The Conformation-dependent Interaction of α2-Macroglobulin with Vascular Endothelial Growth Factor (2000)

α2-Macroglobulin (α2M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2–4 mg/ml). α2M exists in two conformations, a native form and an activated, receptor-recognized form. While α2M binds to numerous cytokines and growth factors, in most cases, the nature of the α2M interaction with these factors is poorly understood. We examined in detail the interaction between α2M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to α2M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of α2M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated α2M and to the exterior of native and proteinase-converted α2M; and 3) VEGF (molecular mass ∼40 kDa) can access the interior of receptor-recognized α2M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of α2M is internalized and degraded by macrophages via the α2M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized α2M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of α2M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca2+ levels. We demonstrate that under physiological conditions, α2M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca2+. α2-Macroglobulin (α2M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2–4 mg/ml). α2M exists in two conformations, a native form and an activated, receptor-recognized form. While α2M binds to numerous cytokines and growth factors, in most cases, the nature of the α2M interaction with these factors is poorly understood. We examined in detail the interaction between α2M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to α2M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of α2M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated α2M and to the exterior of native and proteinase-converted α2M; and 3) VEGF (molecular mass ∼40 kDa) can access the interior of receptor-recognized α2M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of α2M is internalized and degraded by macrophages via the α2M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized α2M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of α2M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca2+ levels. We demonstrate that under physiological conditions, α2M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca2+. native α2-macroglobulin α2-macroglobulin that has been converted to its receptor-recognized form with methylamine porcine pancreatic elastase α2-macroglobulin that has been converted to its receptor-recognized form with PPE complex generated by covalently incorporating VEGF during conversion of α2M to α2M-PPE general term for receptor-recognized forms of α2-macroglobulin vascular endothelial growth factor human umbilical vein endothelial cells intracellular calcium 5,5′-dithiobis(2-nitrobenzoate) interleukin-6 transforming growth factor platelet-derived growth factor The human plasma protein α2-macroglobulin (α2M)1 is a proteinase inhibitor that is unique in its ability to inhibit proteinases of all four major classes (1Barrett A.J. Starkey P.M. Biochem. J. 1973; 133: 709-724Crossref PubMed Scopus (884) Google Scholar). This inhibition process triggers a conformational change in the α2M molecule, converting it to a receptor-recognized so-called “fast” form (α2M*). The term “fast” refers to a marked difference in migration relative to the native molecule (“slow” form) by gel electrophoresis under nondenaturing conditions (2Barrett A.J. Brown M.A. Sayers C.A. Biochem. J. 1979; 181: 401-418Crossref PubMed Scopus (413) Google Scholar). This change in electrophoretic mobility is the result of the more compact structure of the fast form in comparison with the native molecule, which exhibits a unique, “open cage” structure that closes upon interaction with a proteinase. This conformational change can also be induced by ammonia (α2M-NH3) or other small amine nucleophiles, such as methylamine (α2M-MA) (3Gron H. Thogersen I.B. Enghild J.J. Pizzo S.V. Biochem. J. 1996; 318: 539-545Crossref PubMed Scopus (14) Google Scholar, 4Gonias S.L. Reynolds J.A. Pizzo S.V. Biochim. Biophys. Acta. 1982; 705: 306-314Crossref PubMed Scopus (148) Google Scholar). Recent studies have demonstrated that α2M has other important biological activities, including enhancement of antigen presentation (5Osada T. Noro N. Kuroda Y. Ikai A. Biochem. Biophys. Res. Commun. 1987; 146: 26-31Crossref PubMed Scopus (13) Google Scholar, 6Chu C.T. Pizzo S.V. J. Immunol. 1993; 150: 48-58PubMed Google Scholar), promotion of tumoricidal capabilities by macrophages (7Ades E.W. Hinson A. Chapuis-Cellier C. Arnaud P. Scand. J. Immunol. 1982; 15: 109-113Crossref PubMed Scopus (62) Google Scholar), and the binding and possible regulation of cytokine and growth factor activity (8Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar). Recent work has demonstrated various methods of nonproteolytic ligand interaction with α2M (9Gron H. Pizzo S.V. 