The Binding of Receptor-recognized α2-Macroglobulin to the Low Density Lipoprotein Receptor-related Protein and the α2M Signaling Receptor Is Decoupled by Oxidation
1997; Elsevier BV; Volume: 272; Issue: 33 Linguagem: Inglês
10.1074/jbc.272.33.20627
ISSN1083-351X
AutoresSean M. Wu, Cinda M. Boyer, Salvatore V. Pizzo,
Tópico(s)Hemoglobin structure and function
ResumoReceptor-recognized forms of α2-macroglobulin (α2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by α2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl− system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of α2M* using 200 μm hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (K d − 71 ± 12 pm). Surprisingly, oxidation of native α2-macroglobulin (α2M) with 125 μm hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (K d − 0.7 ± 0.15 nm). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of α2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding K d, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native α2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (K d − 71 pm) are the α2M* signaling receptor. Receptor-recognized forms of α2-macroglobulin (α2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by α2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl− system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of α2M* using 200 μm hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (K d − 71 ± 12 pm). Surprisingly, oxidation of native α2-macroglobulin (α2M) with 125 μm hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (K d − 0.7 ± 0.15 nm). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of α2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding K d, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native α2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (K d − 71 pm) are the α2M* signaling receptor. α2-Macroglobulin (α2M) 1The abbreviations used are: α2M, α2-macroglobulin; α2M*, the receptor-recognized form of α2M; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; RBF, carboxyl-terminal receptor binding fragment of α2M;125I-BH-α2M,125I-Bolton-Hunter-labeled α2M;125I-BH-α2M*,125I-Bolton-Hunter-labeled α2M*;cis-DDP, cis-dichlorodiamine-platinum(II); HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; Fura-2/AM, 1-[2-(5-carboxyoxazol-1-yl)-6-aminobenzofuran]-5-oxyl-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxylmethyl ester; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. 1The abbreviations used are: α2M, α2-macroglobulin; α2M*, the receptor-recognized form of α2M; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; RBF, carboxyl-terminal receptor binding fragment of α2M;125I-BH-α2M,125I-Bolton-Hunter-labeled α2M;125I-BH-α2M*,125I-Bolton-Hunter-labeled α2M*;cis-DDP, cis-dichlorodiamine-platinum(II); HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; Fura-2/AM, 1-[2-(5-carboxyoxazol-1-yl)-6-aminobenzofuran]-5-oxyl-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxylmethyl ester; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. is a highly conserved, homotetrameric, 720-kDa glycoprotein found in high concentration in the plasma (2–4 mg/ml). It has the unique ability to inhibit all mechanistic classes of proteinases by "entrapping" the proteinase and thereby sterically blocking the access of high molecular weight substrates (reviewed in Refs. 1Chu C.T. Pizzo S.V. Lab. Invest. 1994; 71: 792-812PubMed Google Scholar and 2Sottrup-Jensen L. Putnam F.W. The Plasma Proteins. 5. Academic Press, Orlando, FL1987: 191-291Crossref Google Scholar). Proteinases first cleave the "bait region" of native α2M exposing the internal γ-glutamyl-β-cysteinyl thioester bond. Reaction of the thioester bond with a free amino lysyl residue on the surface of the proteinase results in bond rupture and a major conformational change in native α2M. The resulting molecule is much more compact as evidenced by faster migration on a native acrylamide gel (3Barrett A.J. Brown M.A. Sayers C.A. Biochem. J. 1979; 181: 401-418Crossref PubMed Scopus (413) Google Scholar), electron microscopy (4Gonias S.L. Figler N.L. J. Biol. Chem. 1989; 264: 9565-9570Abstract Full Text PDF PubMed Google Scholar, 5Hussaini I.M. Figler N.L. Gonias S.L. Biochem. J. 1990; 270: 291-295Crossref PubMed Scopus (17) Google Scholar), sedimentation behavior (6Delain E. Pochon F. Barray M. Van Leuven F. Electron Microsc. Rev. 1992; 5: 231-281Crossref PubMed Scopus (48) Google Scholar), and circular dichroism (7Martini J.