The binding of receptor-recognized alpha2-macroglobulin to the low density lipoprotein receptor-related protein and the alpha2M signaling receptor is decoupled by oxidation.

Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) 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 alpha2M*. 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 alpha2M* using 200 microM 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 (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM 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 (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, 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 alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.

␣ 2 -Macroglobulin (␣ 2 M) 1 is a highly conserved, homotet-rameric, 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. 1 and 2). Proteinases first cleave the "bait region" of native ␣ 2 M exposing the internal ␥-glutamyl-␤-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 ␣ 2 M. The resulting molecule is much more compact as evidenced by faster migration on a native acrylamide gel (3), electron microscopy (4,5), sedimentation behavior (6), and circular dichroism (7). 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 ␣ 2 M (␣ 2 M*) 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) (8,9).
LRP is a multiligand receptor that binds to a wide variety of unrelated ligands (reviewed in Ref. 10). Binding of all ligands to LRP can be effectively competed by receptor-associated protein (RAP), which co-purifies with LRP. Prior investigation of ␣ 2 M* binding to LRP has shown that the binding mechanism involves a cluster of positively charged residues on ␣ 2 M* interacting with the second complement-like repeat on LRP, which contains clusters of negatively charged residues (11). Analysis of the receptor-binding site on ␣ 2 M* using monoclonal antibody (12,13) and recombinantly expressed protein (14,15) demonstrates that the carboxyl terminus of ␣ 2 M* is involved in receptor binding.
Although LRP is the only ␣ 2 M* receptor identified to date, some important cellular regulatory functions ascribed to ␣ 2 M* suggest that an alternate receptor must exist. ␣ 2 M*, but not native ␣ 2 M, suppresses the production of superoxide anion (16), enhances the release of prostaglandin E 2 (17,18) and platelet activating factor (19), and stimulates the proliferation of vascular smooth muscle cells (20). Moreover, our laboratory has characterized a novel signaling cascade and found that it does not appear to be LRP-mediated (21)(22)(23). Furthermore, we have identified two classes of ␣ 2 M* binding sites on peritoneal macrophages and human trabecular meshwork cells, both of which demonstrate activation of signaling cascades after exposure to ␣ 2 M* (24). The lower affinity binding site is 10 times more abundant than the high affinity binding site and has clearly been identified as LRP (25,26). 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 ␣ 2 M (RBF) is important for signaling (26).
Very recently, however, some investigators reported that residue 1374 is involved in binding to LRP as well, raising the possibility that the ␣ 2 M* signaling receptor may not be a separate receptor (27). Our previous attempts to study ␣ 2 M* binding to LRP and the signaling receptor using cis-dichlorodiamine-platinum(II) (cis-DDP) modification have shown that cis-DDP modifies a region upstream of the 20-kDa carboxylterminal of ␣ 2 M* and that this modification results in decreased binding to LRP while having no effect on cell signaling (25,28,29). This observation together with other immunochemical studies (12,13,30) suggest that a region outside of RBF may be involved in ␣ 2 M* 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 ␣ 2 M* signaling cascade (23,26). Mutational studies have suggested that LRP and the ␣ 2 M* signaling receptor are distinct entities. To date, however, no data have demonstrated a complete dissociation between ␣ 2 M* binding to LRP and to the signaling receptor.
Previous studies have shown that 25 M sodium hypochlorite completely abolishes the anti-proteinase activity of native ␣ 2 M (31, 32). Its effects on ␣ 2 M* receptor-recognition have not been examined. In this study we demonstrate that hypochlorite oxidation of ␣ 2 M* 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 ␣ 2 M results in the selective exposure of the receptor-recognition site to LRP, the ligand cannot signal, thereby providing direct evidence for the dissociation of ␣ 2 M* binding to LRP from binding to the signaling receptor.
