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J. Biol. Chem., Vol. 277, Issue 15, 13338-13345, April 12, 2002

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Distinct Binding Sites in the Structure of ₂-Macroglobulin Mediate the Interaction with -Amyloid Peptide and Growth Factors^*

Joseph M. Mettenburg, Donna J. Webb^§, and Steven L. Gonias^¶

From the Departments of  Biochemistry and Molecular Genetics, ^§ Cell Biology, and ^¶ Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received for publication, July 18, 2001, and in revised form, December 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

₂-Macroglobulin (₂M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of -amyloid peptide (A). In this study, we demonstrate that A binds selectively to ₂M that has been induced to undergo conformational change by reaction with methylamine. Denatured ₂M subunits, which were immobilized on polyvinylidene difluoride membranes, bound A, suggesting that ₂M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in ₂M is responsible for A binding, we prepared and analyzed defined ₂M fragments and glutathione S-transferase-₂M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314-1365, was identified as the only major A-binding site. Importantly, A did not bind to the previously characterized growth factor-binding site (aa 718-734). Although the A binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of A did not inhibit LRP-mediated clearance of ₂M-MA in mice. Using various methods, we determined that the K_D for the interaction of A with its binding site in the individual ₂M subunit is 0.7-2.4 µM. The capacity of ₂M to bind A and deliver it to LRP may be greater than that predicted by the K_D, because each ₂M subunit may bind A and the bound A may multimerize. These studies suggest a model in which ₂M has three protein interaction sites with distinct specificities, mediating the interaction with A, growth factors, and LRP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Accumulation of -amyloid peptide (A_(1-40) and A_(1-42))¹ in the brain plays a central role in the development and progression of Alzheimer's disease (AD) (1). Mutations in -amyloid precursor protein (APP), which result in increased production of A, are associated with autosomal dominant forms of familial AD in humans (2-4). Mutated forms of human APP may also induce changes consistent with AD when expressed as transgenes in mice (5-8). Furthermore, immunization with A_(1-42) prevents progression of AD in animal model systems and may reverse symptoms by promoting resorption of A-containing plaques (9, 10). These results suggest that A accumulation in the brain is a dynamic and reversible process. Proteins other than antibodies with the capacity to bind A and promote its catabolism may influence disease progression.

₂-Macroglobulin (₂M) is a 718-kDa homotetrameric glycoprotein, which is well characterized as an extracellular proteinase inhibitor (11) and as a carrier of specific growth factors, including transforming growth factor- (TGF-) and nerve growth factor- (NGF-) (12, 13). At least two separate polymorphisms in the A2M gene may be associated with increased risk of late-onset AD. The first involves a region within intron 17, at the 5' splice acceptor site for exon 18 (14). This exon is important because it encodes part of the bait region, where proteinases initiate reaction with ₂M by cleaving susceptible peptide bonds (15, 16), and a segment of the growth factor binding sequence (17-19). In the second A2M gene polymorphism, Val-1000 is replaced by Ile (20). The linkage of A2M gene polymorphisms to late-onset AD remains incompletely understood, because the original observations have been confirmed in only a limited number of populations (21-25) and because there is no molecular explanation regarding how A2M gene mutations may affect ₂M structure, function, and expression.

₂M is expressed by microglia, which accumulate near amyloid plaques (26). Thus, locally synthesized ₂M may affect AD progression by regulating the activity of various proteinases or by binding important growth factors. The previously demonstrated ability of ₂M to bind and neutralize the activity of TGF- (12, 13, 27-29) may be detrimental in AD, because TGF- stimulates A clearance by microglial cells and reduces A accumulation in the brain parenchyma of mice that overexpress human APP (30). Furthermore, TGF- has been reported to antagonize the cytotoxic activity of A (29, 31, 32).

Another mechanism whereby ₂M may regulate AD progression involves its ability to bind A, forming a complex that is internalized by the ₂M receptor, low density lipoprotein receptor-related protein (LRP) and then degraded (33-35). Du et al. (36) originally reported that A_(1-40) and A_(1-42) bind to native ₂M and to ₂M that has been transformed into the LRP-recognized or "activated" conformation by reaction with methylamine (₂M-MA). Narita et al. (33) subsequently reported selective binding of A_(1-40) and A_(1-42) to the activated conformation of ₂M. ₂M-MA apparently binds A_(1-40) and A_(1-42) with equivalent affinity (33). Hughes et al. (37) executed a yeast two-hybrid screen using A_(1-42) as bait and identified a 250-amino acid peptide from the C terminus of ₂M as a strong and specific interactor. The same group also reported experiments confirming the interaction of A with intact ₂M; however, they did not demonstrate that the sequence identified by yeast-two hybrid screen is responsible for the binding of A to intact ₂M.