-L. Pochon F. Biochimie ( Paris ). 1989; 71: 325-332Crossref PubMed Scopus (5) Google Scholar). Consequently the receptor-recognition site is exposed. Small amine nucleophiles, such as methylamine, can initiate this reaction by directly attacking the thioester bond generating the conformational change and the exposure of the receptor-recognition site without bait region cleavage. Receptor-recognized α2M (α2M*) can rapidly eliminate the "entrapped" proteinase from the circulation by binding to a cell surface clearance receptor, the low density lipoprotein receptor-related protein (LRP) (8Strickland 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, 9Kristensen T. Moestrup S.K. Gliemann J. Lone B. Sand O. Sottrup Jensen L. FEBS Lett. 1990; 276: 151-155Crossref PubMed Scopus (255) Google Scholar).LRP is a multiligand receptor that binds to a wide variety of unrelated ligands (reviewed in Ref. 10Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1057) Google Scholar). Binding of all ligands to LRP can be effectively competed by receptor-associated protein (RAP), which co-purifies with LRP. Prior investigation of α2M* binding to LRP has shown that the binding mechanism involves a cluster of positively charged residues on α2M* interacting with the second complement-like repeat on LRP, which contains clusters of negatively charged residues (11Willnow T.E. Goldstein J.L. Orth K. Brown M.S. Herz J. J. Biol. Chem. 1992; 267: 26172-26180Abstract Full Text PDF PubMed Google Scholar). Analysis of the receptor-binding site on α2M* using monoclonal antibody (12Isaacs I.J. Steiner J.P. Roche P.A. Pizzo S.V. Strickland D.K. J. Biol. Chem. 1988; 263: 6709-6714Abstract Full Text PDF PubMed Google Scholar, 13Birkenmeier G. Stigbrand T. J. Immunol. Methods. 1993; 162: 59-67Crossref PubMed Scopus (36) Google Scholar) and recombinantly expressed protein (14Salvesen G. Quan L.T. Enghild J.J. Snipas S. Fey G.H. Pizzo S.V. FEBS Lett. 1992; 313: 198-202Crossref PubMed Scopus (19) Google Scholar, 15Holtet T.L. Nielsen K.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thøgersen H.C. FEBS Lett. 1994; 344: 242-246Crossref PubMed Scopus (17) Google Scholar) demonstrates that the carboxyl terminus of α2M* is involved in receptor binding.Although LRP is the only α2M* receptor identified to date, some important cellular regulatory functions ascribed to α2M* suggest that an alternate receptor must exist. α2M*, but not native α2M, suppresses the production of superoxide anion (16Hoffman M. Feldman S.R. Pizzo S.V. Biochim. Biophys. Acta. 1983; 760: 421-423Crossref PubMed Scopus (41) Google Scholar), enhances the release of prostaglandin E2 (17Hoffman M. Pizzo S.V. Weinberg J.B. Agents Actions. 1988; 25: 360-367Crossref PubMed Scopus (14) Google Scholar, 18Uhing R.J. Martenson C.H. Rubenstein D.S. Hollenbach P.W. Pizzo S.V. Biochim. Biophys. Acta. 1991; 1093: 115-120Crossref PubMed Scopus (13) Google Scholar) and platelet activating factor (19Misra U.K. Pizzo S.V. J. Cell. Biochem. 1996; 61: 39-47Crossref PubMed Scopus (9) Google Scholar), and stimulates the proliferation of vascular smooth muscle cells (20Webb D.J. Hussaini I.M. Weaver A.M. Atkins T.L. Chu C.T. Pizzo S.V. Owens G.K. Gonias S.L. Eur. J. Biochem. 1995; 318: 1-9Google Scholar). Moreover, our laboratory has characterized a novel signaling cascade and found that it does not appear to be LRP-mediated (21Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar, 22Misra U.K. Chu C.T.-C. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar, 23Misra U.K. Chu C.T.-C. Gawdi G. Pizzo S.V. J. Biol Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar). Furthermore, we have identified two classes of α2M* binding sites on peritoneal macrophages and human trabecular meshwork cells, both of which demonstrate activation of signaling cascades after exposure to α2M* (24Howard G.C. Roberts B.C. Epstein D.L. Pizzo S.V. Arch. Biochem. Biophys. 1996; 333: 19-26Crossref PubMed Scopus (14) Google Scholar). The lower affinity binding site is 10 times more abundant than the high affinity binding site and has clearly been identified as LRP (25Howard G.C. Misra U.K. DeCamp D.L. Pizzo S.V. J. Clin. Invest. 