Preparation of Activated ␣ 2 M (␣ 2 M*)-Human native ␣ 2 M was purified according to a previously published protocol (33). Native ␣ 2 M was activated with 200 mM methylamine in a buffer containing 150 mM NaCl, 50 mM Tris, pH 8.0, for 16 -18 h at room temperature in the dark. Unreacted methylamine was removed by dialysis for 48 h with four changes of buffer containing 150 mM NaCl, 25 mM sodium phosphate, pH 7.4. Dialyzed ␣ 2 M* was sterile-filtered using 0.22-m syringe microfilters from Millipore (Bedford, MA), stored at 4°C, and used within 2 weeks. Native ␣ 2 M and ␣ 2 M* were iodinated using either IODO-BEADS or 125 I-Bolton-Hunter reagent according to the manufacturerspecified protocol. Specific activity of 125 I-␣ 2 M* varied from 1000 to 1500 cpm/ng for IODO-BEADS labeling and 500 -700 cpm/ng for 125 I-Bolton-Hunter labeling method. The molecular mass of ␣ 2 M* used in these experiments is 720 kDa (34).
Production of Polyclonal Antisera against RBF-BALB/c mice were immunized three times at 4-week intervals with 50 g of antigen (RBF) and Titermax adjuvant (Vaxcel, Norcross, GA) (35). Two weeks after the third immunization, retro-orbital blood was collected, and polyclonal sera from the individual mice were screened for antigen activity by ELISA with RBF as the coating antigen. The mouse with the best titer was boosted with 50 g of antigen. Four days following the final boost, the mouse was euthanized and bled by cardiac puncture.
Oxidation of Native ␣ 2 M, ␣ 2 M*, and RBF-Oxidation of native ␣ 2 M, ␣ 2 M*, and RBF was performed essentially as described previously (32). In brief, native ␣ 2 M, ␣ 2 M*, and RBF were incubated with sodium hypochlorite (from 1.25 M to 2 mM) for 15 min at 37°C in phosphatebuffered saline. At the end of the incubation, 20 mM of L-methionine was added to the mixture to quench residual oxidants. The sodium hypochlorite concentration was determined by measuring the absorption at a wavelength of 292.5 nm using 206 M Ϫ1 cm Ϫ1 as the extinction coefficient at pH 7.5 (36). To ensure that oxidation did not result in a loss of 125 I label from proteins, trichloroacetic acid precipitation of 250 M hypochlorite-oxidized 125 I-␣ 2 M was performed. No significant loss of labeling (i.e. less than 5%, n ϭ 4) was found.
Spectrophotometric Analysis of Oxidized Native ␣ 2 M, ␣ 2 M*, and RBF-The spectral differences between hypochlorite oxidized native ␣ 2 M, ␣ 2 M*, and RBF, and nonoxidized native ␣ 2 M, ␣ 2 M*, and RBF, were analyzed on a DU 640 spectrophotometer (Beckman Instruments) as described previously (37) with the following modifications. Native ␣ 2 M, ␣ 2 M*, or RBF (0.25 mg/ml) was first oxidized according to the published protocol. 1 ml of each sample was then added to the sample cuvette and measured against nonoxidized native ␣ 2 M, ␣ 2 M*, or RBF (0.25 mg/ml) in the reference cuvette. The absorption difference from 220 nm to 400 nm was calculated. As controls, the absorptions of L-methionine and methionine sulfoxide at these wavelengths were found to be negligible.
Polyacrylamide Gel Electrophoresis (PAGE)-Nondenaturing, nonreducing gradient (5-15%) PAGE, or reducing SDS-PAGE (7.5%) were performed to determine the effects of oxidation on the structure of ␣ 2 M. To visualize the protein bands, gels were fixed in acetic acid with Coomassie Brilliant Blue. Hypochlorite oxidation at a concentration up to 125 M had no effect on the ability of ␣ 2 M to be stained by the dye. Oxidation at greater 250 M, however, appears to decrease the ability of ␣ 2 M to be Coomassie-stained.