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We demonstrate that the binding of VEGF to α2M is and that this binding is by of both α2M and interaction with α2M occurs VEGF is present during proteinase conversion of native α2M to its receptor-recognized form complex binds VEGF in a to the binding of native α2M and to The binding of VEGF for is for the native molecule. studies that VEGF binds to the exterior of native α2M and whereas it binds the VEGF bound to both and α2M-PPE is internalized and degraded by macrophages via the binding of VEGF to these receptor-recognized the physiological by the α2M receptor, the low density lipoprotein receptor-related is binding studies demonstrate that VEGF binds to α2M and at distinct from other growth factor binding the novel nature of the interaction between α2M and VEGF and from and from and from and cells and porcine pancreatic elastase from and from and from The electrophoresis from α2M as Pizzo S.V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The concentration of by with 5,5′-dithiobis(2-nitrobenzoate) G. Enghild J.J. 1993; PubMed Scopus Google Scholar). The protein concentration on mass The 5,5′-dithiobis(2-nitrobenzoate) that more of the in the α2M by native α2M with for at in the by α2M-PPE by α2M with a of PPE for at PPE by the of 1 to binding also with 1 α2M in during conversion to α2M-PPE in to a the of VEGF and PPE from of by gel on a VEGF the of and A.E. Biochem. J. 1973; 133: PubMed Scopus Google Scholar). of α2M-MA, and α2M-PPE with for to at and on nondenaturing which and on a to binding. The of present in to the of α2M-MA, and α2M-PPE The for ligand by to the binding under conditions to binding. macrophages from as C.T. G. Pizzo S.V. Biochem. J. 1993; PubMed Scopus Google Scholar). in at 2 and at at the of cells with and VEGF or in complex with 1 α2M or to well in a of and to at for of the to ligand as The macrophages with for on to the cell The binding from well in a the binding the cells with and in the from macrophages during the to and by at for at for by in the 1 of α2M-MA, or α2M-PPE by in of for at in and at to binding studies with The of α2M-MA, and α2M-PPE in the α2M-MA, and to the in the of an to of VEGF, TGF-β1, or and at for with to and the in for at in a VEGF with 1 of α2M-MA, or α2M-PPE in of for at 1 of in of and 2 at or cells and the for 1 at The at and both the and in a α2M and as C.M. Pizzo S.V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In α2M with for at in The concentration at with an of at A. P. A.J. J. Sci. Full Text PDF PubMed Scopus Google Scholar). the of the to the to binding studies with VEGF as studies with by 1 VEGF in the or absence of of α2M and for in with and in a at for with the by and cells cells a and on a also VEGF with α2M and for at to with on at and in with and for in a at from the and and to with the cells for in the a as C.T. G. Pizzo S.V. Biochem. J. 1993; PubMed Scopus Google Scholar). VEGF in the or absence of 1 of α2M-MA, and α2M-PPE a also In some VEGF with α2M-MA, or α2M-PPE for at to results to the not with various of VEGF from 1 to in with α2M-MA, and all In a of at VEGF with α2M during conversion by PPE in to a covalently under conditions and of demonstrated a to the mass of α2M kDa) and VEGF of VEGF with native α2M-MA, or α2M-PPE interaction of VEGF with α2M-MA, and α2M-PPE at and for to binding to α2M-MA, of the VEGF between and In the of and α2M-MA, and α2M-PPE with VEGF and to gel electrophoresis under native and of in the native at that to the of α2M demonstrated that more VEGF bound to to native α2M or α2M-PPE 2 as of the VEGF binds to α2M-MA, whereas with α2M or The for native α2M that of α2M-PPE and that of The for VEGF bound to native α2M and α2M-PPE be as to relative to VEGF bound to an electrophoresis between 1 and of electrophoresis significant impact on binding or of with α2M-MA, or α2M-PPE during gel very binding. that VEGF binds to all forms of α2M in a and in to VEGF is by macrophages of its interaction with studies demonstrate that VEGF bound to is and degraded by macrophages VEGF and VEGF bound to native α2M are not in this with α2M-PPE that a and binding of VEGF to α2M-PPE with α2M-MA, by a of VEGF is by macrophages of VEGF by α 2 and α 2 VEGF bound to or and by macrophages The VEGF bound to native α2M and VEGF it binding. VEGF bound to or in a VEGF bound to or and by macrophages The VEGF bound to native α2M and VEGF it binding. VEGF bound to or α2M-MA, and α2M-PPE and with a of VEGF in the of a of VEGF, TGF-β1, and in to VEGF binding to distinct or characterized growth factor binding on In the of a of VEGF, of the binding The of or impact on the that and bind to α2M the D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), results that VEGF binds to a distinct from this studies α2M-PPE of α2M or results not studies to VEGF binds to exterior or interior of the α2M molecule. The of VEGF that binds to or to an of VEGF and the of VEGF the VEGF bound to native α2M or is by This that VEGF binds to an exterior of these it is to with results in a of bound VEGF that is by that VEGF binds to the interior of α2M-MA, at a site that is distinct from the binding site on the native of VEGF from by VEGF by with in the of α2M-MA, or VEGF to is as the of growth factor bound to α2M-MA, or α2M-PPE that is for by in a VEGF with in the of α2M-MA, or VEGF to is as the of growth factor bound to α2M-MA, or α2M-PPE that is for by α2M and as under of VEGF with of these forms of α2M at for and to nondenaturing In of these cases, the binding of VEGF to α2M by at with forms of α2M VEGF on in the and absence of α2M-MA, or α2M-PPE for at The for at to in the proliferation or to the cells at the of the proliferation on for the interaction of VEGF with various forms of α2M and the that these are that endothelial cell proliferation not be α2M-MA, α2M-PPE effect on VEGF-induced of α2M high as for native and for receptor-recognized with effect on proliferation and to for a of to ligand The to the ligand The of VEGF a in the up-regulation of as by The of α2M-MA, or α2M-PPE effect on these the been with VEGF for at This that term of VEGF are not by its interaction with native α2M-MA, or α2M-MA, α2M-PPE effect on which that the high α2M C.T. G. Pizzo S.V. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) is not present on endothelial cells. VEGF that been covalently α2M during conversion is in its ability to up-regulate of the more recently demonstrated of α2M is its ability to with cytokines and growth factors in some physiological In cases, such as and both the interaction of these with α2M and the ability of α2M to have been D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 14Danielpour D. Sporn M.B. J. Biol. Chem. 1990; 265: 6973-6977Abstract Full Text PDF PubMed Google Scholar, 16Raines E.W. Bowen-Pope D.F. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3424-3428Crossref PubMed Scopus (70) Google Scholar, 17Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, A. M. J. 1990; PubMed Scopus Google Scholar, S. J. U. U. M. B. Lab. Invest. 1992; Google Scholar). to VEGF, an demonstrated that VEGF to human plasma with α2M (22Soker S. Svahn C.M. Neufeld G. J. Biol. Chem. 1993; 268: 7685-7691Abstract Full Text PDF PubMed Google Scholar). VEGF is important in conditions including rheumatoid in most and its possible role in various forms of its interaction with the highly plasma which has also been implicated in some of these same disease processes (34Cawston T.E. McLaughlin P. Hazleman B.L. Br. J. Rheumatol. 1987; 26: 354-358Crossref PubMed Scopus (16) Google Scholar, 35Pejovic M. Stankovic A. Mitrovic D.R. Br. J. Rheumatol. 1995; 34: 520-524Crossref PubMed Scopus (25) Google Scholar). We upon the the interaction between VEGF and α2M by human native α2M and studying two receptor-recognized forms of and We demonstrate that a of VEGF binds to the receptor-recognized form of α2M (α2M-MA) and with a it does to the native molecule or the proteinase-converted form. The binding to both receptor-recognized forms of α2M results in and via the α2M on This is significant in that it a mechanism by which the biological activity of VEGF bound to receptor-recognized forms of α2M be in In all cases, the interaction is which is with the mechanism of interaction of α2M with other growth factors and cytokines (8Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar, P.A. Saksela O. Harpel P. Rifkin D.B. J. Biol. Chem. 1989; 264: 7210-7216Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, D.J. K.P. LaMarre J. Gonias S.L. Biochem. 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PubMed Scopus Google Scholar, Biochemistry. 1984; PubMed Scopus Google Scholar). the of a proteinase the α2M molecule the of VEGF bound such that the is to that for native results are with PPE a significant of the interior Gonias S.L. Pizzo S.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) of to access to is not in such a and VEGF is to bind to the interior of the molecule. with other growth factors known to bind to such as and that VEGF binds at a distinct from the recently characterized growth factor binding site D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This that different classes of growth factors bind to distinct on α2M or work has demonstrated that of α2M and has on cytokine binding. necrosis factor and basic fibroblast growth factor demonstrate binding to α2M and α2M-MA, whereas the binding of growth TGF-β1, and are Pizzo S.V. J. Immunol. 1998; Google Scholar). We in this that VEGF binding is to this of cytokines in that the interaction with both α2M and is The is significant in that it that during are by and macrophages J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. Biochem. J. 1997; PubMed Scopus Google Scholar, N. Engl. J. Med. 1989; PubMed Scopus Google Scholar), receptor-recognized forms of α2M be in ability to bind and In to the interaction between VEGF and α2M the biological of VEGF, two types of in vitro to VEGF The of these a term examined the effect of α2M and on VEGF-induced The a term up-regulation of intracellular calcium by VEGF in the and absence of α2M and In both the interaction of VEGF with α2M and not have a significant effect on its biological This is α2M is present at high in human plasma as such that some interaction between α2M and VEGF be both are present in plasma or VEGF has been for conditions such as tumor or as a in results that the interaction of native α2M with VEGF not the therapeutic of it is possible that the of VEGF bound to receptor-recognized forms of α2M by low density lipoprotein receptor-related protein VEGF biological activity during forms of α2M are The with to the effect of native or receptor-recognized α2M on to be in of the of these different forms of α2M for VEGF bound to native α2M or receptor-recognized α2M or is in with growth and it is this VEGF that This is by the that covalently VEGF is to induce the nature of VEGF interaction with the various forms of α2M are both novel and This is the of a growth factor binding to different on and fast form is also that a molecule as as VEGF kDa) is to the by We have demonstrated that nonproteolytic can covalently in α2M upon at for (9Gron H. Pizzo S.V. Biochemistry. 1998; 37: 6009-6014Crossref PubMed Scopus (14) Google Scholar). that interaction occurs via at an whereas the interaction is We that VEGF is to the molecule the cage” and bind to the interior of the molecule. studies in this have all been with proteinases and under such as inhibitor (molecular mass of kDa) are to the interior of We demonstrate that is from the of a other can the α2M and with We J. and K. for and and for with of the