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• W2012180376 abstract "We have undertaken a comparative study of the interaction of the three mammalian transforming growth factor-βs (TGF-β) with heparin and heparan sulfate. TGF-β1 and -β2, but not -β3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-β1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-β1 by α2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-β2·α2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-β3 cannot be affected. Comparison of the amino acid sequences of the TGF-β isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-β dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain. We have undertaken a comparative study of the interaction of the three mammalian transforming growth factor-βs (TGF-β) with heparin and heparan sulfate. TGF-β1 and -β2, but not -β3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-β1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-β1 by α2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-β2·α2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-β3 cannot be affected. Comparison of the amino acid sequences of the TGF-β isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-β dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain. A large number of growth factors and cytokines, belonging to structurally and biologically diverse protein families, possess affinity for heparin in vitro. Such affinity has often proved to be an experimental indicator of a physiological interaction with the heparan sulfate (HS) 1The abbreviations used are: HS, heparan sulfate; TGF-β, transforming growth factor-β; HSPG, heparan sulfate proteoglycan; CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; α2M, α2-macroglobulin; LRP, low density lipoprotein receptor-related protein. chains of heparan sulfate proteoglycans (HSPGs), a widespread and abundant family of complex glycoconjugates expressed on the surface of all adherent cells (1David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (374) Google Scholar), and also present within basement membranes and stromal matrices (2Iozzo R.V. Cohen I.R. Grassel S. Murdoch A.D. Biochem. J. 1994; 302: 625-639Crossref PubMed Scopus (341) Google Scholar). Binding to HSPGs in vivo may have an important role in retaining active growth factors/cytokines within a local sphere of action by protecting them from both diffusional and degradative loss. Importantly, however, in the case of an increasing number of such growth factors, e.g. various members of the fibroblast growth factor family, vascular endothelial growth factor, heparin-binding epidermal growth factor, and hepatocyte growth factor, it has been demonstrated that HSPGs have a specific co-receptor role in directly modulating growth factor activation of the respective cell surface signaling receptors (3Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 4Lyon M. Gallagher J.T. Biochem. Soc. Trans. 1994; 22: 365-370Crossref PubMed Scopus (27) Google Scholar). The transforming growth factor-βs (TGF-βs) are important regulators of the growth, differentiation, and adhesion of a wide variety of cells (5Massagué J. Attisano L. Wrana J.L. Trends Cell. Biol. 1994; 4: 172-177Abstract Full Text PDF PubMed Scopus (527) Google Scholar). They are believed to have an important role in natural repair processes, but overexpression or dysregulation can lead to the development of various fibrotic disorders. TGF-β1 has been demonstrated to possess strong heparin binding activity in vitro (6McCaffrey T.A. Falcone D.J. Du B. J. Cell. Physiol. 1992; 152: 430-440Crossref PubMed Scopus (170) Google Scholar). Such interaction protects TGF-β1 from proteolytic degradation in vitro (7McCaffrey T.A. Falcone D.J. Vicente D. Du B. Consigli S. Borth W. J. Cell. Physiol. 