ELISA-Proteins to be tested were incubated in 96-well Immulon plates (Dynatech, Chantily, VA) for 1 h in 0.1 M NaHCO 3 , pH 9.6, at room temperature. Following incubation, each well was washed twice in PBS-Tween (phosphate-buffered saline, 0.05% Tween-20) to remove unbound proteins. 50 l of PBS-Tween with 4% BSA were then added to each well for 0.5 h at room temperature to block nonspecific binding sites. Following incubation, each well was washed twice with PBS-Tween. To each well was then added 50 l of 1:100 dilution of primary antibody against the protein to be tested and then incubation was continued for 1 h at 25°C. Following this incubation, each well was washed twice with PBS-Tween and then 50 l of 1:400 dilution of anti-mouse IgG-horseradish peroxidase-conjugated antibody was added. After 1-h incubation at 25°C, the unbound anti-mouse IgG antibody was removed by washing with PBS-Tween and 50 l of ophenylenediamine dihydrochloride substrate solution was added to each well. The enzyme-substrate reaction was allowed to proceed for 15 min and then the absorbance at a wavelength of 450 nm was determined with a microplate reader (Molecular Devices, Menlo Park, CA). Since oxidation may alter the binding of oxidized ␣ 2 M to the plate, we compared the quantity of the oxidized proteins bound to the plate with that of the nonoxidized protein. We found no statistically significant difference (n ϭ 3) in the amount of proteins bound between oxidized and nonoxidized ␣ 2 M.
Macrophage Harvesting-Pathogen-free female C57BL/6 mice were obtained from Charles River Laboratories (Raleigh, NC). Peritoneal macrophages were obtained as described previously (38). Thioglycollate-elicited peritoneal macrophages were harvested by peritoneal larvage using 5 ml of 150 mM NaCl, 20 mM HEPES, pH 7.4. The macrophages were pelleted by centrifugation at ϳ800 ϫ g for 5 min and suspended in RPMI 1640 medium containing 12.5 units/ml penicillin, 6.254 mg/ml streptomycin, and 10% fetal bovine serum. Cell viability was assayed by the trypan blue method (Hausser Scientific, Horsham, PA).
Cell Surface Binding Assay-Macrophages were plated in 24-well cell culture plates (Becton Dickinson, Lincoln, Park, NJ) at 1 ϫ 10 6 cells/well (with a maximum of 7% variation between wells, n ϭ 8) and incubated for 3 h at 37°C in a humidified 5% CO 2 incubator. These plates were then cooled to 4°C, and unbound cells were removed by rinsing three times with ice-cold HBSS containing 20 mM HEPES, 5% BSA, pH 7.4 (buffer A). As a control for nonspecific ␣ 2 M* binding, some of the wells were rinsed three times with ice-cold HBSS without CaCl 2 , MgCl 2 , MgSO 4 containing 20 mM HEPES, 5% BSA, and 5 mM EDTA, pH 7.4 (buffer B) to assess calcium-independent binding. For RAP compe-tition studies, radiolabeled ligands (2 nM) were added to each well in the presence or absence of unlabeled RAP. For oxidized native ␣ 2 M and oxidized ␣ 2 M* competition binding studies, various concentrations of unlabeled inhibitors were added to each well followed immediately by the addition of radiolabeled ligands (0.5 nM for 125 I-␣ 2 M* and 11 nM for 125 I-RBF). Cells were then incubated at 4°C for 12-16 h. Unbound ligand was removed from the wells, and the cell monolayer was rinsed twice with ice-cold buffer A or B. Cells in each well were then solubilized with 0.1 M NaOH, 0.5% SDS at room temperature for Ͼ 5 h before transferring to polystyrene tubes to be counted in a ␥-counter (LKB-Wallac, CliniGamma 1272). Specific binding of ␣ 2 M* to cells was determined by subtracting calcium-independent binding, which averaged less than 10% over four experiments, from total binding. To verify that calcium-dependent binding represents binding to both the signaling receptor and LRP, radioligand competition binding experiments in the presence of 100-fold excess unlabeled ligands were performed (n ϭ 3). No difference was observed between binding in the presence of buffer B or with 100-fold excess unlabeled ligand.
Calcium Signaling Studies-The measurement of intracellular calcium level, [Ca 2ϩ ] i , was performed according to a previously published protocol (21). Briefly, macrophages were plated on glass coverslips at a density of 400,000 cells/cm 2 . The cells were then incubated for 16 -18 h in a humidified 5% CO 2 incubator at 37°C. After addition of 4 M of Fura-2/AM, cells were incubated for another 30 min in the dark at 25°C. The coverslips containing adherent cells were then washed twice with HBSS without calcium and mounted on a digital imaging microscope. After obtaining a stable base-line level of [Ca 2ϩ ] i , ligands were added and the level of [Ca 2ϩ ] i was immediately measured. Typical base-line [Ca 2ϩ ] i was approximately 100 -150 nM. A positive response was typically 2-4-fold increase over base line. All ligands and buffers were tested negative (Ͻ0.01 enzyme units/ml) for endotoxin using the Limulus amebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA).