The growth factor binding site in ₂M is contained within a 16-amino acid peptide located ~500 amino acids N-terminal to the A-binding site identified by yeast-two hybrid screen (19). The growth factor binding sequence is composed mainly of hydrophobic amino acids with two potentially important acidic residues. TGF-, platelet-derived growth factor-BB (PDGF-BB), and NGF- all interact with the growth factor-binding site in ₂M (18, 19), despite the fact that these proteins demonstrate limited sequence identity. Based on this promiscuous behavior, we hypothesized that the growth factor-binding site in ₂M may also function as an A-binding site.

To test our hypothesis, we undertook a comprehensive molecular analysis to identify sequences in ₂M with A binding activity. Our results demonstrate that a single sequence, located near the C terminus of the ₂M subunit, constitutes the only significant A-binding site. Importantly, this sequence is entirely distinct from the growth factor-binding site. The LRP recognition sequence is also located near the C terminus of the ₂M subunit (38-42); however, our evidence indicates that the LRP recognition site and the A binding sequence are distinct. Thus, in addition to the bait region, the ₂M subunit has at least three distinct "protein interaction sites" with distinct binding specificities. These sites mediate interactions with growth factors, A and LRP.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Proteins and Reagents-- ₂M was purified from human plasma by the method of Imber and Pizzo (43). ₂M-MA was prepared by dialyzing ₂M against 200 mM methylamine-HCl in 50 mM Tris-HCl, pH 8.2, for 12 h at 22 °C and then exhaustively against 20 mM sodium phosphate, 150 mM NaCl, pH 7.4. Modification of ₂M by methylamine was confirmed by demonstrating the characteristic increase in ₂M electrophoretic mobility by non-denaturing PAGE (15). ₂M-MA was radioiodinated using IODO-BEADs (Pierce) and stored at 4 °C for no more than 2 weeks. The specific activity was 0.5-1.0 µCi/µg. Receptor-associated protein (RAP), which blocks binding of ₂M-MA to LRP (65), was expressed as a glutathione S-transferase (GST) fusion protein in bacteria and purified by chromatography on glutathione-Sepharose. A_(1-40) was purchased from Bachem and radioiodinated using ¹²⁵I-labeled Bolton-Hunter reagent (di-iodinated, PerkinElmer Life Sciences). Biotinylated A_(1-40) was prepared by reacting A_(1-40) with 4 µM sulfo-N-hydroxysuccinimide biotin (Pierce) for 2 h at 4 °C in siliconized tubes. The reaction mixture was dialyzed extensively against water. Biotinylated A was stored for up to 1 month at 4 °C or frozen at 80 °C and thawed once without affecting its ability to bind to ₂M. GST-specific IgG, bovine serum albumin (BSA, greater than 99% pure), dithiothreitol (DTT), and iodoacetamide were from Sigma Chemical Co. Bis(sulfosuccinimidyl) suberate (BS³) and horseradish peroxidase-conjugated avidin were from Pierce. Polyclonal A-specific rabbit antibody was from Zymed Laboratories Inc.

Methods for Defined Fragmentation of ₂M-- When ₂M is treated with papain under mildly acidic conditions, an 18-kDa fragment is released from the C terminus of each ₂M subunit (aa 1314-1451) (38). The 18-kDa fragment includes the intact receptor-binding site and is thus referred to as the receptor binding fragment (RBF). The residual 600-kDa ₂M remnant retains the major structural features of the parent molecule (45). To obtain the 18- and 600-kDa ₂M fragments, 4.0 µM ₂M-MA was treated with 2.4 µM papain in 50 mM sodium acetate, 1 mM cysteine, pH 5.0, for 20 h at 22 °C. The pH of the reaction mixture was increased to 7.4, and the products were purified by molecular exclusion chromatography on Ultrogel AcA-22.