1994; 159: 51-59Crossref PubMed Scopus (99) Google Scholar), and also prevents the formation of inactive complexes with α2-macroglobulin (α2M) (8McCaffrey T.A. Falcone D.J. Brayton C.F. Agarwal L.A. Welt F.G.P. Weksler B.B J. Cell. Biol. 1989; 109: 441-448Crossref PubMed Scopus (191) Google Scholar). The physiological significance of an interaction restricted to heparin, a GAG released only upon the degranulation of activated mast cells, is unclear. Although TGF-β does bind to a number of proteoglycan species in vivo, namely the cell surface HS-containing proteoglycan, betaglycan (the type III TGF-β receptor) (9Cheifetz S. Massagué J. J. Biol. Chem. 1989; 264: 12025-12028Abstract Full Text PDF PubMed Google Scholar), as well as various members of the family of small secreted chondroitin/dermatan sulfate (CS/DS) proteoglycans (i.e. decorin, biglycan, and fibromodulin) (10Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1299) Google Scholar, 11Hildebrand A. Romaris M. Rasmussen L.M. Heinegård D. Twardzik D.R. Border W.A. Ruoslahti E. Biochem. J. 1994; 302: 524-527Crossref Scopus (863) Google Scholar), these associations are mediated principally, if not solely, by protein-protein rather than protein-GAG interactions. In addition to TGF-β1, there are two other mammalian isoforms, TGF-β2 and -β3, and also two distinct, but poorly characterized, non-mammalian isoforms, TGF-β4 and -β5 (identified in chick andXenopus, respectively). TGF-βs 1–3 possess very high levels of amino acid sequence homology within the mature bioactive molecule (>70% amino acid identity between isoform pairs with the majority of changes being conservative), including conservation, in both number and position, of the eight cysteine residues that contribute to the compact folding of the monomer, as well as the single cysteine residue involved in disulfide-bonded dimerization. Interestingly, however, there is little if any interspecies variation within each individual isoform. This has led to the proposition that the individual isoforms have closely related tertiary structures, which have diverged sufficiently to behave as functionally distinct species. Indeed, there is substantial evidence pointing to significant differences in biological behavior between the mammalian isoforms, bothin vitro and in vivo. There are differential patterns of isoform expression during fetal development (12Gatherer D. ten Dijke P. Baird D.T. Akhurst R.J. Development. 1990; 110: 445-460PubMed Google Scholar, 13Pelton R.W. Saxena B. Jones M. Moses H.L. Gold L.I. J. Cell. Biol. 1991; 115: 1091-1105Crossref PubMed Scopus (636) Google Scholar) and in the adult (14Miller D.A. Lee A. Matsui Y. Chen E.Y. Moses H.L. Derynck R. Mol. Endocrinol. 1989; 3: 1926-1934Crossref PubMed Scopus (150) Google Scholar, 15Miller D.A. Lee A. Pelton R.W. Chen E.Y. Moses H.L. Derynck R. Mol. Endocrinol. 1989; 3: 1108-1114Crossref PubMed Scopus (205) Google Scholar), and indeed the existence of distinctive regulatory elements in the promoter regions of the respective genes (16Roberts A.B. Kim S.J. Noma T. Glick A.B. Lafyatis R. Lechleider R. Jakowlew S.B. Geiser A. O'Reilly M.A. Danielpour D. Sporn M.B. CIBA Found. Symp. 1991; 157: 7-28PubMed Google Scholar) is suggestive of a capacity for independent isoform regulation. The isoforms also display marked specificities or differential potencies in various in vitro biological systems. For example, TGF-β3 is more potent than the -β1 or -β2 isoforms in inhibiting DNA synthesis in human keratinocytes in vitro (17Graycar J.L. Miller D.A. Arrick B.A. Lyons R.M. Moses H.L. Derynck R. Mol. Endocrinol. 1989; 3: 1977-1986Crossref PubMed Scopus (223) Google Scholar). While TGF-β2, but not -β1, can stimulate mesoderm induction inXenopus embryos (18Roberts A.B. Kondaiah P. Rosa F. Watanabe S. Good P. Danielpour D. Roche N.S. Rebbert M.L. Dawid I.B. Sporn M.B. Growth Factors. 1990; 3: 277-286Crossref PubMed Scopus (49) Google Scholar), TGF-β2 specifically has little or no antiproliferative effect on vascular endothelial cells in vitro (19Jennings J.C. Mohan S. Linkhart T.A. Widstrom R. Baylink D.J. J. Cell. Physiol. 