke P. Purchio A.F. Soloff M.S. Gentry L.E. Mol. Endocrinol. 1992; 6: 694-702Crossref PubMed Scopus (28) Google Scholar). In brief, thrombospondin (9 μg; amount chosen based on our previous studies of latent TGF-β activation by thrombospondin) was incubated with LAP (28 ng) in 100 μl of serum-free DMEM for 1 h at room temperature. TGF-β (2 ng in 2 μl) was then added to the appropriate samples, followed by an additional incubation for 1 h at room temperature. Samples to which no TGF-β was added, samples containing TGF-β alone, and samples in which LAP and thrombospondin were not incubated together prior to addition of TGF-β were incubated at the same temperature for the same extent of time to minimize variations due to loss of protein to the tube or loss of TGF-β activity over time. Immediately following the second incubation, samples were tested for TGF-β activity as described above. The search for a sequence in LAP complementary to the thrombospondin-1 sequence KRFK was performed through computer analysis utilizing a computer program designed to identify patterns of inverted hydropathy (25Maier C.C. Moseley H.N.B. Zhou S.-R. Whitaker J.N. Blalock J.E. Immunomethods. 1994; 5: 107-113Crossref PubMed Scopus (23) Google Scholar). The parameter settings used were as follows: 1) search a window size of five amino acids (hits are searched for in a window of five residues, sliding the window down the sequence one amino acid at a time); 2) average chain complementarity set at 1.2 (this value represents the average of the differences in the hydropathic scores of aligned amino acids for the window size selected; the closer to 0, the better the complementarity); and 3) the cutoff point for considering if two amino acids are opposite set to 2.0 (the absolute value of the two aligned residues added together is denoted as the cutoff). Previous results indicated that an antibody raised against the amino terminus of LAP could block thrombospondin-mediated activation of latent TGF-β (13Schultz-Cherry S. Ribeiro S.M.F. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar), suggesting a possible interaction of thrombospondin with the LAP portion of the latent complex. During the course of our studies, Yang et al. (45Yang Y. Dignam J.D. Gentry L. Biochemistry. 1997; 36: 11923-11932Crossref PubMed Scopus (22) Google Scholar) reported that recombinant dimeric LAP binds to immobilized thrombospondin. Since the presence of LAP is both necessary and sufficient to confer latency on TGF-β and since TGF-β associated with thrombospondin-1 is in its active state, one would predict that LAP would not be present in biologically active thrombospondin-1·TGF-β complexes. However, human platelet thrombospondin-1 that has TGF-β bioactivity (11Murphy-Ullrich J.E. Schultz-Cherry S. Hook M. Mol. Biol. Cell. 1992; 3: 181-188Crossref PubMed Scopus (222) Google Scholar) also contains detectable LAP, suggesting that LAP may potentially be associated with thrombospondin-1·TGF-β complexes (Fig. 1A). Furthermore, LAP isolated by immunoprecipitation from media conditioned by BAE cells in culture co-purifies with thrombospondin-1, as detected by Western blotting (Fig. 1B). These observations show that in biological fluids, thrombospondin and LAP can exist in complexes. Furthermore, these data suggest the possibility that active TGF-β can form a ternary complex with thrombospondin-1 and LAP. Since previous observations showed that thrombospondin-1 contains associated TGF-β activity, we hypothesized that LAP, thrombospondin, and TGF-β may form ternary complexes that maintain bioactivity. To investigate this hypothesis, it was determined whether removal of thrombospondin molecules that had associated LAP resulted in depletion of TGF-β activity present in the thrombospondin-1 preparation. Thrombospondin-associated TGF-β activity was measured prior to and following immunodepletion of LAP-associated thrombospondin-1 with anti-LAP antibodies coupled to Sepharose beads. Equal concentrations of protein in both the starting and immunodepleted materials were evaluated for TGF-β activity. As shown in Fig. 2A, thrombospondin-1 that had been depleted of LAP by immunoprecipitation with anti-LAP antibodies was correspondingly depleted of TGF-β activity. Immunodepletion of LAP from the thrombospondin-1 samples was confirmed by our inability to detect LAP on Western blots of treated samples (Fig. 2A). To further investigate the hypothesis that ternary complexes containing