Limited Mutations in Full-length Tetrameric Human α2-Macroglobulin Abrogate Binding of Platelet-derived Growth Factor-BB and Transforming Growth Factor-β1

α2-Macroglobulin (α2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1), and carries β-amyloid peptide. α2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant α2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the α2M subunit. However, because intact α2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the α2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length α2M. These mutations did not perturb the tetrameric structure of α2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact α2M. E730R did not alter TGF-β1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-β1 binding associated with α2M conformational change. These studies demonstrate that growth factor binding to intact α2M is specific, involving a defined region of the α2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences. α2-Macroglobulin (α2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1), and carries β-amyloid peptide. α2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant α2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the α2M subunit. However, because intact α2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the α2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length α2M. These mutations did not perturb the tetrameric structure of α2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact α2M. E730R did not alter TGF-β1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-β1 binding associated with α2M conformational change. These studies demonstrate that growth factor binding to intact α2M is specific, involving a defined region of the α2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences. α2-Macroglobulin (α2M) 2The abbreviations used are: α2M, α2-macroglobulin; rα2M, recombinant human α2M; rα2M(RR), rα2M in which Glu714 and Asp719 are converted into Arg; rα2M(RRR), rα2M in which Glu714, Asp719, and Glu730 are converted into Arg; PDGF-BB, platelet-derived growth factor-BB; TGF-β1, transforming growth factor-β1; LRP-1, low density lipoprotein-related protein-1; MA, methylamine; GST, glutathione S-transferase; BSA, bovine serum albumin; BS3, bis(sulfosuccinimidyl) suberate; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase. is a 718-kDa homotetrameric glycoprotein that functions as a protease inhibitor and as a carrier of growth factors in the blood and in other extracellular spaces (1LaMarre J. Wollenberg G.K. Gonias S.L. Hayes M.A. Lab. Investig. 1991; 65: 3-14PubMed Google Scholar). Proteases react with α2M by cleaving any of a number of target peptide bonds near the center of the α2M subunit, in the “bait region” (2Barrett A.J. Starkey P.M. Biochem. J. 1973; 133: 709-724Crossref PubMed Scopus (886) Google Scholar). Cleavage in the bait region induces a major conformational change in α2M that entraps the reacting protease within the hollow core of the cylindrical α2M structure (2Barrett A.J. Starkey P.M. Biochem. J. 1973; 133: 709-724Crossref PubMed Scopus (886) Google Scholar, 3Barrett A.J. Brown M.A. Sayers C.A. Biochem. J. 1979; 181: 401-418Crossref PubMed Scopus (413) Google Scholar, 4Gonias S.L. Exp. Hematol. 1992; 20: 302-311PubMed Google Scholar). α2M conformational change reveals the recognition site for the α2M receptor/low density lipoprotein receptor-related protein (LRP-1), which is cryptic in the native α2M conformation (5Moestrup S.K. Holtet T.L. Etzerodt M. Thogersen H.C. Nykjaer A. Andreasen P.A. Rasmussen H.H. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1993; 268: 13691-13696Abstract Full Text PDF PubMed Google Scholar, 6Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar). For this reason, conformationally transformed α2M is referred to as “activated.” α2M thiol ester bonds, which are formed by the side chains of Cys949 and Glu952, also become exposed during α2M conformational change and may react with lysine residues of the attacking protease to form covalent linkages; however, these linkages are not necessary to stabilize the α2M-protease complex because the trapping mechanism is essentially irreversible under physiological conditions (7Feinman R.D. Wang D. Windwer S.R. Wu K. Ann. N. Y. Acad. Sci. 1983; 421: 178-187Crossref PubMed Scopus (6) Google Scholar, 8Salvesen G.S. Sayers C.A. Barrett A.J. Biochem. J. 1981; 195: 453-461Crossref PubMed Scopus (136) Google Scholar). α2M conformational change may be induced in the absence of proteases by aminolysis of the thiol ester bonds with small primary amines such as methylamine (MA) (9Bjork I. Fish W.W. Biochem. J. 1982; 207: 347-356Crossref PubMed Scopus (91) Google Scholar, 10Gonias S.L. Reynolds J.A. Pizzo S.V. Biochim. Biophys. Acta. 1982; 705: 306-314Crossref PubMed Scopus (148) Google Scholar). One growth factor reported to interact with α2M is platelet-derived growth factor (PDGF), a potent stimulator of mesenchymal cell mitogenesis and