Data Analysis-In direct binding studies, the B max and K d were derived from the x intercept and the slope of the Scatchard plot, respectively. These numbers were also verified using least square analysis based on single-site binding using the Systat 5.0 computer program. The apparent dissociation constant for the unlabeled ligand, K i , was determined using the equation K i ϭ IC 50 /(1 ϩ L/K d ), where L and K d are the concentration and the dissociation constant for the radiolabeled protein, respectively (37). The concentrations of radiolabeled proteins used in oxidized native ␣ 2 M and oxidized ␣ 2 M* cold competition experiments equal the dissociation constant. This reduces the above equation to K i ϭ IC 50 /2.

RESULTS
Spectrophotometric Absorption Analysis of Oxidized Native ␣ 2 M, ␣ 2 M*, and RBF-To quantitate the concentration dependence of native ␣ 2 M, ␣ 2 M*, and RBF modifications, the spectrophotometric absorption differences between oxidized and nonoxidized native ␣ 2 M, ␣ 2 M*, and RBF from 220 nm to 400 nm were determined. As shown in Fig. 1, the peak difference between oxidized and nonoxidized native ␣ 2 M and ␣ 2 M* occurred at approximately 242 nm and, to a lesser extent, at 300 nm . The absorption at 242 nm corresponds to a mixture of species with chloramine modification of amino terminus and lysine side chain being the most abundant as determined by trinitrobenzene sulfonate titration (39,40). The absorption at 300 nm corresponds to the formation of dichloramine (41). The hypochlorite oxidation of RBF resulted in minimal modification (Fig. 1C). This is not unexpected since our previous attempts to modify RBF using cis-DDP and hydrogen peroxide, which react at the same residues, have both failed to affect RBF (42)(43)(44). Fig. 1D shows the concentration dependence of native ␣ 2 M, ␣ 2 M*, and RBF oxidation at 242 nm . Both native ␣ 2 M and ␣ 2 M* were equally susceptible to hypochlorite oxidation at all concentrations. Moreover, this modification plateaued at an oxidant concentration of approximately 2 mM. On the other hand, RBF oxidation showed minimal protein modification even at high oxidant concentrations.
Binding Competition of Oxidized ␣ 2 M* to Macrophages-The effect of hypochlorite modification on the receptor binding properties of ␣ 2 M* was determined. Two different sets of experiments were performed. The first set showed that oxidation significantly decreased the ability of ␣ 2 M* to compete for the binding of nonoxidized ␣ 2 M* ( Fig. 2A). This effect appeared to take place only when ␣ 2 M* was oxidized at a hypochlorite concentration of at least 37.5 M. At 75 M hypochlorite, oxidized ␣ 2 M* was unable to compete for more than 90% of the binding of nonoxidized ␣ 2 M*. The second set of experiments showed that oxidation can abolish the RAP-sensitive binding of ␣ 2 M* (Fig. 2B). Consistent with the results in the first set of experiments, no significant effect on ␣ 2 M* binding occurred when the protein was oxidized by less than 37.5 M hypochlorite. At concentrations equal to or greater than 37.5 M, pronounced inhibition of ␣ 2 M* binding was observed. The slight increase in the amount of RAP-insensitive binding when the protein is modified by high concentration of hypochlorite pos- sibly reflects an increase in electrostatic interaction between oxidized ␣ 2 M* and the cell surface.