1988; 137: 167-172Crossref PubMed Scopus (156) Google Scholar). Also, exogenously added TGF-β3 appears to have a specific ability to reduce scar formation during wound healing in the adult animal (20Shah M. Foreman D.M. Ferguson M.W.J. J. Cell. Sci. 1995; 108: 985-1002Crossref PubMed Google Scholar). These differences in biological activity may be due, in part, to the known differences in the receptor binding properties of the isoforms (e.g. the reduced affinity of TGF-β2 for the type II receptor protein (21Lin H.Y. Moustakas A. Knaus P. Wells R.G. Henis Y.I. Lodish H.F. J. Biol. Chem. 1995; 270: 2747-2754Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar)), although other interactive specificities may also be important. In the light of these known isoformic differences, together with the previously described affinity of TGF-β1 for heparin, we were interested in further elucidating the nature and extent of GAG-TGF-β interactions. Human platelet TGF-β1, recombinant human TGF-β2, and mouse anti-human pan TGF-β monoclonal antibody were purchased from Genzyme (West Malling, United Kingdom (UK)). Recombinant human TGF-β3 was a generous gift from Oncogene Science (Uniondale, NY). α2-Macroglobulin was obtained from Boehringer Mannheim (Lewes, UK). Normal rat kidney fibroblasts (NRK 49F) and mink lung epithelial cells (Mv1Lu CCL-64) were from the American Type Culture Collection (ATCC). Bovine lung heparin, whale shark cartilage CS, bovine mucosal DS, bovine kidney HS, heparin conjugated to cross-linked 4% agarose and heparinase I (Flavobacterium heparinum; EC 4.2.2.7) were all purchased from Sigma (Poole, UK). Heparinase II (F. heparinum; no EC number assigned) and heparinase III (F. heparinum; EC 4.2.2.8) were obtained from Grampian Enzymes (Aberdeen, UK). Porcine mucosal HS was a gift from NV Organon (Oss, The Netherlands). Decorin proteoglycan was generously provided by Dr. H. Pearson (University of Alberta, Edmonton, Canada). De-N-sulfated, re-N-acetylated heparin, and selectively de-6-O-sulfated heparin were kindly provided by Dr. B. Mulloy (National Institute for Biological Standards and Control, Potters Bar, Herts., UK). Selectively de-2-O-sulfated heparin was a gift from Dr. B. Casu (Instituto Chimica and Biochimica “G. Ronzini,” Milan, Italy). Cell surface HSPGs were purified from rat liver by the method of Lyon and Gallagher (22Lyon M. Gallagher J.T. Biochem. J. 1991; 273: 415-422Crossref PubMed Scopus (44) Google Scholar). HS chains were liberated by alkaline elimination using 50 mm NaOH, 1 m sodium borohydride at 45 °C for 48 h. After neutralization with acetic acid the HS was precipitated by the addition of four volumes of 95% (v/v) ethanol at −20 °C overnight. HS concentrations were quantified using an Alcian Blue-binding microassay (23Bartold P.M. Page R.C. Anal. Biochem. 1985; 150: 320-324Crossref PubMed Scopus (59) Google Scholar), relative to a standard curve obtained with bovine kidney HS. Samples were exhaustively digested with heparinases I, II, and III. The resulting disaccharide products were then resolved by strong anion-exchange chromatography on a 5-μm particle size Spherisorb SAX-HPLC column (Technicol, Stockport, UK), essentially as described by Lyon et al. (24Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar). Disaccharides were detected by UV absorbance at 232 nm. Batches of TGF-β were routinely assayed for their biological activity through their ability to inhibit the incorporation of [3H]thymidine into mink lung epithelial cells. Briefly, Mv1Lu CCL-64 cells were plated out at 104 cells/well of a 24-well plate (Costar, High Wycombe, UK) in Dulbecco's modified Eagle's medium/F-12 medium (Life Technologies, Inc., Paisley, UK) containing 5% (v/v) donor calf serum. After 4 h, cultures were treated with a range of TGF-β concentrations and then pulsed with 0.5 μCi/ml [3H]thymidine (NEN Life Science Products, Stevenage, UK) for 2 h. The incorporation of 3H radiolabel into trichloroacetic acid-insoluble cellular material was then determined. Samples of TGF-β1, -β2, and -β3 (50 ng of each) were individually applied to 0.3-ml volume columns of heparin-agarose and Sepharose CL4B (as a control) in 0.3 ml of 0.05m NaCl, 100 μg of bovine serum albumin/ml, 10 mm Tris-HCl, pH 7.0. The sample was recycled through the columns five times and then left on the column for an additional 1 h at room temperature before washing with the same solution to remove all non-binding material. Columns were then sequentially step eluted with 2 ml each of 0.1, 0.15, 0.2, 0.25, 0.5, and 2.0 m NaCl in 10 mm Tris-HCl, pH 7.0, containing 100 μg of bovine serum albumin/ml. The individual eluates were assayed for their TGF-β content by immunodetection, as described below. Samples of TGF-β1, -β2, and -β3 (50 ng each) were individually incubated with 1 μg (quantified as HS) of rat liver HSPG in 20 μl of phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20, for 2 h at room temperature. The mixtures were then chromatographed on a TSK-3000 PW (7.8 mm × 30 cm; Toyo Soda Manufacturing Co. Ltd., Tokyo, Japan) HPLC size exclusion column eluted with PBS, 0.1% (v/v) Tween 20 at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed for their TGF-β content. The column was calibrated using dextran blue (V o) and sodium dichromate (V T). The interaction of TGF-β3 (50 ng) and α2M (750 μg) was analyzed in an identical manner. Chromatography fractions were loaded into the wells of a dot blot apparatus (Bio-Rad Laboratories, Hemel Hempstead, UK) and adsorbed by filtration under vacuum onto Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) washed through with PBS. The nitrocellulose was removed, blocked overnight in PBS containing 1% (w/v) bovine serum albumin and then washed three times with PBS, 0.05% (v/v) Tween 20. After incubation with a 1:200 dilution of mouse anti-human pan TGF-β monoclonal antibody for 2 h at 4 °C, the membrane was washed three times with PBS, 0.05% (v/v) Tween 20, and then further incubated with a 1:1000 dilution of horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dako, High Wycombe, UK) for 1.5 h. The membrane was finally washed six times in succession with PBS, 0.05% (v/v) Tween 20, and the presence of TGF-β was then visualized by enhanced chemoluminescence (ECL) following the manufacturer's (Amersham International, Amersham, UK) protocol. The resulting dot blot images were then scanned and analyzed using a model GS-700 Imaging Densitometer (Bio-Rad) operating in transmittance mode, yielding semi-quantitative measurements of TGF-β levels in arbitrary units of darkness per unit area. Small (35 mm) bacteriological dishes (Nunc, Life Technologies, Inc., Paisley, UK) were overlaid with 0.5 ml of molten 5% (w/v) agar (Difco Laboratories, West Molesey, UK) in water and allowed to set. An additional 0.75 ml of molten 5% (w/v) agar in water was diluted with 4.25 ml of assay medium (Dulbecco's modified Eagle's medium; Life Technologies, Inc.) supplemented with 5% (v/v) fetal calf serum (Life Technologies, Inc.), 10 ng of epidermal growth factor/ml, 1 mm sodium pyruvate, and a standard concentration of non-essential amino acids. Four milliliters of this warm agar mixture was then added to a 1-ml suspension of 5 × 104 NRK 49F cells in assay medium (i.e. a final agar concentration of 0.6% (w/v)). In test cases the cell suspensions also contained 0.25 ng/ml (a suboptimal concentration) of individual TGF-β isoforms, with or without the addition of various glycosaminoglycans over a range of concentrations. After thorough mixing, 1.5 ml of the agar-suspended cells were plated out onto the agar-coated dishes. Cells were then incubated at 37 °C in a humidified atmosphere of 5% CO2(v/v) in air for 7 days. After this period the number of colonies comprising greater than 50 cells were counted under a Leitz/Diavert inverted microscope (25De Larco J.E. Todaro G.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4001-4005Crossref PubMed Scopus (1199) Google Scholar, 26Roberts A.B. Anzano M.A. Lamb L.C. Smith J.M. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5339-5343Crossref PubMed Scopus (720) Google Scholar). For experiments performed in the absence of α2M, a serum-free assay (27Rizzino A. Ruff E. Rizzino H. Cancer Res. 1986; 46: 2816-2820PubMed Google Scholar) was adopted in which the fetal calf serum in the assay medium was replaced by a mixture of 10 μg of insulin, 5 μg of transferrin, 0.3 mg of high density lipoprotein, and 1 mg of bovine serum albumin per ml of medium. Interaction with heparin was assessed by affinity chromatography on heparin-agarose columns. Because of the known ability of TGF-β to bind strongly and nonspecifically to many surfaces, the chromatography was performed in the presence of a relatively high concentration of serum albumin. Samples were also run in parallel on control columns of Sepharose CL-4B. The in vitro biological activity of each TGF-β isoform batch (i.e.. the ability to inhibit the incorporation of [3H] thymidine by mink lung epithelial cells) was tested beforehand to confirm that an essentially active and native conformation was being analyzed (data not shown). TGF-β1 demonstrated a strong interaction with heparin-agarose (Fig. 1 A), confirming the original observation of McCaffrey et al. (6McCaffrey T.A. Falcone D.J. Du B. J. Cell. Physiol. 1992; 152: 430-440Crossref PubMed Scopus (170) Google Scholar). The bound fraction was predominantly released by a 0.5 m NaCl step, although a proportion required a higher ionic strength for elution. The behavior of TGF-β2 was consistently more complex showing marked nonspecific binding, as evidenced by its behavior on the Sepharose CL-4B column. Nevertheless, a small but consistent fraction of the TGF-β2 bound specifically to the heparin-agarose column requiring 0.5m NaCl, or higher, for elution (Fig. 1 B). A significant proportion of both TGF-β1 and -β2 failed to bind to the heparin-agarose. This was a reproducible, although quantitatively variable, phenomenon, which has been observed with radiolabeled TGF-β1 (6McCaffrey T.A. Falcone D.J. Du B. J. Cell. Physiol. 1992; 152: 430-440Crossref PubMed Scopus (170) Google Scholar), and probably reflects a continual process of denaturation and loss of activity with time of TGF-β stocks. Biologically active TGF-β3 displayed no discernible affinity for heparin at physiological ionic strength or above (Fig. 1 C). This was confirmed by the inability of TGF-β3, either covalently coupled to Affi-Gel 10 (Bio-Rad) or non-covalently adsorbed onto nitrocellulose, to capture [3H]heparin (NEN Life Science Products) (data not shown), and also by heparin's apparent inability to afford any protection to TGF-β3 from digestion by trypsin (data not shown). Affinity for heparan sulfate was assessed by rapid chromatography of a preincubated mixture of TGF-β and purified rat liver HSPG (approximately 25-fold molar excess of HS chains), at physiological ionic strength and pH, on a size-exclusion HPLC column. Intact HSPGs, rather than HS chains, were used so as to maximize the potential size difference between free TGF-β and any complex formed. Whereas a significant fraction of both TGF-β1 and -β2 eluted as a high molecular weight complex in the presence of the liver HSPG, there was no evidence of any such complex formed with TGF-β3 (Fig.2). Thus the differential binding properties of the TGF-β isoforms were identical for both heparin and liver HSPG. This similarity suggests that the major binding site within the HSPG resides in the HS chains and not the core protein. Although TGF-β does bind specifically and with high affinity to the core protein of the betaglycan HSPG, the latter species is essentially absent from the purified liver HSPG preparation employed here (28Pierce A. Lyon M. Hampson I.N. Cowling G.J. Gallagher J.T. J. Biol. Chem. 1992; 267: 3894-3900Abstract Full Text PDF PubMed Google Scholar). Also, betaglycan interacts non-discriminately with all three TGF-βs (29Cheifetz S. Bassols A. Stanley K. Ohta M. Greenberger J. Massague J. J. Biol. Chem. 1988; 263: 10783-10789Abstract Full Text PDF PubMed Google Scholar). All three TGF-β isoforms stimulate to a similar extent the anchorage-independent proliferation of colonies of NRK 49F fibroblasts in soft agar suspension, primarily through the stimulation of the endogenous synthesis and secretion of extracellular matrix macromolecules, especially fibronectin. Using suboptimal concentrations of the TGF-β isoforms (0.25 ng/ml, equivalent to approximately 10 pm), it was observed that the addition of heparin markedly potentiated the activity of TGF-β1, with the optimal heparin concentration of 1 μg/ml eliciting a 6.6-fold potentiation (Fig. 3 A). This effect was specific in that there was no discernible effect on cellular proliferation induced by either TGF-β2 or -β3 over the same range of heparin concentrations (at a higher concentration of 10 μg/ml, a barely significant potentiation of TGF-β2 was seen) (Fig. 3 A). Heparin alone, in the absence of TGF-β, has no independent stimulatory effect on colony growth (Fig. 3 A). In contrast to the effects of heparin, a porcine mucosal heparan sulfate preparation failed to potentiate any of the TGF-β isoforms, including TGF-β1 (Fig. 3 B). Interestingly, however, HS chains derived from the liver HSPG did markedly potentiate the activity of TGF-β1 (Fig. 3 C), and to an extent comparable with that of heparin. This effect is consistent with the earlier premise that the TGF-β binding activity of intact liver HSPG resides in the HS chains and not the core protein. The apparent reduction in potentiation of TGF-β1 at the highest concentrations of heparin (Fig. 3 A) and liver HS (Fig. 3 C) may be due to a competing direct antagonistic effect on the cells of these GAGs, apparent only at higher concentrations, or an inhibitory sequestration of the TGF-β at very high molar ratios of GAG to TGF-β. Protection assays also indicate that both heparin and liver HS protect TGF-β1 from tryptic degradation, whereas porcine mucosal HS does not (data not shown), implying that their ability to potentiate TGF-β1 activity is positively correlated with direct high affinity binding. The foregoing data indicate that of theN-sulfated GAGs tested the most potent were found to be heparin and rat liver HS, while the lower sulfated porcine mucosal HS had little effect (see Table I for structural comparisons). Compared with heparin, other classes of sulfated GAGs were relatively ineffective. CS had no discernible effect, while a mucosal DS, although having a potentiating effect at the highest concentration tested (1 μg/ml), was approximately 10-fold less potent (data not shown). However, as this DS preparation was found to be contaminated to at least 3–4% with highly sulfated HS and/or heparin, the specificity of this level of activity is uncertain. Indeed, McCaffrey et al. (8McCaffrey T.A. Falcone D.J. Brayton C.F. Agarwal L.A. Welt F.G.P. Weksler B.B J. Cell. Biol. 1989; 109: 441-448Crossref PubMed Scopus (191) Google Scholar) observed no apparent interaction of TGF-β1 with DS by agarose electrophoresis. However, a weak inherent DS binding activity of heparin/HS-binding proteins is not unusual, presumably because DS also contains both iduronate and variable sulfation which may partially satisfy the binding requirements.Table IComparative sulfate contents of the various heparin, heparan sulphate and modified heparin speciesSulfates per 100 disaccharidesRat liver HS1-aData derived from Lyon et al. (24).Porcine mucosal HSBovine lung heparin1-bThe various modified heparins were obtained from different sources and therefore probably derive from different batches of bovine lung heparin, which may differ slightly in composition.Modified bovine lung heparins1-bThe various modified heparins were obtained from different sources and therefore probably derive from different batches of bovine lung heparin, which may differ slightly in composition.De-N-sulfated, re-N-acetylatedDe-6-O-sulfatedDe-2-O-sulfatedN-Sulfates60.743.597.72.498.291.46-O-Sulfates34.019.492.485.34.280.22-O-Sulfates38.418.289.390.554.72.2Total sulfates1-cAdditional minor contributions from 3-O-sulfate groups are not included.133.181.1279.4178.2157.1173.8Compositions were determined as described under “Experimental Procedures.”1-a Data derived from Lyon et al. (24Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar).1-b The various modified heparins were obtained from different sources and therefore probably derive from different batches of bovine lung heparin, which may differ slightly in composition.1-c Additional minor contributions from 3-O-sulfate groups are not included. Open table in a new tab Compositions were