chemotaxis and a regulator of inflammation and wound healing in vivo (11Meyer-Ingold W. Eichner W. Cell Biol. Int. 1995; 19: 389-398Crossref PubMed Scopus (52) Google Scholar). Biologically active PDGF is formed by homodimerization of A-chains or B-chains or by heterodimerization of the A- and B-chains to form PDGF-AB (11Meyer-Ingold W. Eichner W. Cell Biol. Int. 1995; 19: 389-398Crossref PubMed Scopus (52) Google Scholar). Recently, PDGF C-chains and D-chains also have been described (12Fredriksson L. Li H. Eriksson U. Cytokine Growth Factor Rev. 2004; 15: 197-204Crossref PubMed Scopus (600) Google Scholar). The PDGFs mediate biological activities by binding to the PDGF α-receptor or β-receptor; the specificity of this interaction is dictated by the subunit composition of the PDGF (12Fredriksson L. Li H. Eriksson U. Cytokine Growth Factor Rev. 2004; 15: 197-204Crossref PubMed Scopus (600) Google Scholar). The interaction of PDGF with α2M was first discovered by analysis of PDGF carrier proteins in the plasma (13Huang J.S. Huang S.S. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 342-346Crossref PubMed Scopus (142) Google Scholar, 14Raines E.W. Bowen-Pope D.F. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3424-3428Crossref PubMed Scopus (70) Google Scholar). Subsequent studies demonstrated binding of and PDGF-AB to α2M, not J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The interaction of with α2M binding to A. M. P.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). binding to α2M was also discovered by analysis of carrier proteins in the plasma J. Biol. Chem. Full Text PDF PubMed Google Scholar). in complex with α2M biological the complex was referred to as a for the complex in which the and binds protein J. J. J. PubMed Google Scholar). growth factors in the inflammation and the to J. Rev. Biochem. PubMed Scopus Google Scholar). These factors are also in J. Rev. Biochem. PubMed Scopus Google Scholar). The which is may be by proteases, or in active growth factor J.S. J. Cell Sci. PubMed Scopus Google Scholar). active with native α2M, which is not the is and may be at of or inflammation J. Biol. Chem. Full Text PDF PubMed Google Scholar, Gonias S.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of TGF-β1 and is by α2M conformational growth factors with to α2M that is by methylamine or by proteases Gonias S.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S.L. J. Hayes M.A. Ann. N. Y. Acad. Sci. PubMed Scopus Google Scholar). native α2M and forms of α2M are TGF-β1 binding the of α2M conformation in growth factor binding S.L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Gonias S.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Gonias S.L. Sci. PubMed Scopus Google Scholar). The of growth factors also that sequences in the structure of α2M may be for the To growth factor-binding in the structure of α2M, we a of overlapping glutathione proteins and defined a binding site for TGF-β1 between and in the α2M subunit J. Gonias S.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Gonias S.L. Sci. PubMed Scopus Google Scholar). proteins the sequence also and growth that these binding may be or overlapping S.L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we synthetic and a putative binding site for and to between and A. Gonias S.L. Sci. PubMed Scopus Google Scholar, S. Gonias S.L. PubMed Scopus Google Scholar). The growth factor binding for of and the blocked binding of TGF-β1 to that the and to or overlapping on the growth factor surface S. Gonias S.L. PubMed Scopus Google Scholar). studies with full-length recombinant α2M have to of the structure and of this Cys949 is to of thiol α2M is expressed in conformationally transformed PubMed Scopus Google Scholar). of also thiol ester and reaction with K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the α2M bait region α2M of protease entrapment and induces in subunit J. Biol. 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The forms of rα2M and have demonstrated that binding is eliminated in TGF-β1 binding is not blocked by any of these however, the increase in TGF-β1 binding that of α2M to the conformation is eliminated in These studies have demonstrated that growth factor binding to α2M a interaction involving defined within the α2M of different growth factors may overlapping and and bovine serum was The was was and was The of was by active site with PubMed Scopus Google Scholar). was with to the was by into for TGF-β1 and to the of and A. Biochem. Biophys. PubMed Scopus Google Scholar). and to and to and at for The of was The of was was human plasma to the of and Pizzo Pizzo S.V. J. Biol. Chem. 1981; Full Text PDF PubMed Google Scholar). The of α2M was by and the by the at an of Biochem. J. PubMed Scopus Google Scholar). To form α2M, native α2M was in at by at of native α2M with was by the increase in in (9Bjork I. Fish W.W. Biochem. 