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• W2078595904 abstract "The transforming growth factors-β (TGF-β) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-β isoforms TGF-β3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-β3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-β3. In addition, the aggregation of TGF-β3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-β3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH ∼3.9, a monomer ⇄ dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8.5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-β3 dissolved in solution and aggregated TGF-β3. Aggregated TGF-β3 showed a higher β-sheet content and lower β-turn and random coil contributions compared with monomeric TGF-β3. Both the solution structure and the aggregate structure of TGF-β3 were different from the crystal structure. This was in contrast to TGF-β2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-β3 conformation in solution in the range of 2.3 < pH < 11.0 was reversible. Aggregation of TGF-β3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, c NaCl = 164 mm) TGF-β3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations. The transforming growth factors-β (TGF-β) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-β isoforms TGF-β3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-β3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-β3. In addition, the aggregation of TGF-β3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-β3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH ∼3.9, a monomer ⇄ dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8.5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-β3 dissolved in solution and aggregated TGF-β3. Aggregated TGF-β3 showed a higher β-sheet content and lower β-turn and random coil contributions compared with monomeric TGF-β3. Both the solution structure and the aggregate structure of TGF-β3 were different from the crystal structure. This was in contrast to TGF-β2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-β3 conformation in solution in the range of 2.3 < pH < 11.0 was reversible. Aggregation of TGF-β3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, c NaCl = 164 mm) TGF-β3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations. transforming growth factor-β 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid 2-(cyclohexylamino)ethanesulfonic acid Transforming growth factors-β (TGF-β)1 are multifunctional cytokines used for cellular communication. They are called growth factors for historical reasons (1Sporn M.B. Roberts A.B. Wakefield L.M. de Crombrugghe B. J. Cell Biol. 1987; 105: 1039-1045Crossref PubMed Scopus (1000) Google Scholar) but their main function is to control cell proliferation and differentiation (2Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar, 3Moses H.L. Yang E.Y. Pietenpol J.A. Cell. 1990; 63: 245-247Abstract Full Text PDF PubMed Scopus (876) Google Scholar) and to stimulate the synthesis of extracellular matrix proteins (4Massagué J. Annu. Rev. Cell. Biol. 1990; 6: 597-641Crossref PubMed Scopus (2996) Google Scholar). TGF-β plays a major role in the response of cells and tissues to injury (5Grande J.P. Proc. Soc. Exp. Biol. Med. 1997; 214: 27-40Crossref PubMed Google Scholar, 6Border W.A. Ruoslahti E. J. Clin. Invest. 