1996; 97: 1193-1203Crossref PubMed Scopus (39) Google Scholar, 26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The identity of the signaling receptor remains elusive; however, using site-directed mutagenesis, we have found that a lysine residue (1374, human numbering) within a 20-kDa fragment constituting the carboxyl terminus of α2M (RBF) is important for signaling (26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Very recently, however, some investigators reported that residue 1374 is involved in binding to LRP as well, raising the possibility that the α2M* signaling receptor may not be a separate receptor (27Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thøgersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Our previous attempts to study α2M* binding to LRP and the signaling receptor usingcis-dichlorodiamine-platinum(II) (cis-DDP) modification have shown that cis-DDP modifies a region upstream of the 20-kDa carboxyl-terminal of α2M* and that this modification results in decreased binding to LRP while having no effect on cell signaling (25Howard G.C. Misra U.K. DeCamp D.L. Pizzo S.V. J. Clin. Invest. 1996; 97: 1193-1203Crossref PubMed Scopus (39) Google Scholar, 28Enghild J.J. Thøgersen I.B. Roche P.A. Pizzo S.V. Biochemistry. 1989; 28: 1406-1412Crossref PubMed Scopus (59) Google Scholar, 29Roche P.A. Strickland D.K. Enghild J.J. Pizzo S.V. J. Biol. Chem. 1988; 263: 6715-6721Abstract Full Text PDF PubMed Google Scholar). This observation together with other immunochemical studies (12Isaacs I.J. Steiner J.P. Roche P.A. Pizzo S.V. Strickland D.K. J. Biol. Chem. 1988; 263: 6709-6714Abstract Full Text PDF PubMed Google Scholar, 13Birkenmeier G. Stigbrand T. J. Immunol. Methods. 1993; 162: 59-67Crossref PubMed Scopus (36) Google Scholar, 30Marynen P. Van Leuvan F. Cassiman J. Van den Berghe H. J. Immunol. 1981; 127: 1782-1786PubMed Google Scholar) suggest that a region outside of RBF may be involved in α2M* binding to LRP. Further characterization of the receptor-binding sites using RBF demonstrated that this fragment both binds to LRP and retains the ability to induce α2M* signaling cascade (23Misra U.K. Chu C.T.-C. Gawdi G. Pizzo S.V. J. Biol Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar, 26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Mutational studies have suggested that LRP and the α2M* signaling receptor are distinct entities. To date, however, no data have demonstrated a complete dissociation between α2M* binding to LRP and to the signaling receptor.Previous studies have shown that 25 μm sodium hypochlorite completely abolishes the anti-proteinase activity of native α2M (31Reddy V.Y. Pizzo S.V. Weiss S.J. J. Biol. Chem. 1989; 264: 13801-13809Abstract Full Text PDF PubMed Google Scholar, 32Reddy V.Y. Desrochers P.E. Pizzo S.V. Gonias S.L. Sahakian J.A. Levine R.L. Weiss S.J. J. Biol. Chem. 1994; 269: 4683-4691Abstract Full Text PDF PubMed Google Scholar). Its effects on α2M* receptor-recognition have not been examined. In this study we demonstrate that hypochlorite oxidation of α2M* completely destroys its ability to bind to LRP without affecting its ability to bind to the signaling receptor. This modification occurs predominantly outside of the carboxyl-terminal 20 kDa, consistent with our previous finding that the cis-DDP-sensitive site is upstream of RBF. Surprisingly, we also found that although hypochlorite oxidation of native α2M results in the selective exposure of the receptor-recognition site to LRP, the ligand cannot signal, thereby providing direct evidence for the dissociation of α2M* binding to LRP from binding to the signaling receptor.DISCUSSIONIn this study we demonstrate that hypochlorite oxidation of native α2M or α2M* can generate exposure of the receptor binding sites to either LRP or the α2M signaling receptor, respectively. Oxidation of α2M* by 200 μm hypochlorite completely abolished its binding to LRP without affecting its ability to bind to the high affinity sites or the signaling receptor. Oxidation of native α2M by 125 μm hypochlorite resulted in the exposure of the previously buried receptor-binding site to LRP without exposing the binding site to the signaling receptor. Oxidation of RBF showed no decrease in its ability to bind to cell surface receptors, supporting our earlier work showing that the oxidation-sensitive site in α2M* is outside of the carboxyl-terminal 20-kDa receptor-binding domain. Studies of the mechanism of oxidative exposure of the LRP-binding site in native α2M suggested that protein unfolding may be responsible for this phenomenon. These experiments provide strong proof for the existence of two distinct α2M* receptors and the presence of two independent receptor-binding regions on α2M*.Our earlier studies using cis-DDP modification of α2M* and RBF have shown that thecis-DDP-sensitive site in α2M* is outside of the 20-kDa carboxyl terminus and appears identical to the oxidation-sensitive site (28Enghild J.J. Thøgersen I.B. Roche P.A. Pizzo S.V. Biochemistry. 1989; 28: 1406-1412Crossref PubMed Scopus (59) Google Scholar, 29Roche P.A. Strickland D.K. Enghild J.J. Pizzo S.V. J. Biol. Chem. 1988; 263: 6715-6721Abstract Full Text PDF PubMed Google Scholar, 37Pizzo S.V. Roche P.A. Feldman S.A. Gonias S.L. Biochem. J. 1986; 238: 217-225Crossref PubMed Scopus (23) Google Scholar). Although cis-DDP and oxidation are capable of modifying similar residues, such modification caused only a 4–5-fold decrease in the binding affinity of α2M* for LRP. Subsequent studies demonstrate that a lysine 1370 mutant has decreased binding to LRP, and a lysine 1374 mutant is unable to activate the signaling cascade (26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This has been the best evidence for the existence of two distinct α2M* receptors; however, recent work by Nielsen et al. (27Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thøgersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) suggested that lysine 1374 mutants also have decreased binding to LRP.To investigate further the identity of the two classes of binding sites, we searched for ligands that could exclusively bind to either class of binding sites and tested their abilities to signal. Hypochlorite is a potent oxidant of native α2M (31Reddy V.Y. Pizzo S.V. Weiss S.J. J. Biol. Chem. 1989; 264: 13801-13809Abstract Full Text PDF PubMed Google Scholar, 32Reddy V.Y. Desrochers P.E. Pizzo S.V. Gonias S.L. Sahakian J.A. Levine R.L. Weiss S.J. J. Biol. Chem. 1994; 269: 4683-4691Abstract Full Text PDF PubMed Google Scholar). Treatment of native α2M with 25 μmhypochlorite resulted in complete destruction of its anti-proteinase activity. We hypothesized that hypochlorite could also inhibit the ability of α2M* to bind to cell surface receptors. In this study, we show that hypochlorite treatment completely eliminated the RAP-sensitive binding of α2M* to macrophages without affecting its ability to activate the signal transduction cascade or to bind to the high affinity cell surface receptors. This confirms and extends our previous observation that RAP competes for the binding of α2M* to the low affinity sites but is unable to inhibit the ability of α2M* to signal or to bind to the high affinity sites (24Howard G.C. Roberts B.C. Epstein D.L. Pizzo S.V. Arch. Biochem. Biophys. 1996; 333: 19-26Crossref PubMed Scopus (14) Google Scholar, 25Howard G.C. Misra U.K. DeCamp D.L. Pizzo S.V. J. Clin. Invest. 1996; 97: 1193-1203Crossref PubMed Scopus (39) Google Scholar, 26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Since hypochlorite oxidation is also able to cause the exposure of LRP binding sites in native α2M without inducing its ability to signal or to bind to the high affinity sites, our studies provide the best direct evidence to date that the high affinity sites represent the α2M* signaling receptors.The oxidative exposure of the α2M-binding site to LRP but not to the signaling receptor, is unique in a number of ways. All of the known naturally occurring α-macroglobulins or recombinantly expressed receptor binding fragments activate the signaling cascade (21Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar, 23Misra U.K. Chu C.T.-C. Gawdi G. Pizzo S.V. J. Biol Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar, 46Misra U.K. Gawdi G. Pizzo S.V. J. Cell. Biochem. 