determined as described under “Experimental Procedures.” To try and elucidate the relative importance of the different sulfate groups in heparin for the binding and subsequent potentiation of TGF-β1 activity, a series of selectively desulfated heparins (see Table I for structural comparisons) were tested for their effect in the soft agar colony growth assay. The results, although complex, suggested that the specific loss of N-sulfates had a greater effect than the selective loss of either 2-O- or 6-O-sulfates. At a heparin concentration of 0.1 μg/ml (giving a 4-fold potentiation of TGF-β1 activity), de-N-sulfation, with replacement by N-acetyl groups, resulted in a 95 ± 3% reduction in activity. Removal of 2-O- or 6-O-sulfates resulted in lesser, although similar reductions of 48.5 ± 10.8% and 51.5 ± 4.5% respectively (although the 2-O-sulfates had been more selectively removed than the 6-O-sulfates; see Table I). In all cases a 10-fold increase in concentration of the modified heparins increased the level of potentiation. De-2-O- and de-6-O-sulfated heparins at 1 μg/ml gave 79.9 ± 1.3% and 88.7 ± 3%, respectively, of the potentiation observed with 0.1 μg/ml heparin, although the de-N-sulfated derivative still only achieved 56.5 ± 9% (all % values being the mean of triplicate determinations ± S.E.). This complex behavior suggests that the TGF-β1 binding site in heparin/liver HS probably involves a combination of specific structural determinants of which N-sulfation is a major contributor. The NRK 49F colony growth assay is normally performed in the presence of 5% (v/v) fetal calf serum (25De Larco J.E. Todaro G.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4001-4005Crossref PubMed Scopus (1199) Google Scholar,26Roberts A.B. Anzano M.A. Lamb L.C. Smith J.M. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5339-5343Crossref PubMed Scopus (720) Google Scholar). The requirement for serum can, however, be replaced by a defined protein mixture (comprising insulin, transferrin, high density lipoprotein, and serum albumin) (27Rizzino A. Ruff E. Rizzino H. Cancer Res. 1986; 46: 2816-2820PubMed Google Scholar) in which the cells remain responsive to stimulation by all three TGF-β isoforms (data not shown). Interestingly, under these serum-free conditions, the potentiating effect of exogenous heparin upon TGF-β1 is lost, and the behavior of TGF-β1 becomes comparable to that of TGF-β2 (Fig.4 A). This suggests that the potentiating effect is not due to a direct heparin-mediated enhancement of TGF-β1 binding to its receptor, but to a modulation by heparin of a TGF-β1 neutralizing activity present in serum. The most likely candidate molecule is α2M, which is known to be the major TGF-β-binding protein in serum (30O'Connor-McCourt M.D. Wakefield L.M. J. Biol. Chem. 1987; 262: 14090-14099Abstract Full Text PDF PubMed Google Scholar) and forms non-covalent complexes in which the TGF-β is rendered latent (30O'Connor-McCourt M.D. Wakefield L.M. J. Biol. Chem. 1987; 262: 14090-14099Abstract Full Text PDF PubMed Google Scholar, 31Huang S.S. O'Grady P. Huang J.S. J. Biol. Chem. 1988; 263: 1535-1541Abstract Full Text PDF PubMed Google Scholar). The addition of 750 μg/ml α2M to the serum-free cell system inhibited the activities of all the TGF-β isoforms by between 43% (TGF-β3) and 68% (TGF-β1 and -β2) (Fig. 4 B). Heparin alone at 1 μg/ml had no effect (Fig. 4 B). However, the combination of heparin and α2M elicited differential responses. Whereas the activity of TGF-β3 remained completely suppressed, that of TGF-β1 was markedly restored by heparin to 84% of control levels, although the activity of TGF-" @default.
• W2012180376 created "2016-06-24" @default.
• W2012180376 creator A5037822891 @default.
• W2012180376 creator A5048560058 @default.
• W2012180376 creator A5078514469 @default.
• W2012180376 date "1997-07-01" @default.
• W2012180376 modified "2023-10-01" @default.
• W2012180376 title "The Interaction of the Transforming Growth Factor-βs with Heparin/Heparan Sulfate Is Isoform-specific" @default.
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