Binding Competition of Oxidized Native ␣ 2 M to Macrophages-As a control for the binding of oxidized proteins to the cell surface, we also determined the ability of oxidized native ␣ 2 M to compete for the binding of ␣ 2 M*. Since native ␣ 2 M does not bind to either LRP or the signaling receptor, we expect that oxidized native ␣ 2 M also would not bind to either receptor. Fig.  3A shows that oxidation of native ␣ 2 M with either 12.5 or 25 M hypochlorite resulted in an increased ability of oxidized native ␣ 2 M to compete for the binding of ␣ 2 M*, suggesting that its receptor-recognition site had been exposed. Oxidation with concentrations of hypochlorite greater than 25 M resulted in a concentration-dependent decrease in its ability to compete for ␣ 2 M* binding (data not shown). These findings are confirmed in Fig. 3B showing that native ␣ 2 M that has been oxidized with hypochlorite can bind specifically to LRP, since its binding can be competed by RAP. Figs. 2 and 3, the iodinated proteins used in the binding experiments were labeled with IODO-BEADS. Since this method can potentially oxidize ␣ 2 M, we performed additional experiments using 125 I-Bolton-Hunter-labeled native ␣ 2 M and ␣ 2 M* ( 125 I-BH-␣ 2 M, 125 I-BH-␣ 2 M*, respectively) to verify the results. Fig. 4A shows that oxidation of 125 I-BH-␣ 2 M* at a hypochlorite concentration greater than 200 M resulted in the complete absence of binding to LRP. Fig. 4B shows that oxidation by as little as 25 M of hypochlorite can enhance the binding of 125 I-BH-␣ 2 M. Maximal enhancement, however, occur at a hypochlorite concentration of 125 M. Oxidation appear to effect 125 I-BH-␣ 2 M similarly compared with IODO-BEADS-labeled ␣ 2 M; however, higher concentrations of oxidants were necessary to achieve the same results. Given the greater resistance to oxidation by 125 I-BH-␣ 2 M as demonstrated by receptor binding, we compared the susceptibility of Bolton-Hunter or IODO-BEADS-labeled ␣ 2 M to structural damage by oxidation as a mean of determining which labeling method gave a product that is more represent- ative of the unlabeled protein. A previous study has shown that ␣ 2 M oxidation results in fracturing of the protein along its dimeric axis (32). Fig. 4C shows the tetramer to dimer transition of ␣ 2 M upon oxidation. IODO-BEADS-labeled ␣ 2 M appear to be more sensitive to oxidation compared with unlabeled or Bolton-Hunter labeled ␣ 2 M confirming the results from receptor binding.

Comparison of the Effects of Different Labeling Methods on Oxidized ␣ 2 M Binding to Macrophages-In
Competition Binding of Oxidized RBF to Macrophages-Since RBF binds to both LRP and the signaling receptor, we investigated whether oxidation altered its receptor binding properties. Consistent with the spectral analyses data, oxidation at a hypochlorite concentration up to 125 M had no significant effect on the ability of RBF to compete for the binding of nonoxidized RBF to cell surface receptors (Fig. 5). Additional calcium signaling experiments revealed that oxidation had no effect on the ability of RBF to signal (data not shown).
Intracellular Calcium Signaling Studies of Oxidized Native ␣ 2 M and ␣ 2 M*-Since our cell surface binding studies showed that oxidation can completely abolish the RAP-sensitive binding of oxidized ␣ 2 M* and expose the receptor-recognition site of native ␣ 2 M to LRP, we investigated whether oxidized native ␣ 2 M and oxidized ␣ 2 M* also increase intracellular calcium levels by binding to the ␣ 2 M* signaling receptor. Fig. 6A shows that oxidation at a concentration of hypochlorite up to 200 M had no effect on the ability of ␣ 2 M* to generate an increase in [Ca 2ϩ ] i . The intracellular calcium rose from base-line levels between 100 and 150 nM to peak levels between 400 and 500 nM within the first 10 s of cell exposure to the ligands. Boiled ␣ 2 M* generated no increase in [Ca 2ϩ ] i . To determine whether oxidized native ␣ 2 M also bound to the signaling receptor, we measured the changes in intracellular calcium levels when cells were treated with native ␣ 2 M that was oxidized with 50 and 125 M hypochlorite. These concentrations correspond to the concentrations that generated the maximal exposure of LRP binding sites. Fig. 6B shows that no increase in [Ca 2ϩ ] i was observed with the addition of either oxidized or nonoxidized native ␣ 2 M.