1996; 61: 61-71Crossref PubMed Google Scholar). RBF mutant 1374 is the first ligand that does not induce a signal, yet it still binds to the high affinity site, albeit with lower affinity. Our hypochlorite oxidized native α2M is the first ligand produced that is incapable of signaling and binding to the high affinity sites. The fact that it is still capable of binding to LRP suggests that the binding site on α2M* for the signaling receptor is distinct from the LRP binding site. That hypochlorite oxidation can selectively expose only the LRP binding sites in native α2M or the signaling receptor binding sites in α2M* demonstrates that the ability of α2M* to bind to its two receptors can be uncoupled. Efforts are currently being made using oxidized α2M* to isolate and purify the signaling receptor.Our investigation of the mechanism that may explain the oxidative exposure of LRP binding site in native α2M suggests that partial protein unfolding may be responsible. Earlier works by Davieset al. (47Davies K.J.A. Free Radical Biol. Med. 1986; 2: 155-173Crossref Scopus (247) Google Scholar, 48Davies K.J.A. J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 49Davies K.J.A. Delsignore M.D. Lin S.W. J. Biol. Chem. 1987; 262: 9902-9907Abstract Full Text PDF PubMed Google Scholar, 50Davies K.J.A. Delsignore M.E. J. Biol. Chem. 1987; 262: 9908-9913Abstract Full Text PDF PubMed Google Scholar) have shown that protein oxidation results in a partial unfolding of the protein secondary structure, which results in greater susceptibiliy to intracellular degradation by proteosomes. Similar finding has been resported by Ossanna et al. (51Ossanna P.J. Test S.T. Matheson N.R. Regiani S. Weiss S.J. J. Clin. Invest. 1986; 77: 1939-1951Crossref PubMed Scopus (140) Google Scholar) showing that extracellular proteins such as α1-antitrypsin may undergo oxidative inactivation resulting in partial protein unfolding and greater susceptibility to proteinase digestion.It is interesting that the exposure of the LRP binding site is dependent on the concentration of hypochlorite used to treat α2M and on the labeling method. With IODO-BEADS® labeling, the amount of hypochlorite needed to generate the exposure of LRP-binding sites begins with as little as 5 μm and peaks at 25 μm. This is in marked contrast with α2M that has been labeled with Bolton-Hunter reagent, which generates LRP binding sites with as little as 25 μmof hypochlorite but does not peak until 125 μm. At hypochlorite concentrations greater than 125 μm, oxidized α2M binding to LRP decreased with the concentration of the oxidant. The results obtained from the two labeling methods raise important questions regarding the effects of radiolabeling on receptor binding. Radiolabeling with IODO-BEADS® involves oxidation of tyrosine residues where as Bolton-Hunter labeling modifies amino terminus and lysine side chains. It is possible that the Bolton-Hunter reagent may protect lysine residues from hypochlorite oxidation, thereby generating a ligand that is more resistant to oxidation. Our results, however, show that Bolton-Hunter-labeled α2M has similar susceptibility to oxidation as unlabeled α2M, whereas IODO-BEADS®-labeled α2M is significantly more susceptible to oxidation. This suggests that receptor binding studies with α2M should use the Bolton-Hunter labeling method to minimize protein oxidation.The selective exposure of the LRP binding site in oxidized α2M suggests that the two receptor binding regions have distinct properties. We performed an ELISA using polyclonal antisera against RBF to determine if unfolding of the oxidized native α2M is associated with an increase in the exposure of RBF. We found that oxidation of α2M at greater than 12.5 μm hypochlorite results in full recognition of RBF by polyclonal antibodies. This exposure, however, is not associated with the ability of the ligand to signal. It is possible that recognition by the signaling receptor requires a more stringent three-dimensional conformation in the receptor binding domain of α2M* than recognition by LRP. This is supported by data showing that residues important for LRP binding appear to fall within a short consensus sequence having a predominance of positively charged residues, while the receptor binding region for the signaling receptor appears to require participation by residues from an exposed helix and from other regions of RBF (9Kristensen T. Moestrup S.K. Gliemann J. Lone B. Sand O. Sottrup Jensen L. FEBS Lett. 1990; 276: 151-155Crossref PubMed Scopus (255) Google Scholar, 26Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 27Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thøgersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). It is also possible that the binding site to the signaling receptor is exposed by oxidation but quickly destroyed; however, the fact that binding to the signal receptor is retained in oxidized α2M* even when the protein is treated with 200 μm hypochlorite suggests otherwise.Oxidative inactivation of α2M* receptor binding to LRP suggests an interesting pathophysiological process that may occur during inflammation. α2M is ubiquitous in serum and extracellular fluids (6Delain E. Pochon F. Barray M. Van Leuven F. Electron Microsc. Rev. 1992; 5: 231-281Crossref PubMed Scopus (48) Google Scholar, 52Petersen C.M. Dan. Med. Bull. 1993; 40: 409-446PubMed Google Scholar). During inflammation, neutrophils secrete hypochlorite and proteinases as a defense mechanism against invading foreign organisms (40Weiss S.J. Klein R.K. Slivka A. Wei M. J. Clin. Invest. 1982; 70: 598-607Crossref PubMed Scopus (672) Google Scholar, 51Ossanna P.J. Test S.T. Matheson N.R. Regiani S. Weiss S.J. J. Clin. Invest. 1986; 77: 1939-1951Crossref PubMed Scopus (140) Google Scholar, 53Thomas E.L. Jefferson M.M. Grisham M.B. Biochemistry. 1982; 21: 6299-6308Crossref PubMed Scopus (108) Google Scholar, 54Thomas E.L. Infect. Immun. 1979; 25: 117-120Crossref PubMed Google Scholar, 55Thomas E.L. Infect. Immun. 1979; 23: 522-531Crossref PubMed Google Scholar). In the presence of oxidants α2M that has reacted with proteinase will lose its ability to bind to its endocytic receptor (LRP) while retaining its ability to signal. This may have significant pathophysiological consequences given that α2M* signaling has been associated with increased production of prostaglandins and platelet-activating factor as well as increased mitogenesis in vascular smooth muscle cells (17Hoffman M. Pizzo S.V. Weinberg J.B. Agents Actions. 1988; 25: 360-367Crossref PubMed Scopus (14) Google Scholar, 18Uhing R.J. Martenson C.H. Rubenstein D.S. Hollenbach P.W. Pizzo S.V. Biochim. Biophys. Acta. 1991; 1093: 115-120Crossref PubMed Scopus (13) Google Scholar, 19Misra U.K. Pizzo S.V. J. Cell. Biochem. 1996; 61: 39-47Crossref PubMed Scopus (9) Google Scholar, 20Webb D.J. Hussaini I.M. Weaver A.M. Atkins T.L. Chu C.T. Pizzo S.V. Owens G.K. Gonias S.L. Eur. J. Biochem. 1995; 318: 1-9Google Scholar). α2M that has not reacted with proteinase will lose its anti-proteinase capacity and the ability to bind to the signaling receptor. The physiological significance of these mechanisms is highlighted by the finding that activated neutrophils can create an environment that contains 124 μm hypochlorite in 2 h (40Weiss S.J. Klein R.K. Slivka A. Wei M. J. Clin. Invest. 1982; 70: 598-607Crossref PubMed Scopus (672) Google Scholar, 51Ossanna P.J. Test S.T. Matheson N.R. Regiani S. Weiss S.J. J. Clin. Invest. 1986; 77: 1939-1951Crossref PubMed Scopus (140) Google Scholar) and that oxidized α2M* can be isolated from inflammatory lesions in humans (56Ohlsson K. Tegner H. Eur. J. Clin. Invest. 1975; 5: 221-227Crossref PubMed Google Scholar). Further investigation of the ability of α2M to inhibit proteinases, bind to cell surface receptors, and carry cytokines in the presence of oxidants should provide novel insights into the biological role of this complex molecule during inflammation. α2-Macroglobulin (α2M) 1The abbreviations used are: α2M, α2-macroglobulin; α2M*, the receptor-recognized form of α2M; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; RBF, carboxyl-terminal receptor binding fragment of α2M;125I-BH-α2M,125I-Bolton-Hunter-labeled α2M;125I-BH-α2M*,125I-Bolton-Hunter-labeled α2M*;cis-DDP, cis-dichlorodiamine-platinum(II); HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; Fura-2/AM, 1-[2-(5-carboxyoxazol-1-yl)-6-aminobenzofuran]-5-oxyl-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxylmethyl ester; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. 1The abbreviations used are: α2M, α2-macroglobulin; α2M*, the receptor-recognized form of α2M; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; RBF, carboxyl-terminal receptor binding fragment of α2M;125I-BH-α2M,125I-Bolton-Hunter-labeled α2M;125I-BH-α2M*,125I-Bolton-Hunter-labeled α2M*;cis-DDP, cis-dichlorodiamine-platinum(II); HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; Fura-2/AM, 1-[2-(5-carboxyoxazol-1-yl)-6-aminobenzofuran]-5-oxyl-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxylmethyl ester; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. is a highly conserved, homotetrameric, 720-kD
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