1992; 90: 1-7Crossref PubMed Scopus (1042) Google Scholar). Five isoforms of TGF-β are known; however, only three of them are expressed in mammalians. All isoforms show a highly homologous sequence (>70% of conserved residues). For a given isoform the homology between proteins from different species is >95%. The isoforms have similar biological activities but exhibit differences in potency depending on the target cell examined (7Cox D.A. Cell Biol. Int. 1995; 19: 357-371Crossref PubMed Scopus (81) Google Scholar, 8Roberts A.B. Sporn M.B. Adv. Cancer Res. 1988; 51: 107-145Crossref PubMed Scopus (358) Google Scholar). TGF-β is produced by virtually all cell types as inactive precursors (1Sporn M.B. Roberts A.B. Wakefield L.M. de Crombrugghe B. J. Cell Biol. 1987; 105: 1039-1045Crossref PubMed Scopus (1000) Google Scholar, 9Bailly S. Brand C. Chambaz E.M. Feige J.J. J. Biol. Chem. 1997; 272: 16329-16334Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and receptors for TGF-β are universally distributed throughout the body. The inactive precursor is first cleaved into a latent complex, which upon activation by acidification represents the regulation step of the signaling process of TGF-β (7Cox D.A. Cell Biol. Int. 1995; 19: 357-371Crossref PubMed Scopus (81) Google Scholar, 10Koppa S.D. Drug Ther. 1994; 2: 34-37Google Scholar). The activation of latent TGF-βin vivo by osteoclasts during bone resorption may be linked to the acidification (pH < 3) of the osteoclast pericellular space (11Gleizes P.E. Munger J.S. Nunes I. Harpel J.G. Mazzieri R. Noguera I. Rifkin D.B. Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (220) Google Scholar, 12Bonewald L.F. Oreffo R.O. Lee C.H. Park-Snyder S. Twardzik D. Mundy G.R. Endocrinology. 1997; 138: 657-666Crossref PubMed Scopus (23) Google Scholar).The in vivo activation seems, however, to be more often caused by proteolytic cleavage involving plasmin (13Grainger D.J. Kemp P.R. Liu A.C. Lawn R.M. Metcalfe J.C. Nature. 1994; 370: 460-462Crossref PubMed Scopus (338) Google Scholar) or calpain (14Abe M. Oda N. Sato Y. J. Cell. Physiol. 1998; 174: 186-193Crossref PubMed Scopus (65) Google Scholar). A further mechanism is the enzymatic deglycosylation of the mannose 6-phosphate of the latency associated peptide (15Miyazono K. Heldin C.H. Nature. 1989; 338: 158-160Crossref PubMed Scopus (213) Google Scholar). In addition, protease-independent conformational changes of the latent complex following binding to thrombospondin (16Schultz-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 (371) Google Scholar, 17Chambaz E.M. Souchelnitskiy S. Pellerin S. Defaye G. Cochet C. Feige J.J. Horm. Res. 1996; 45: 222-226Crossref PubMed Scopus (27) Google Scholar) also leads to release of active TGF-β in vivo.TGF-β2 was the first isoform for which the crystal structure could be solved with x-ray crystallography (18Daopin S. Piez K.A. Ogawa Y. Davies D.R. Science. 1992; 257: 369-373Crossref PubMed Scopus (374) Google Scholar, 19Daopin S. Li M. Davies D.R. Proteins. 1993; 17: 176-192Crossref PubMed Scopus (45) Google Scholar, 20Schlünegger M.P. Grütter M.G. Nature. 1992; 358: 430-434Crossref PubMed Scopus (284) Google Scholar, 21Schlünegger M.P. Cerletti N. Cox D.A. McMaster G.K. Schmitz A. Grütter M.G. FEBS Lett. 1992; 303: 91-93Crossref PubMed Scopus (20) Google Scholar, 22Schlünegger M.P. Grutter M.G. J. Mol. Biol. 1993; 231: 445-458Crossref PubMed Scopus (61) Google Scholar). Next, the solution structure of TGF-β1 was determined by heteronuclear magnetic resonance spectroscopy (23Archer S.J. Bax A. Roberts A.B. Sporn M.B. Ogawa Y. Piez K.A. Weatherbee J.A. Tsang M.L. Lucas R. Zheng B.L. Wenker J. Torchia D.A. Biochemistry. 1993; 32: 1164-1171Crossref PubMed Scopus (53) Google Scholar) and was found to be very similar to the crystal structure of TGF-β2. The detailed comparison revealed only small differences (mainly in the β-turns) which, however, could play an important role in receptor binding and isoform recognition (24Hinck A.P. Archer S.J. Qian S.W. Roberts A.B. Sporn M.B. Weatherbee J.A. Tsang M.L. Lucas R. Zhang B.L. Wenker J. Torchia D.A. Biochemistry. 1996; 35: 8517-8534Crossref PubMed Scopus (148) Google Scholar). The crystal structure of TGF-β3 was also solved recently (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar). Compared with the TGF-β2 crystal no essential differences in the tertiary structure were observed. Minor deviations were detected in the N-terminal α-helix and in the β-sheet loop regions. The well established differences in the biological activity of TGF-β2 and TGF-β3 (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar) seem to depend on differences in the surface side chains rather than the tertiary structure. Alternatively, it could be argued that despite the rather similar crystal structures, the two peptides assume different structures in solution. This problem was investigated here with CD spectroscopy.The biologically active TGF-βs are homodimers consisting of two identical chains connected via a single interchain disulfide bridge (C77–C77), the latter being exposed to solvent. Heterodimeric TGF-β1,2 and TGF-β2,3 are also known but rather unusual (26Ogawa Y. Schmidt D.K. Dasch J.R. Chang R.J. Glaser C.B. J. Biol. Chem. 1992; 267: 2325-2328Abstract Full Text PDF PubMed Google Scholar). Reduction of the active dimer results in the formation of inactive monomers. Activity is not recovered by simple reoxidation of the protein (27Archer S.J. Bax A. Roberts A.B. Sporn M.B. Ogawa Y. Piez K.A. Weatherbee J.A. Tsang M.L. Lucas R. Zheng B.L. Wenker J. Torchia D.A. Biochemistry. 1993; 32: 1152-1163Crossref PubMed Scopus (40) Google Scholar). The 8 other cysteine residues form 4 intrachain disulfide bridges, leading to a structural feature called “TGF-β knot.” The knot is almost inaccessible to solvent and stabilizes the monomer structure. The monomer exhibits an elongated nonglobular structure with dimensions of about 60 × 20 × 15 Å3. As there is only a single interchain disulfide bridge between two monomers, hydrophobic interactions between the interface areas are supposed to be of major importance in stabilizing the dimer (18Daopin S. Piez K.A. Ogawa Y. Davies D.R. Science. 1992; 257: 369-373Crossref PubMed Scopus (374) Google Scholar, 19Daopin S. Li M. Davies D.R. Proteins. 1993; 17: 176-192Crossref PubMed Scopus (45) Google Scholar, 20Schlünegger M.P. Grütter M.G. Nature. 1992; 358: 430-434Crossref PubMed Scopus (284) Google Scholar, 21Schlünegger M.P. Cerletti N. Cox D.A. McMaster G.K. Schmitz A. Grütter M.G. FEBS Lett. 1992; 303: 91-93Crossref PubMed Scopus (20) Google Scholar, 22Schlünegger M.P. Grutter M.G. J. Mol. Biol. 1993; 231: 445-458Crossref PubMed Scopus (61) Google Scholar). In addition, the dimer is further stabilized by a network of hydrogen bonds, including several water molecules, located at well defined positions in the hydrophilic cavities surrounding the intersubunit disulfide bridge.In contrast to TGF-β1 and TGF-β2, TGF-β3 shows a strong tendency to aggregate at physiological pH, making spectroscopic measurements at pH 7.0 rather difficult. Inspection of the crystal structure of TGF-β3 reveals many hydrophobic residues on its surface, which could explain its low solubility. In view of the functional differences between TGF-β2 and TGF-β3 and also of the therapeutic potential of TGF-β3, a detailed characterization of TGF-β3 solubility and aggregation is required.In this study we have characterized the solution structure of TGF-β3 under a variety of conditions with CD spectroscopy. The experimental CD spectra were deconvoluted into its secondary structural elements and compared with the predictions derived from the TGF-β3 and TGF-β2 crystal structures. In addition, the details of the aggregation process and the structural changes accompanying aggregation were investigated. To this purpose, CD spectra were recorded as a function of pH, protein concentration, and salt concentration. Analytical ultracentrifugation measurements were performed to study the size of the aggregates while titrating TGF-β3 from acidic to basic conditions and vice versa. UV spectroscopy was used to monitor the turbidity of the TGF-β3 solutions as a result of protein aggregation at different pH and salt conditions.DISCUSSIONCD and UV spectroscopy as well as ultracentrifugation studies demonstrate that TGF-β3 is soluble in monomeric form at pH ≤ 3.8 and pH ≥ 9.7. In contrast, the peptide aggregates at intermediate pH values with the aggregation maximum occurring at 6.8 ≤ pH ≤ 8.2 (cf. Fig. 2). Based on the amino acid sequence (24Hinck A.P. Archer S.J. Qian S.W. Roberts A.B. Sporn M.B. Weatherbee J.A. Tsang M.L. Lucas R. Zhang B.L. Wenker J. Torchia D.A. Biochemistry. 1996; 35: 8517-8534Crossref PubMed Scopus (148) Google Scholar) and on the known pK values of amino acids free in solution (neglecting pK shifts induced by intrachain interactions), it is possible to calculate the net charge of TGF-β3 as a function of pH using the Henderson-Hasselbach equation. An isoelectric point is estimated for pH = pI ≈ 6.8. The solubility of TGF-β3 at low and high pH values could thus be explained by its large positive or negative electric charge, respectively, at these pH extremes. In contrast, aggregation at physiological pH could be induced by the rather hydrophobic surface of the electrically neutral protein. Analogous calculations for TGF-β1 and TGF-β2 yield isoelectric points of pI ∼ 9.5 and pI ∼ 8.5, respectively. In fact, TGF-β1 and TGF-β2 are distinctly more soluble than TGF-β3 under physiological conditions. The good solubility of TGF-β3 at low pH could explain its prominent rolein vivo in processes involving acidification of the surroundings. The most striking examples are: (i) bone remodeling with pH < 3.0 around the osteoclasts (34ten Dijke P. Iwata K.K. Goddard C. Pieler C. Canalis E. McCarthy T.L. Centrella M. Mol. Cell. Biol. 1990; 10: 4473-4479Crossref PubMed Google Scholar, 35Yang N.N. Bryant H.U. Hardikar S. Sato M. Galvin R.J. Glasebrook A.L. Termine J.D. Endocrinology. 1996; 137: 2075-2084Crossref PubMed Scopus (124) Google Scholar, 36Roth D.A. Gold L.I. Han V.K. McCarthy J.G. Sung J.J. Wisoff J.H. Longaker M.T. Plast. Reconstr. Surg. 1997; 99 (Discussion 310–316): 300-309Crossref PubMed Scopus (133) Google Scholar, 37Kloen P. Gebhardt M.C. Perez-Atayde A. Rosenberg A.E. Springfield D.S. Gold L.I. Mankin H.J. Cancer. 1997; 80: 2230-2239Crossref PubMed Scopus (84) Google Scholar); (ii) inflammation where lysosomal release can locally lower the pH under 5.0 (38Levine J.H. Moses H.L. Gold L.I. Nanney L.B. Am. J. Pathol. 1993; 143: 368-380PubMed Google Scholar, 39Schmid P. Cox D. Bilbe G. McMaster G. Morrison C. Stahelin H. Luscher N. Seiler W. J. Pathol. 1993; 171: 191-197Crossref PubMed Scopus (114) Google Scholar, 40Frank S. Madlener M. Werner S. J. Biol. Chem. 1996; 271: 10188-10193Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar); and (iii) the ubiquitous expression of TGF-β3 in gastric tissues and its strong influence in gastric cancers, in contrast to TGF-β1, which is localized principally in parietal cells, and TGF-β2, which is present exclusively in chief cells (41Naef M. Ishiwata T. Friess H. Buchler M.W. Gold L.I. Korc M. Int. J. Cancer. 1997; 71: 131-137Crossref PubMed Scopus (56) Google Scholar).TGF-β2 was the first TGF-β isoform to be crystallized (at pH 4.5). The x-ray analysis yielded a structure with 11% α-helix, 36% β-sheet, 8% helical turns, 3% 310-helical turn and 42% random coil (22Schlünegger M.P. Grutter M.G. J. Mol. Biol. 1993; 231: 445-458Crossref PubMed Scopus (61) Google Scholar). In 1996, the crystal structure of TGF-β3 was also solved (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar). Comparison with TGF-β2 revealed, however, only small differences, mainly in the β-loop regions (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar). Because the percentages of structural elements were not specified by Mittl et al. (1996), TGF-β2 was taken as the starting point for the simulation of the CD spectra. Based on reference CD spectra of 18 globular proteins (31Yang J.T. Wu C.S. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Crossref PubMed Scopus (1730) Google Scholar) and using the percentages of the crystal structure, the CD spectrum shown by the dotted line in Fig.4 was calculated. The theoretical spectrum is quite different from the experimental spectrum (solid line), indicating that the TGF-β2/TGF-β3 crystal structure is not a good model for TGF-β3 in solution. A much better fit to the experimental spectrum at pH 4.4 is given by a simulation containing 4% α-helix, 66% β-sheet, 8% β-turns, and 22% random coil (♦ in Fig. 4). Compared with the crystal structure, the α-helical content is reduced and the contribution of β-structures is clearly enhanced. The displacement in the wavelength of the two spectra is due to spectral distortions caused by light scattering of the TGF-β3 aggregates.The simulation of CD spectra is a multiparameter fit, and the relevance of the structural parameters is often subject to criticism. However, CD simulations are biased in sensitivity toward α-helical structures. The decrease of α-helix of TGF-β3 in solution compared with the crystal structure is unambiguous and clearly beyond the error of the numerical approach. A possible explanation for the reduced α-helical content of TGF-β3 in solution is the presence of glycine (Gly-63) in the α-helical region of TGF-β3 between amino acids 57 and 68. This glycine, which confers to the protein backbone additional flexibility, is not present in the α-helical regions of TGF-β1 and TGF-β2 (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar).CD spectra of TGF-β3 in solution at pH 1.9 have been reported previously, indicating a much larger helix content (28Runser S. Cerletti N. Biotechnol. Appl. Biochem. 1995; 22: 39-53PubMed Google Scholar). We have not been able to confirm these results, which were probably caused by excessive smoothing of the spectra. The present findings are, however, in agreement with 2D-NMR studies of TGF-β3, which suggest an increased molecular flexibility in comparison with TGF-β1. 2M. J. Blommers and T. Arvinte, unpublished results. The considerable structural difference between TGF-β3 in solution and the crystal structure suggests a rather flexible conformation that can adjust itself to external constraints. It could explain the different receptor specificity of TGF-β2 and TGF-β3 despite similar x-ray structures.We have also recorded CD spectra of TGF-β2 in solution at pH 2.96 (data not shown) and have compared them with theoretical spectra calculated on the basis of the crystal structure. In contrast to TGF-β3, a good agreement between the experimental and theoretical spectral shapes was found for TGF-β2. The CD results for TGF-β2 may serve as a positive control for the sensitivity of CD spectroscopy to detect conformational changes for the problem at hand. They emphasize that TGF-β2 adopts the same structure in the crystal and in solution, whereas TGF-β3 reveals two different conformations.The CD spectra of TGF-β3 further demonstrate that the conformation of this protein varies with the pH of the solution. A first conformational change occurs at pH ∼ 4.4 and is accompanied by TGF-β3 aggregation; the second transition begins at pH ∼ 9.8 and is associated with the deprotonation of the solvent-accessible tyrosine residues (8 of 16; see above). At the same time the aggregation process is reversed. The pH-induced aggregation can also be detected with fluorescence spectroscopy using the TGF-β3 Trp residues as intrinsic markers. Aggregation leads to an increase in the steady state polarization and an increase of the fluorescence life time. 3T. Arvinte, unpublished results. A deconvolution of the CD spectra was attempted in the pH ranges of 2.2 ≤ pH ≤ 6.0 and 9 ≤ pH ≤ 10.4 where aggregation was not too pronounced. The relative contributions of the different secondary structures are summarized in Fig. 5. The most prominent change is the increase in β-structure around pH 4.4, which is reversed at pH ∼ 9.8.The destabilization of the β-structure at pH ∼ 9.8 is compensated by an increase in α-helix.Figure 5Variation of the secondary structure of TGF-β3, as a function of pH as derived from CD simulations. ♦, α-helix; ○, β-sheet; ■, β-turns; ●, random coil. In the pH range of 4.5 ≤ pH ≤ 9.8, indicated by thedashed lines, the protein solutions are turbid.View Large Image Figure ViewerDownload (PPT)Concluding RemarksIn conclusion, the solubility of TGF-β3 under physiological conditions (pH 7.0) is low and distinctly smaller than that of TGF-β1 and TGF-β2. TGF-β3 has a high tendency to adsorb to hydrophobic surfaces and to form large aggregates. At extreme pH values (pH < 2.3 or pH > 11.3) TGF-β3 is monomeric in solution, whereas at pH ∼ 3.9 a monomer ⇄ dimer equilibrium was detected by ultracentrifugation. The addition of salt greatly reduces the solubility of TGF-β3 and enhances its tendency to aggregate. The structure of TGF-β3 in solution is different from the crystal structure; notably, the helix content is reduced, and the β-structure content is increased. The change in conformation of TGF-β3 in solution could explain the different receptor specificity of TGF-β3 compared with TGF-β2 despite their very similar x-ray structure. Transforming growth factors-β (TGF-β)1 are multifunctional cytokines used for cellular communication. They are called growth factors for historical reasons (1Sporn M.B. Roberts A.B. Wakefield L.M. de Crombrugghe B. J. Cell Biol. 1987; 105: 1039-1045Crossref PubMed Scopus (1000) Google Scholar) but their main function is to control cell proliferation and differentiation (2Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar, 3Moses H.L. Yang E.Y. Pietenpol J.A. Cell. 1990; 63: 245-247Abstract Full Text PDF PubMed Scopus (876) Google Scholar) and to stimulate the synthesis of extracellular matrix proteins (4Massagué J. Annu. Rev. Cell. Biol. 1990; 6: 597-641Crossref PubMed Scopus (2996) Google Scholar). TGF-β plays a major role in the response of cells and tissues to injury (5Grande J.P. Proc. Soc. Exp. Biol. Med. 1997; 214: 27-40Crossref PubMed Google Scholar, 6Border W.A. Ruoslahti E. J. Clin. Invest. 1992; 90: 1-7Crossref PubMed Scopus (1042) Google Scholar). Five isoforms of TGF-β are known; however, only three of them are expressed in mammalians. All isoforms show a highly homologous sequence (>70% of conserved residues). For a given isoform the homology between proteins from different species is >95%. The isoforms have similar biological activities but exhibit differences in potency depending on the target cell examined (7Cox D.A. Cell Biol. Int. 1995; 19: 357-371Crossref PubMed Scopus (81) Google Scholar, 8Roberts A.B. Sporn M.B. Adv. Cancer Res. 1988; 51: 107-145Crossref PubMed Scopus (358) Google Scholar). TGF-β is produced by virtually all cell types as inactive precursors (1Sporn M.B. Roberts A.B. Wakefield L.M. de Crombrugghe B. J. Cell Biol. 1987; 105: 1039-1045Crossref PubMed Scopus (1000) Google Scholar, 9Bailly S. Brand C. Chambaz E.M. Feige J.J. J. Biol. Chem. 1997; 272: 16329-16334Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and receptors for TGF-β are universally distributed throughout the body. The inactive precursor is first cleaved into a latent complex, which upon activation by acidification represents the regulation step of the signaling process of TGF-β (7Cox D.A. Cell Biol. Int. 1995; 19: 357-371Crossref PubMed Scopus (81) Google Scholar, 10Koppa S.D. Drug Ther. 1994; 2: 34-37Google Scholar). The activation of latent TGF-βin vivo by osteoclasts during bone resorption may be linked to the acidification (pH < 3) of the osteoclast pericellular space (11Gleizes P.E. Munger J.S. Nunes I. Harpel J.G. Mazzieri R. Noguera I. Rifkin D.B. Stem Cells. 