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Glu730 for was with the native and forms of α2M and rα2M The to of was by native α2M low of and of conformational change as the binding of α2M for Gonias S.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with binding was reaction with we binding of to α2M and by and mutation of a single was to binding to rα2M The in that the absence of binding was not to of the mutation of Glu714 and Asp719, in rα2M(RR), to binding of to rα2M(RRR), which mutation of was not with the with a to binding of to we plasma α2M and on to was for with the as a with that not with α2M. was by of with plasma α2M, and the of to the of α2M was with proteins the with and and to of with plasma α2M was by with was of the with plasma α2M and was by of the a to that mutation of Glu730 binding to α2M, we the of Gonias S.L. J. Biol. Chem. Full Text PDF PubMed Google in which to is used to stabilize complex to The reaction under and the of complex is to the of complex the binding of to plasma α2M and the absence of BS3, a low of plasma α2M complex was covalent to Gonias S.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). to the of associated with plasma α2M binding to was not with or of α2M on binds to the PDGF and induces of the which is the primary by analysis with L. S. Gonias S.L. J. Cell Biochem. 2004; PubMed Scopus Google Scholar). previously demonstrated that plasma α2M binds and binding of the growth factor to a PDGF protein S.L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we the of plasma α2M and rα2M (E730R) to PDGF in to with for or in the of plasma α2M or Cell to analysis to as a for the absence of α2M, induced of a major with an of with the of PDGF plasma α2M and to alter the of of was with plasma α2M, PDGF was of was not because are and J.A. J. Biol. 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PubMed Scopus Google Scholar). we the binding of TGF-β1 to rα2M(RRR), which mutation of Glu730 with Glu714 and in in native with native plasma α2M; however, to demonstrate the increase in TGF-β1 binding that reaction with The studies in and demonstrated that induces conformational change we the studies as that mutation of the the of the binding site for TGF-β1 in α2M that conformational change. To the binding of TGF-β1 to in a model we plasma α2M in was to the in the and absence of native or plasma α2M or is in native plasma α2M binding of to the α2M by was by plasma α2M. The native form of binding by however, to increase the of to TGF-β1 binding to the α2M the in The α2M subunit the bait thiol ester L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. S. PubMed Scopus Google Scholar, J. Biol. Chem. 1979; Full Text PDF PubMed Google which the binding of growth factors S.L. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Gonias S.L. J. Biol. Chem. 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Full Text Full Text PDF PubMed Scopus Google Scholar, A. Gonias S.L. Sci. PubMed Scopus Google Scholar, S. Gonias S.L. PubMed Scopus Google we that a surface as a binding for with that specificity in the of studies in α2M, the surface may be within the of α2M. For α2M binds two of the of rα2M and growth factor binding is J. Hayes M.A. Gonias S.L. Biochem. J. 1992; PubMed Scopus Google Scholar). Based on the structure of we a residues within the of Glu730 completely blocked the on as to to that the mutation did not to become was by that The site in full-length tetrameric α2M is complex studies with proteins and synthetic to of synthetic peptides, the peptide that binds TGF-β1 Glu714 and of these residues in the protein TGF-β1 binding S. Gonias S.L. PubMed Scopus Google however, mutation of Glu714 and Asp719 in intact to alter TGF-β1 Glu714 and Asp719 the binding of TGF-β1 to was the mutation to the binding of TGF-β1 to native α2M. These that may a in the binding of TGF-β1 to the conformation of not an for the low binding of TGF-β1 to native α2M of The mechanism by which native α2M binds TGF-β1 an that the of TGF-β1 in the plasma is associated with native α2M and J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of α2M in the plasma is (2Barrett A.J. Starkey P.M. Biochem. J. 1973; 133: 709-724Crossref PubMed Scopus (886) Google the for TGF-β1 The of this is that binding of growth such as PDGF-BB, to α2M a interaction as to nonspecific studies in which we have α2M to a factor an to the mechanism of of α2M in cell and in For by binding to and by the interaction of with the interaction α2M may M. M. J. J.A. W. J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). α2M cell by binding growth factors such as TGF-β1 and by signaling involving these growth factors Gonias S.L. J. PubMed Scopus Google Scholar). may an for between these two are with which to S. Gonias S.L. Biochem. Biophys. PubMed Scopus Google Scholar). forms of full-length α2M may also be to for in the α2M L. L. L. K. H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google and for forms of α2M demonstrate potent to J. L. Gonias S.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar).