The Binding of Receptor-recognized alpha 2-Macroglobulin to the Low Density Lipoprotein Receptor-related Protein and the alpha 2M Signaling Receptor Is Decoupled by Oxidation*

(Received for publication, March 4, 1997, and in revised form, May 21, 1997)

Sean M. Wu , Cinda M. Boyer and Salvatore V. Pizzo Dagger

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Receptor-recognized forms of alpha 2-macroglobulin (alpha 2M*) 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 alpha 2M*. 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 alpha 2M* 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 (Kd - 71 ± 12 pM). Surprisingly, oxidation of native alpha 2-macroglobulin (alpha 2M) 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 (Kd - 0.7 ± 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha 2M (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 alpha 2M 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 alpha 2M* signaling receptor.


INTRODUCTION

alpha 2-Macroglobulin (alpha 2M)1 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. 1 and 2). Proteinases first cleave the "bait region" of native alpha 2M exposing the internal gamma -glutamyl-beta -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 alpha 2M. 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 alpha 2M (alpha 2M*) 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 alpha 2M* binding to LRP has shown that the binding mechanism involves a cluster of positively charged residues on alpha 2M* interacting with the second complement-like repeat on LRP, which contains clusters of negatively charged residues (11). Analysis of the receptor-binding site on alpha 2M* using monoclonal antibody (12, 13) and recombinantly expressed protein (14, 15) demonstrates that the carboxyl terminus of alpha 2M* is involved in receptor binding.

Although LRP is the only alpha 2M* receptor identified to date, some important cellular regulatory functions ascribed to alpha 2M* suggest that an alternate receptor must exist. alpha 2M*, but not native alpha 2M, suppresses the production of superoxide anion (16), enhances the release of prostaglandin E2 (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 (21-23). Furthermore, we have identified two classes of alpha 2M* binding sites on peritoneal macrophages and human trabecular meshwork cells, both of which demonstrate activation of signaling cascades after exposure to alpha 2M* (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 alpha 2M (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 alpha 2M* signaling receptor may not be a separate receptor (27). Our previous attempts to study alpha 2M* 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 carboxyl-terminal of alpha 2M* 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 alpha 2M* 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 alpha 2M* signaling cascade (23, 26). Mutational studies have suggested that LRP and the alpha 2M* signaling receptor are distinct entities. To date, however, no data have demonstrated a complete dissociation between alpha 2M* 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 alpha 2M (31, 32). Its effects on alpha 2M* receptor-recognition have not been examined. In this study we demonstrate that hypochlorite oxidation of alpha 2M* 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 alpha 2M results in the selective exposure of the receptor-recognition site to LRP, the ligand cannot signal, thereby providing direct evidence for the dissociation of alpha 2M* binding to LRP from binding to the signaling receptor.


EXPERIMENTAL PROCEDURES

Materials

Recombinant RAP construct was a kind gift of Dr. Joachim Herz (University of Texas, Southwestern, Dallas, TX). Recombinant RBF construct was prepared as in (14). The production of both recombinant RAP and RBF is described in detail in Ref. 26. RPMI 1640, fetal bovine serum, Hanks' balanced salt solution (HBSS), and HBSS without CaCl2, MgCl2, MgSO4 were purchased from Life Technologies, Inc. Bovine serum albumin (BSA), HEPES, sodium hypochlorite, L-methionine, and EDTA were purchased from Sigma. Tris base was purchased from Boehringer Mannheim. PD-10® column was purchased from Pharmacia Biotech Inc. (Uppsala, Sweden). Carrier-free 125I and 125I-Bolton-Hunter reagent for protein iodination was obtained from NEN Life Science Products. 1-[2-(5-Carboxyoxazol-1-yl)-6-aminobenzofuran]-5-oxyl-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxylmethyl ester (Fura-2/AM) was purchased from Molecular Probes (Eugene, OR). BCA protein assay kit, IODO-BEADS®, and 10,000 molecular weight cut-off Slide-A-Lyzer® dialysis cassettes were purchased from Pierce. All other reagents were of the highest quality commercially available.

Preparation of Activated alpha 2M (alpha 2M*)

Human native alpha 2M was purified according to a previously published protocol (33). Native alpha 2M 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 alpha 2M* was sterile-filtered using 0.22-µm syringe microfilters from Millipore (Bedford, MA), stored at 4 °C, and used within 2 weeks. Native alpha 2M and alpha 2M* were iodinated using either IODO-BEADS® or 125I-Bolton-Hunter reagent according to the manufacturer-specified protocol. Specific activity of 125I-alpha 2M* varied from 1000 to 1500 cpm/ng for IODO-BEADS® labeling and 500-700 cpm/ng for 125I-Bolton-Hunter labeling method. The molecular mass of alpha 2M* 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 µg 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 µg of antigen. Four days following the final boost, the mouse was euthanized and bled by cardiac puncture.

Oxidation of Native alpha 2M, alpha 2M*, and RBF

Oxidation of native alpha 2M, alpha 2M*, and RBF was performed essentially as described previously (32). In brief, native alpha 2M, alpha 2M*, and RBF were incubated with sodium hypochlorite (from 1.25 µM to 2 mM) for 15 min at 37 °C in phosphate-buffered 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-1 cm-1 as the extinction coefficient at pH 7.5 (36). To ensure that oxidation did not result in a loss of 125I label from proteins, trichloroacetic acid precipitation of 250 µM hypochlorite-oxidized 125I-alpha 2M was performed. No significant loss of labeling (i.e. less than 5%, n = 4) was found.

Spectrophotometric Analysis of Oxidized Native alpha 2M, alpha 2M*, and RBF

The spectral differences between hypochlorite oxidized native alpha 2M, alpha 2M*, and RBF, and nonoxidized native alpha 2M, alpha 2M*, and RBF, were analyzed on a DU® 640 spectrophotometer (Beckman Instruments) as described previously (37) with the following modifications. Native alpha 2M, alpha 2M*, 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 alpha 2M, alpha 2M*, or RBF (0.25 mg/ml) in the reference cuvette. The absorption difference from lambda 220 nm to lambda 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 alpha 2M. To visualize the protein bands, gels were fixed in acetic acid with Coomassie Brilliant Blue. Hypochlorite oxidation at a concentration up to 125 µM had no effect on the ability of alpha 2M to be stained by the dye. Oxidation at greater 250 µM, however, appears to decrease the ability of alpha 2M 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 NaHCO3, 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 µl 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 µl 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 µl 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 µl of o-phenylenediamine 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 alpha 2M 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 = 3) in the amount of proteins bound between oxidized and nonoxidized alpha 2M.

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 ~800 × 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 × 106 cells/well (with a maximum of 7% variation between wells, n = 8) and incubated for 3 h at 37 °C in a humidified 5% CO2 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 alpha 2M* binding, some of the wells were rinsed three times with ice-cold HBSS without CaCl2, MgCl2, MgSO4 containing 20 mM HEPES, 5% BSA, and 5 mM EDTA, pH 7.4 (buffer B) to assess calcium-independent binding. For RAP competition studies, radiolabeled ligands (2 nM) were added to each well in the presence or absence of unlabeled RAP. For oxidized native alpha 2M and oxidized alpha 2M* 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 125I-alpha 2M* and 11 nM for 125I-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 > 5 h before transferring to polystyrene tubes to be counted in a gamma -counter (LKB-Wallac, CliniGamma 1272). Specific binding of alpha 2M* 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 = 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, [Ca2+]i, was performed according to a previously published protocol (21). Briefly, macrophages were plated on glass coverslips at a density of 400,000 cells/cm2. The cells were then incubated for 16-18 h in a humidified 5% CO2 incubator at 37 °C. After addition of 4 µM 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 [Ca2+]i, ligands were added and the level of [Ca2+]i was immediately measured. Typical base-line [Ca2+]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 (<0.01 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 Bmax and Kd 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, Ki, was determined using the equation Ki = IC50/(1 + L/Kd), where L and Kd are the concentration and the dissociation constant for the radiolabeled protein, respectively (37). The concentrations of radiolabeled proteins used in oxidized native alpha 2M and oxidized alpha 2M* cold competition experiments equal the dissociation constant. This reduces the above equation to Ki = IC50/2.


RESULTS

Spectrophotometric Absorption Analysis of Oxidized Native alpha 2M, alpha 2M*, and RBF

To quantitate the concentration dependence of native alpha 2M, alpha 2M*, and RBF modifications, the spectrophotometric absorption differences between oxidized and nonoxidized native alpha 2M, alpha 2M*, and RBF from lambda 220 nm to lambda 400 nm were determined. As shown in Fig. 1, the peak difference between oxidized and nonoxidized native alpha 2M and alpha 2M* occurred at approximately lambda 242 nm and, to a lesser extent, at lambda 300 nm. The absorption at lambda 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 lambda 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 (42-44). Fig. 1D shows the concentration dependence of native alpha 2M, alpha 2M*, and RBF oxidation at lambda 242 nm. Both native alpha 2M and alpha 2M* 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.


Fig. 1. Difference spectra analyses of oxidized native alpha 2M, alpha 2M*, and RBF. alpha 2M* (A) and native alpha 2M (B) were oxidized using 12.5 (single line) or 37.5 (open square or circle) µM hypochlorite for 15 min at 37 °C. Following incubation, 20 mM of L-methionine was added to quench residual oxidants. The spectra of oxidized proteins were measured from wavelengths of 220-400 nm using nonoxidized alpha 2M* or native alpha 2M spectra as base lines. The absorption spectrum of RBF (C) oxidized with 50 µM of hypochlorite was also measured. The concentration dependence of alpha 2M* (open square), native alpha 2M (open circle), and RBF (filled circle) modification by hypochlorite is shown in D. These data represent the average of two independent experiments performed in duplicate.
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Binding Competition of Oxidized alpha 2M* to Macrophages

The effect of hypochlorite modification on the receptor binding properties of alpha 2M* was determined. Two different sets of experiments were performed. The first set showed that oxidation significantly decreased the ability of alpha 2M* to compete for the binding of nonoxidized alpha 2M* (Fig. 2A). This effect appeared to take place only when alpha 2M* was oxidized at a hypochlorite concentration of at least 37.5 µM. At 75 µM hypochlorite, oxidized alpha 2M* was unable to compete for more than 90% of the binding of nonoxidized alpha 2M*. The second set of experiments showed that oxidation can abolish the RAP-sensitive binding of alpha 2M* (Fig. 2B). Consistent with the results in the first set of experiments, no significant effect on alpha 2M* binding occurred when the protein was oxidized by less than 37.5 µM hypochlorite. At concentrations equal to or greater than 37.5 µM, pronounced inhibition of alpha 2M* binding was observed. The slight increase in the amount of RAP-insensitive binding when the protein is modified by high concentration of hypochlorite possibly reflects an increase in electrostatic interaction between oxidized alpha 2M* and the cell surface.


Fig. 2. Binding competition of oxidized and 125I-oxidized alpha 2M* to peritoneal macrophages. A, 125I-alpha 2M* was added to macrophages followed by the addition of the indicated concentrations of excess unlabeled alpha 2M* that was oxidized at 0 (filled circle), 25 (open circle), 37.5 (filled square), 50 (open square), and 75 (filled triangle) µM hypochlorite for 15 min. Following incubation, cells were rinsed twice to remove unbound ligands and solubilized in SDS/NaOH solution before gamma -counting. B, 125I-alpha 2M* (2 nM) that was oxidized at the indicated concentrations of hypochlorite was added to each well in the absence (filled bar) or presence (open bar) of 250-fold molar excess of unlabeled RAP. Nonspecific binding was subtracted from total binding as described under "Experimental Procedures." These data represent the mean of two independent experiments performed in triplicate. The S.E. is less than ±9% for all points in A and as indicated by error bars in B.
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Binding Competition of Oxidized Native alpha 2M to Macrophages

As a control for the binding of oxidized proteins to the cell surface, we also determined the ability of oxidized native alpha 2M to compete for the binding of alpha 2M*. Since native alpha 2M does not bind to either LRP or the signaling receptor, we expect that oxidized native alpha 2M also would not bind to either receptor. Fig. 3A shows that oxidation of native alpha 2M with either 12.5 or 25 µM hypochlorite resulted in an increased ability of oxidized native alpha 2M to compete for the binding of alpha 2M*, suggesting that its receptor-recognition site had been exposed. Oxidation with concentrations of hypochlorite greater than 25 µM resulted in a concentration-dependent decrease in its ability to compete for alpha 2M* binding (data not shown). These findings are confirmed in Fig. 3B showing that native alpha 2M that has been oxidized with hypochlorite can bind specifically to LRP, since its binding can be competed by RAP.


Fig. 3. Binding competition of oxidized and 125I-oxidized native alpha 2M to peritoneal macrophages. A, 125I-alpha 2M* was added to each well followed by the addition of indicated concentrations of excess unlabeled native alpha 2M that has been oxidized at 0 (open circle), 12.5 (open square), and 25 (filled square) µM hypochlorite for 15 min. For comparison, alpha 2M* (filled circle) was also added at the same concentrations. B, 125I-native alpha 2M (2 nM) that was oxidized at the indicated concentrations of hypochlorite for 15 min was added to each well in the absence (filled bar) or presence (open bar) of 250-fold molar excess of unlabeled RAP. Nonspecific binding was subtracted from total binding as described previously. These data represent the mean of two independent experiments performed in triplicate. The S.E. is less than ±10% for all points in A and as indicated by error bars in B.
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Comparison of the Effects of Different Labeling Methods on Oxidized alpha 2M Binding to Macrophages

In Figs. 2 and 3, the iodinated proteins used in the binding experiments were labeled with IODO-BEADS®. Since this method can potentially oxidize alpha 2M, we performed additional experiments using 125I-Bolton-Hunter-labeled native alpha 2M and alpha 2M* (125I-BH-alpha 2M, 125I-BH-alpha 2M*, respectively) to verify the results. Fig. 4A shows that oxidation of 125I-BH-alpha 2M* at a hypochlorite concentration greater than 200 µM resulted in the complete absence of binding to LRP. Fig. 4B shows that oxidation by as little as 25 µM of hypochlorite can enhance the binding of 125I-BH-alpha 2M. Maximal enhancement, however, occur at a hypochlorite concentration of 125 µM. Oxidation appear to effect 125I-BH-alpha 2M similarly compared with IODO-BEADS®-labeled alpha 2M; however, higher concentrations of oxidants were necessary to achieve the same results. Given the greater resistance to oxidation by 125I-BH-alpha 2M as demonstrated by receptor binding, we compared the susceptibility of Bolton-Hunter or IODO-BEADS®-labeled alpha 2M to structural damage by oxidation as a mean of determining which labeling method gave a product that is more representative of the unlabeled protein. A previous study has shown that alpha 2M oxidation results in fracturing of the protein along its dimeric axis (32). Fig. 4C shows the tetramer to dimer transition of alpha 2M upon oxidation. IODO-BEADS®-labeled alpha 2M appear to be more sensitive to oxidation compared with unlabeled or Bolton-Hunter labeled alpha 2M confirming the results from receptor binding.


Fig. 4. Comparison of the effects of labeling method on oxidized alpha 2M binding to macrophages. A, 125I-BH-alpha 2M* (2 nM) that was oxidized at the indicated concentrations of hypochlorite for 15 min was added to each well in the absence (filled bar) or presence (open bar) of 250-fold molar excess of unlabeled RAP. Following incubation for 16 h at 4 °C, cells were rinsed, solubilized, and counted in a gamma -counter. B, similar experiments were performed as in A except that 125I-BH-native alpha 2M was added to each well. C, native alpha 2M, Bolton-Hunter, or IODO-BEADS®-labeled native alpha 2M were oxidized with the indicated concentrations of hypochlorite and analyzed on nondenaturing, nonreducing, 5-15% gradient PAGE. The upper and lower bands represent tetrameric and dimeric forms of alpha 2M, respectively. Binding data represent the mean of three independent experiments performed in triplicate. Electrophoresis results are the representative data of two independent experiments.
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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 µM 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).


Fig. 5. Binding compeititon of oxidized RBF to peritoneal macrophages. 125I-RBF was added to macrophages in wells followed by the addition of indicated concentrations of excess unlabeled RBF that had been oxidized at 0 (filled circle), 25 (open circle), 50 (filled square), and 125 (open square) µM hypochlorite for 15 min. After 16-h incubation at 4 °C, cells were rinsed twice to remove unbound ligands and solubilized in SDS/NaOH solution before gamma -counting. Nonspecific binding was subtracted from total binding as described previously. The data shown are obtained from a representative experiment of two that have been performed in triplicate. The S.E. is less than ±9% at all points.
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Intracellular Calcium Signaling Studies of Oxidized Native alpha 2M and alpha 2M*

Since our cell surface binding studies showed that oxidation can completely abolish the RAP-sensitive binding of oxidized alpha 2M* and expose the receptor-recognition site of native alpha 2M to LRP, we investigated whether oxidized native alpha 2M and oxidized alpha 2M* also increase intracellular calcium levels by binding to the alpha 2M* signaling receptor. Fig. 6A shows that oxidation at a concentration of hypochlorite up to 200 µM had no effect on the ability of alpha 2M* to generate an increase in [Ca2+]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 alpha 2M* generated no increase in [Ca2+]i. To determine whether oxidized native alpha 2M also bound to the signaling receptor, we measured the changes in intracellular calcium levels when cells were treated with native alpha 2M that was oxidized with 50 and 125 µM hypochlorite. These concentrations correspond to the concentrations that generated the maximal exposure of LRP binding sites. Fig. 6B shows that no increase in [Ca2+]i was observed with the addition of either oxidized or nonoxidized native alpha 2M.


Fig. 6. Calcium signaling study of oxidized native alpha 2M and alpha 2M*. Murine peritoneal macrophages were prepared as described under "Experimental Procedures." After the addition of Fura-2/AM, coverslips containing adherent cells were mounted on a digital imaging microscope followed by the addition of ligands (20 nM). A, changes in intracellular calcium levels, [Ca2+]i, upon the addition of nonoxidized alpha 2M* (filled circle) or alpha 2M* that has been oxidized with 75 (open square) or 200 (filled triangle) µM hypochlorite. Boiled alpha 2M* (open circle) was added as a negative control. The arrow indicates the time of addition of ligands. B, changes in [Ca2+]i upon the addition of nonoxidized native alpha 2M (filled circle) or native alpha 2M that has been oxidized with 50 (open square) or 125 (filled square) µM hypochlorite. Boiled native alpha 2M (open circle) was added as a negative control.
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Reduced SDS-PAGE of Trypsin-treated Oxidized Native alpha 2M

To determine the mechanism responsible for the oxidative exposure of the receptor-recognition site of native alpha 2M for LRP, we performed a SDS-PAGE under reducing conditions of oxidized native alpha 2M that was treated with 20-fold molar excess of trypsin. We hypothesized that if the exposure of the receptor-recognition site is associated with the unfolding of the protein, then oxidized native alpha 2M should be more susceptible to digestion by trypsin. Fig. 7 shows that as the concentration of hypochlorite used to modify native alpha 2M increased, fewer large molecular weight protein bands remained, indicating that the oxidized protein is digested more efficiently by trypsin. Oxidation at up to 5 µM hypochlorite appeared to have no effect on the susceptibility of native alpha 2M toward trypsin. However, at oxidant concentrations greater than 5 µM, native alpha 2M 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 alpha 2M has been oxidized by at least an oxidant concentration of 5 µM. To investigate whether other mechanisms may account for the oxidative exposure of the receptor-binding site of native alpha 2M for LRP, we performed thioester bond titration of oxidized native alpha 2M. Thioester bond rupture results in exposure of the receptor-recognition site, analogous to the reaction of methylamine with native alpha 2M. 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 alpha 2M oxidation (32).


Fig. 7. Reduced SDS-PAGE of trypsin-treated native alpha 2M. Native alpha 2M oxidized at various concentrations of hypochlorite was incubated with 20-fold excess trypsin for 20 min at 37 °C. At the end of incubation, the reaction was stopped with suicide substrate p-nitrophenyl p'-guanidinobenzoate, and 20 µl of the mixture was loaded into each lane. Lane 1, molecular weight standard. Lane 2, trypsin-treated native alpha 2M. Lanes 3-10, trypsin-treat native alpha 2M that had been oxidized with 1.25 (lane 3), 1.5 (lane 4), 5 (lane 5), 12.5 (lane 6), 25 (lane 7), 37.5 (lane 8), 50 (lane 9), and 75 (lane 10) µM hypochlorite. The most prominent bands at 85 and 95 kDa represent fragments IVa and IVb from the cleavage of the alpha 2M bait region by trypsin. Other bands represent additional cleavages at sites other than the alpha 2M bait region.
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ELISA of Anti-RBF Antibodies against Oxidized Native alpha 2M

To further probe whether unfolding of the alpha 2M secondary structure may result in exposure of RBF, we performed an ELISA using polyclonal antisera raised against RBF. The carboxyl-terminal 20 kDa of alpha 2M 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 alpha 2M 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 alpha 2M was partially recognized by the anti-RBF antibodies whereas RBF and alpha 2M* were both fully recognized. Oxidized native alpha 2M was also fully recognized by anti-RBF antibodies; however, this occurred only after it was oxidized by at least 12.5 µM hypochlorite.


Fig. 8. ELISA of mouse anti-RBF polyclonal antisera against RBF, native alpha 2M, alpha 2M*, and oxidized native alpha 2M. Polyclonal antisera raised against RBF (filled bar) or normal mouse serum (open bar) were incubated in 96-well plates coated with either BSA (a), RBF (b), native alpha 2M (c), alpha 2M* (d), and native alpha 2M oxidized at 5 (e), 12.5 (f), 25 (g), and 50 (h) µM hypochlorite. Following incubation, plates were washed twice with PBS-Tween 20 to remove unbound proteins. Anti-mouse IgG antibody conjugated with horseradish peroxidase was then added to each well and allowed incubate for 1 h at 25 °C. Following incubation, o-phenylenediamine dihydrochloride substrate solution was added, and the plates were read at lambda 450 nm for 15 min. These data represent the mean and S.E. of two independent experiments performed in triplicate.
[View Larger Version of this Image (18K GIF file)]

Direct Binding of Oxidized Native alpha 2M and alpha 2M* to Macrophage Cell Surface

Since alpha 2M* has two different Kd and Bmax values, we performed a concentration-dependent binding experiment using oxidized 125I-BH-alpha 2M and 125I-BH-alpha 2M* to determine whether the binding of 125I-oxidized alpha 2M* to the signaling receptor corresponds with its binding to the high affinity site and whether the absence of binding by 125I-oxidized native alpha 2M corresponds with its binding to the low affinity site. Fig. 9A shows the results from direct binding experiments of 125I-oxidized alpha 2M* to macrophages. The Scatchard analysis (A, inset) shows that only a single class of high affinity (Kd ~ 83 pM) binding sites exist with a Bmax of ~ 5.4 fmol per million cells. These numbers were verified using Systat® analysis program (Kd - 71 ± 12 pM, Bmax - 6.2 ± 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 Kd or the Bmax. The binding of 125I-oxidized native alpha 2M to macrophages is shown in Fig. 9B. The Scatchard analysis (B, inset) shows a single class of lower affinity (Kd ~ 0.6 nM) binding sites with a Bmax of approximately 55 fmol/million cells. Analysis using Systat® shows good agreement with these numbers (Kd: 0.7 ± 0.15 nM; Bmax: 57 ± 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 alpha 2M together with the signal transduction studies described above provide direct evidence that the high affinity sites represent the signaling receptor.


Fig. 9. Direct binding of oxidized alpha 2M* and oxidized native alpha 2M to peritoneal macrophages. A, 125I-BH-alpha 2M* oxidized with 200 µM hypochlorite were added to each well at the indicated concentrations in the presence (open circle) or absence (closed circle) of 100-fold excess RAP and allowed to incubate for 16 h at 4 °C. Following incubation, cells were washed twice with ice-cold buffer and solubilized in SDS/NaOH before gamma -counting. The Scatchard plot of 125I-BH-alpha 2M* binding in the absence of RAP is presented in the inset. B, experiments were performed as in A except that 125I-BH-native alpha 2M oxidized with 125 µM of hypochlorite was added to each well at the indicated concentrations in the presence (open circle) or absence (closed circle) of RAP. The Scatchard plot of 125I-BH-native alpha 2M binding in the absence of RAP is presented in the inset. The binding curves represent fitted data to a single class of binding sites using the SYSTAT® program. Each data point represents the mean of two independent experiments performed in triplicate. The S.E. is less than ±12% at all points. Insets: B, bound; F, free.
[View Larger Version of this Image (12K GIF file)]


DISCUSSION

In this study we demonstrate that hypochlorite oxidation of native alpha 2M or alpha 2M* can generate exposure of the receptor binding sites to either LRP or the alpha 2M signaling receptor, respectively. Oxidation of alpha 2M* 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 alpha 2M 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 alpha 2M* 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 alpha 2M suggested that protein unfolding may be responsible for this phenomenon. These experiments provide strong proof for the existence of two distinct alpha 2M* receptors and the presence of two independent receptor-binding regions on alpha 2M*.

Our earlier studies using cis-DDP modification of alpha 2M* and RBF have shown that the cis-DDP-sensitive site in alpha 2M* 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 alpha 2M* 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 alpha 2M* 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 alpha 2M (31, 32). Treatment of native alpha 2M with 25 µM hypochlorite resulted in complete destruction of its anti-proteinase activity. We hypothesized that hypochlorite could also inhibit the ability of alpha 2M* to bind to cell surface receptors. In this study, we show that hypochlorite treatment completely eliminated the RAP-sensitive binding of alpha 2M* 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 alpha 2M* to the low affinity sites but is unable to inhibit the ability of alpha 2M* 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 alpha 2M 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 alpha 2M* signaling receptors.

The oxidative exposure of the alpha 2M-binding site to LRP but not to the signaling receptor, is unique in a number of ways. All of the known naturally occurring alpha -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 alpha 2M 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 alpha 2M* for the signaling receptor is distinct from the LRP binding site. That hypochlorite oxidation can selectively expose only the LRP binding sites in native alpha 2M or the signaling receptor binding sites in alpha 2M* demonstrates that the ability of alpha 2M* to bind to its two receptors can be uncoupled. Efforts are currently being made using oxidized alpha 2M* to isolate and purify the signaling receptor.

Our investigation of the mechanism that may explain the oxidative exposure of LRP binding site in native alpha 2M suggests that partial protein unfolding may be responsible. Earlier works by Davies et al. (47-50) 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 alpha 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 alpha 2M 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 alpha 2M that has been labeled with Bolton-Hunter reagent, which generates LRP binding sites with as little as 25 µM of hypochlorite but does not peak until 125 µM. At hypochlorite concentrations greater than 125 µM, oxidized alpha 2M 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 alpha 2M has similar susceptibility to oxidation as unlabeled alpha 2M, whereas IODO-BEADS®-labeled alpha 2M is significantly more susceptible to oxidation. This suggests that receptor binding studies with alpha 2M should use the Bolton-Hunter labeling method to minimize protein oxidation.

The selective exposure of the LRP binding site in oxidized alpha 2M 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 alpha 2M is associated with an increase in the exposure of RBF. We found that oxidation of alpha 2M 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 alpha 2M* 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 alpha 2M* even when the protein is treated with 200 µM hypochlorite suggests otherwise.

Oxidative inactivation of alpha 2M* receptor binding to LRP suggests an interesting pathophysiological process that may occur during inflammation. alpha 2M 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, 53-55). In the presence of oxidants alpha 2M 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 alpha 2M* signaling has been associated with increased production of prostaglandins and platelet-activating factor as well as increased mitogenesis in vascular smooth muscle cells (17-20). alpha 2M 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 (40, 51) and that oxidized alpha 2M* can be isolated from inflammatory lesions in humans (56). Further investigation of the ability of alpha 2M 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.


FOOTNOTES

*   This work was supported by NHLBI Grant HL-24066.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pathology, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-3528; Fax: 919-684-8689.
1   The abbreviations used are: alpha 2M, alpha 2-macroglobulin; alpha 2M*, the receptor-recognized form of alpha 2M; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; RBF, carboxyl-terminal receptor binding fragment of alpha 2M; 125I-BH-alpha 2M, 125I-Bolton-Hunter-labeled alpha 2M; 125I-BH-alpha 2M*, 125I-Bolton-Hunter-labeled alpha 2M*; 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.

ACKNOWLEDGEMENTS

We thank Tammy Moser and Drs. George Cianciolo, Hanne Grøn, and Yizhi Liang for their comments on the manuscript and Marie Thomas for her assistance with preparation of the manuscript.


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