thrombospondin, TGF-β, and LAP retain TGF-β activity, these proteins were incubated under conditions that allow them to form binary and/or ternary complexes, and the resulting TGF-β activity was measured (Fig. 2B). As expected, incubation of active TGF-β with LAP resulted in inactivation of the growth factor, indicating that the latent TGF-β complex was re-formed under these conditions. However, when TGF-β was incubated with preformed complexes of thrombospondin and LAP (Fig. 2B), LAP failed to inactivate TGF-β. These observations show that LAP complexes with thrombospondin in biological fluids and that active TGF-β, thrombospondin, and LAP can form ternary complexes. These data also show that interactions of thrombospondin with LAP alter the ability of this precursor portion of TGF-β to confer latency on active TGF-β. Polyclonal antibodies raised against a peptide from the LAP amino terminus (amino acids 81–94) inhibit latent TGF-β activation by thrombospondin-1 (13Schultz-Cherry S. Ribeiro S.M.F. Gentry L. Murphy-Ullrich J.E. J. Biol. Chem. 1994; 269: 26775-26782Abstract Full Text PDF PubMed Google Scholar). This observation and the presence of LAP in biologically active thrombospondin-1·TGF-β complexes suggest that the thrombospondin-1 sequence (KRFK) responsible for activation of latent TGF-β might interact with LAP. To test this hypothesis, we chose an approach based on co-immunoprecipitation. When thrombospondin-1 and LAP were incubated together for 1 h at room temperature, they formed complexes that were immunoprecipitated by both polyclonal antibodies against LAP (Fig. 3, A and B,third lanes) and monoclonal antibodies against thrombospondin (Fig. 3, C and D, third lanes). This association between thrombospondin and LAP was competitively inhibited by preincubation of LAP with the thrombospondin-derived peptide KRFK. Partial inhibition occurred when the peptide was present at a 10–100-fold molar excess relative to the thrombospondin concentration, whereas complete inhibition was observed when the peptide was present at a 1000-fold molar excess (Fig. 3,A and C, fourth through seventh lanes). These data suggest a role for the thrombospondin-1 activation sequence KRFK in LAP binding. The inability of the related sequence KRAK, which does not activate latent TGF-β (15Schultz-Cherry S. Chen H. Mosher D.F. Misenheimer T.M. Krutzsch H.C. Roberts D.D. Murphy-Ullrich J.E. J. Biol. Chem. 1995; 270: 7304-7310Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar), to inhibit complex formation between the two proteins (Fig. 3, B and D, sixth lane) provides evidence that this interaction between the thrombospondin-1 sequence KRFK and LAP is specific. Furthermore, data from this experiment also suggest that the interaction between the KRFK sequence and LAP is relevant for the ability of thrombospondin to activate latent TGF-β since the inactive KRFK homologue in thrombospondin-2 (TRIR) had no inhibitory effect on complex formation between thrombospondin-1 and LAP (Fig. 3, B and D,fifth lanes). These data show that thrombospondin-1 binds to the LAP portion of the latent TGF-β molecule. This binding is apparently mediated by the TGF-β-activating sequence KRFK in thrombospondin-1 and may be part of the mechanism by which thrombospondin-1 activates latent TGF-β. To identify the sequence on LAP responsible for binding the thrombospondin-1 sequence KRFK, we used the molecular recognition theory as a strategy to identify sequences in LAP complementary to the KRFK sequence in thrombospondin-1 that could potentially form a binding site (25Maier C.C. Moseley H.N.B. Zhou S.-R. Whitaker J.N. Blalock J.E. Immunomethods. 1994; 5: 107-113Crossref PubMed Scopus (23) Google Scholar, 26Blalock J.E. Smith E.M. Biochem. Biophys. Res. Commun. 1984; 121: 203-207Crossref PubMed Scopus (183) Google Scholar, 27Bost K.L. Smith E.M. Blalock J.E. Proc. Natl. Acad. Sci. U" @default.
• W1978202313 created "2016-06-24" @default.
• W1978202313 creator A5001280592 @default.
• W1978202313 creator A5017416196 @default.
• W1978202313 creator A5030036413 @default.
• W1978202313 creator A5066849326 @default.
• W1978202313 creator A5088615597 @default.
• W1978202313 date "1999-05-01" @default.
• W1978202313 modified "2023-10-10" @default.
• W1978202313 title "The Activation Sequence of Thrombospondin-1 Interacts with the Latency-associated Peptide to Regulate Activation of Latent Transforming Growth Factor-β" @default.
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