Reduced SDS-PAGE of Trypsin-treated Oxidized Native ␣ 2 M-To determine the mechanism responsible for the oxidative exposure of the receptor-recognition site of native ␣ 2 M for LRP, we performed a SDS-PAGE under reducing conditions of oxidized native ␣ 2 M that was treated with 20-fold molar excess of trypsin. We hypothesized that if the exposure of the receptorrecognition site is associated with the unfolding of the protein, then oxidized native ␣ 2 M should be more susceptible to digestion by trypsin. Fig. 7 shows that as the concentration of hypochlorite used to modify native ␣ 2 M increased, fewer large molecular weight protein bands remained, indicating that the oxidized protein is digested more efficiently by trypsin. Oxidation at up to 5 M hypochlorite appeared to have no effect on the susceptibility of native ␣ 2 M toward trypsin. However, at oxidant concentrations greater than 5 M, native ␣ 2 M became more susceptible to tryptic digestion. This is in agreement with our receptor binding data showing that exposure of the receptor-recognition site begins only after native ␣ 2 M has been oxidized by at least an oxidant concentration of 5 M. To investigate whether other mechanisms may account for the oxidative exposure of the receptor-binding site of native ␣ 2 M for LRP, we performed thioester bond titration of oxidized native ␣ 2 M. Thioester bond rupture results in exposure of the receptorrecognition site, analogous to the reaction of methylamine with native ␣ 2 M. This could complicate our interpretations of these results. In data not presented, we found no thioester bond rupture by hypochlorite oxidation. These data are consistent with previous studies of native ␣ 2 M oxidation (32).

ELISA of Anti-RBF Antibodies against Oxidized Native
␣ 2 M-To further probe whether unfolding of the ␣ 2 M secondary structure may result in exposure of RBF, we performed an ELISA using polyclonal antisera raised against RBF. The carboxyl-terminal 20 kDa of ␣ 2 M is partially exposed in the native conformation (45), and therefore, it is not unexpected that it would be partially recognized by a polyclonal antisera to RBF. Upon exposure of native ␣ 2 M to proteinase or methylamine, this region becomes more exposed so that it binds to cell surface receptors and should, therefore, show greater recognition by antibodies raised against RBF. Fig. 8 shows that native ␣ 2 M was partially recognized by the anti-RBF antibodies whereas RBF and ␣ 2 M* were both fully recognized. Oxidized native ␣ 2 M was also fully recognized by anti-RBF antibodies; however, this occurred only after it was oxidized by at least 12.5 M hypochlorite.
Direct Binding of Oxidized Native ␣ 2 M and ␣ 2 M* to Macro- phage Cell Surface-Since ␣ 2 M* has two different K d and B max values, we performed a concentration-dependent binding experiment using oxidized 125 I-BH-␣ 2 M and 125 I-BH-␣ 2 M* to determine whether the binding of 125 I-oxidized ␣ 2 M* to the signaling receptor corresponds with its binding to the high affinity site and whether the absence of binding by 125 I-oxidized native ␣ 2 M corresponds with its binding to the low affinity site. Fig.  9A shows the results from direct binding experiments of 125 Ioxidized ␣ 2 M* to macrophages. The Scatchard analysis (A, inset) shows that only a single class of high affinity (K d ϳ 83 pM) binding sites exist with a B max of ϳ 5.4 fmol per million cells. These numbers were verified using Systat analysis program (K d Ϫ 71 Ϯ 12 pM, B max Ϫ 6.2 Ϯ 2.1 fmol/million cells). Binding to the lower affinity LRP is absent since the addition of 100-fold excess RAP did not alter the K d or the B max . The binding of 125 I-oxidized native ␣ 2 M to macrophages is shown in Fig. 9B. The Scatchard analysis (B, inset) shows a single class of lower affinity (K d ϳ 0.6 nM) binding sites with a B max of approximately 55 fmol/million cells. Analysis using Systat shows good agreement with these numbers (K d : 0.7 Ϯ 0.15 nM; B max : 57 Ϯ 9 fmol/million cells). This binding is entirely due to the LRP receptor since the addition of 100-fold excess RAP completely abolished specific ligand binding. The absence of binding to the high affinity sites by oxidized native ␣ 2 M together with the signal transduction studies described above provide direct evidence that the high affinity sites represent the signaling receptor. DISCUSSION In this study we demonstrate that hypochlorite oxidation of native ␣ 2 M or ␣ 2 M* can generate exposure of the receptor binding sites to either LRP or the ␣ 2 M signaling receptor, respectively. Oxidation of ␣ 2 M* by 200 M 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 ␣ 2 M by 125 M 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 ␣ 2 M* is outside of the carboxylterminal 20-kDa receptor-binding domain. Studies of the mechanism of oxidative exposure of the LRP-binding site in native ␣ 2 M suggested that protein unfolding may be responsible for this phenomenon. These experiments provide strong proof for the existence of two distinct ␣ 2 M* receptors and the presence of two independent receptor-binding regions on ␣ 2 M*.
Our earlier studies using cis-DDP modification of ␣ 2 M* and RBF have shown that the cis-DDP-sensitive site in ␣ 2 M* is outside of the 20-kDa carboxyl terminus and appears identical to the oxidation-sensitive site (28,29,37). 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 ␣ 2 M* 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 (26). This has been the best evidence for the existence of two distinct ␣ 2 M* receptors; however, recent work by Nielsen et al. (27) 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 ␣ 2 M (31, 32). Treatment of native ␣ 2 M with 25 M hypochlorite resulted in complete destruction of its anti-proteinase activity. We hypothesized that hypochlorite could also inhibit the ability of ␣ 2 M* to bind to cell surface receptors. In this study, we show that hypochlorite treatment completely eliminated the RAP-sensitive binding of ␣ 2 M* 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 ␣ 2 M* to the low affinity sites but is unable to inhibit the ability of ␣ 2 M* to signal or to bind to the high affinity sites (24 -26). Since hypochlorite oxidation is also able to cause the exposure of LRP binding sites in native ␣ 2 M 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 ␣ 2 M* signaling receptors.
The oxidative exposure of the ␣ 2 M-binding site to LRP but not to the signaling receptor, is unique in a number of ways. All of the known naturally occurring ␣-macroglobulins or recombinantly expressed receptor binding fragments activate the signaling cascade (21,23,46). 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 ␣ 2 M 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 ␣ 2 M* for the signaling receptor is distinct from the LRP binding site. That hypochlorite oxidation can selectively expose only the LRP binding sites in native ␣ 2 M or the signaling receptor binding sites in ␣ 2 M* demonstrates that the ability of ␣ 2 M* to bind to its two receptors can be uncoupled. Efforts are currently being made using oxidized ␣ 2 M* to isolate and purify the signaling receptor.
Our investigation of the mechanism that may explain the oxidative exposure of LRP binding site in native ␣ 2 M suggests that partial protein unfolding may be responsible. Earlier works by Davies et al. (47)(48)(49)(50) 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. (51) showing that extracellular proteins such as ␣ 1 -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 ␣ 2 M 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 M and peaks at 25 M. This is in marked contrast with ␣ 2 M that has been labeled with Bolton-Hunter reagent, which generates LRP binding sites with as little as 25 M of hypochlorite but does not peak until 125 M. At hypochlorite concentrations greater than 125 M, oxidized ␣ 2 M 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 ␣ 2 M has similar susceptibility to oxidation as unlabeled ␣ 2 M, whereas IODO-BEADS-labeled ␣ 2 M is significantly more susceptible to oxidation. This suggests that receptor binding studies with ␣ 2 M should use the Bolton-Hunter labeling method to minimize protein oxidation.
The selective exposure of the LRP binding site in oxidized ␣ 2 M 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 ␣ 2 M is associated with an increase in the exposure of RBF. We found that oxidation of ␣ 2 M at greater than 12.5 M 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 ␣ 2 M* 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 (9,26,27). 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 ␣ 2 M* even when the protein is treated with 200 M hypochlorite suggests otherwise.
Oxidative inactivation of ␣ 2 M* receptor binding to LRP suggests an interesting pathophysiological process that may occur during inflammation. ␣ 2 M is ubiquitous in serum and extracellular fluids (6,52). During inflammation, neutrophils secrete hypochlorite and proteinases as a defense mechanism against invading foreign organisms (40,51,(53)(54)(55). In the presence of oxidants ␣ 2 M 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 ␣ 2 M* signaling has been associated with increased production of prostaglandins and platelet-activating factor as well as increased mitogenesis in vascular smooth muscle cells (17)(18)(19)(20). ␣ 2 M 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 M hypochlorite in 2 h (40,51) and that oxidized ␣ 2 M* can be isolated from inflammatory lesions in humans (56). Further investigation of the ability of ␣ 2 M 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.