1997; 15: 190-197Crossref PubMed Scopus (220) Google Scholar, 12Bonewald L.F. Oreffo R.O. Lee C.H. Park-Snyder S. Twardzik D. Mundy G.R. Endocrinology. 1997; 138: 657-666Crossref PubMed Scopus (23) Google Scholar). The in vivo activation seems, however, to be more often caused by proteolytic cleavage involving plasmin (13Grainger D.J. Kemp P.R. Liu A.C. Lawn R.M. Metcalfe J.C. Nature. 1994; 370: 460-462Crossref PubMed Scopus (338) Google Scholar) or calpain (14Abe M. Oda N. Sato Y. J. Cell. Physiol. 1998; 174: 186-193Crossref PubMed Scopus (65) Google Scholar). A further mechanism is the enzymatic deglycosylation of the mannose 6-phosphate of the latency associated peptide (15Miyazono K. Heldin C.H. Nature. 1989; 338: 158-160Crossref PubMed Scopus (213) Google Scholar). In addition, protease-independent conformational changes of the latent complex following binding to thrombospondin (16Schultz-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 (371) Google Scholar, 17Chambaz E.M. Souchelnitskiy S. Pellerin S. Defaye G. Cochet C. Feige J.J. Horm. Res. 1996; 45: 222-226Crossref PubMed Scopus (27) Google Scholar) also leads to release of active TGF-β in vivo. TGF-β2 was the first isoform for which the crystal structure could be solved with x-ray crystallography (18Daopin S. Piez K.A. Ogawa Y. Davies D.R. Science. 1992; 257: 369-373Crossref PubMed Scopus (374) Google Scholar, 19Daopin S. Li M. Davies D.R. Proteins. 1993; 17: 176-192Crossref PubMed Scopus (45) Google Scholar, 20Schlünegger M.P. Grütter M.G. Nature. 1992; 358: 430-434Crossref PubMed Scopus (284) Google Scholar, 21Schlünegger M.P. Cerletti N. Cox D.A. McMaster G.K. Schmitz A. Grütter M.G. FEBS Lett. 1992; 303: 91-93Crossref PubMed Scopus (20) Google Scholar, 22Schlünegger M.P. Grutter M.G. J. Mol. Biol. 1993; 231: 445-458Crossref PubMed Scopus (61) Google Scholar). Next, the solution structure of TGF-β1 was determined by heteronuclear magnetic resonance spectroscopy (23Archer S.J. Bax A. Roberts A.B. Sporn M.B. Ogawa Y. Piez K.A. Weatherbee J.A. Tsang M.L. Lucas R. Zheng B.L. Wenker J. Torchia D.A. Biochemistry. 1993; 32: 1164-1171Crossref PubMed Scopus (53) Google Scholar) and was found to be very similar to the crystal structure of TGF-β2. The detailed comparison revealed only small differences (mainly in the β-turns) which, however, could play an important role in receptor binding and isoform recognition (24Hinck A.P. Archer S.J. Qian S.W. Roberts A.B. Sporn M.B. Weatherbee J.A. Tsang M.L. Lucas R. Zhang B.L. Wenker J. Torchia D.A. Biochemistry. 1996; 35: 8517-8534Crossref PubMed Scopus (148) Google Scholar). The crystal structure of TGF-β3 was also solved recently (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar). Compared with the TGF-β2 crystal no essential differences in the tertiary structure were observed. Minor deviations were detected in the N-terminal α-helix and in the β-sheet loop regions. The well established differences in the biological activity of TGF-β2 and TGF-β3 (25Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar) seem to depend on differences in the surface side chains rather than the tertiary structure. Alternatively, it could be argued that despite the rather similar crystal structures, the two peptides assume different structures in solution. This problem was investig" @default.
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• W2078595904 title "Conformation and Self-association of Human Recombinant Transforming Growth Factor-β3 in Aqueous Solutions" @default.
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