Each ₂M subunit has a single thiol ester bond formed by the side chains of Cys-949 and Gln-952 (11, 46, 47). When ₂M is heated in the presence of SDS, the thiol esters react internally, and, as a result, the ₂M peptide backbone is cleaved (47, 48). The products include a 120-kDa N-terminal heat fragment and a 60-kDa C-terminal heat fragment. To produce ₂M heat fragments, native ₂M was incubated at 100 °C in 2% SDS (w/v) and 14 mM DTT for the indicated periods of time. The products were treated with iodoacetamide (70 mM) and subjected to SDS-PAGE.

₂M-peptide-GST Fusion Proteins-- Six previously described fusion proteins, which collectively encode amino acids 99-1451 of the human ₂M sequence, were expressed in BL-21 cells (17, 18). These fusion proteins include: FP1 (aa 99-392), FP2 (aa 341-590), FP3 (aa 591-774), FP4 (aa 775-1059), FP5 (aa 1030-1279), and FP6 (aa 1242-1451). Constructs encoding new GST fusion proteins, including FP6a (aa 1242-1365), FP6b (aa 1242-1400), and FP6c (aa 1365-1451), were generated using PCR and the intact A2M cDNA in pBluescript as a template. The oligonucleotides included recognition sequences for BamHI and EcoRI, to allow direct cloning into the vector, pGEX-2T. Final constructs were subjected to sequence analysis to verify proper orientation and reading frame.

Two constructs, labeled FP6d, correspond in sequence exactly to the 18-kDa RBF (aa 1314-1451). In FP6d-AA, Lys residues at aa 1370 and 1374, which are critical for LRP binding (39, 40), were mutated to Ala, using the QuikChange system (Stratagene). In FP6d-AR, Lys-1370 was mutated to Ala, and Lys-1374 was mutated to Arg.

All of the fusion proteins were partially purified from induced bacterial suspensions by selective detergent extraction, as previously described (17). The resulting preparations yielded clearly defined bands, with the correct molecular masses when assessed by Coomassie Blue staining of SDS gels or immunoblot analysis with GST-specific antibody. FP3, FP6, and FP6d-AA were purified to homogeneity by chromatography on glutathione-Sepharose. Fig. 1 shows the sequences of the GST fusion proteins used in this investigation.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Sequences of GST-2M peptide fusion proteins. The 18-kDa RBF was purified after proteolytic dissociation from intact 2M-MA. All of the other GST fusion proteins were expressed in bacteria. The mutations in FP6d are in amino acids 1370 and 1374. The vertical lines show the relationship between the sequences of the fusion proteins and the amino acids that constitute the  helix, which is the center of the LRP recognition sequence in 2M.

Non-denaturing PAGE Analysis of A Binding to ₂M-- ¹²⁵I-A (2.5 nM) was incubated with native ₂M, ₂M-MA, or the purified 600-kDa fragment (0.3-1.0 µM) in 20 mM sodium phosphate, 150 mM NaCl, pH 7.4, for 2 h at 37 °C. In some experiments, increasing concentrations of the 18-kDa RBF (0.2-2.8 µM) were co-incubated with ¹²⁵I-A and ₂M-MA. Reaction mixtures were subjected to non-denaturing PAGE, using the buffer system described by Van Leuven et al. (49). ¹²⁵I-A binding to ₂M was detected as radioactivity co-migrating with the ₂M band. In control experiments, free ¹²⁵I-A did not migrate near ₂M. To quantitate ¹²⁵I-A binding to ₂M, gels were subjected to PhosphorImager analysis using ImageQuant software. Non-denaturing PAGE preserves non-covalent interactions; however, the amount of binding detected may be influenced by dissociation of protein complexes during electrophoresis (13).

Determination of Apparent Equilibrium Dissociation Constants-- Because A binding to ₂M is reversible and probably subject to rapid dissociation when methods such as non-denaturing PAGE or chromatography are used, we utilized the BS³ rapid cross-linking method to determine the apparent K_D for the binding of A to ₂M-MA. This method has been used previously to determine K_D values for the interaction of ₂M with multiple growth factors and cytokines (12, 13, 50).

Increasing concentrations of ₂M-MA were incubated with 25 nM ¹²⁵I-A for 2 h at 37 °C. Freshly dissolved BS³ (5 mM) or vehicle (H₂O) was then added for 5 min. Cross-linking reactions were quickly terminated by rapid acidification, followed by transfer to buffered SDS. Under pseudo-first order conditions, a constant fraction of the non-covalent ¹²⁵I-A·₂M-MA complex is covalently stabilized by the BS³ (13). To quantitate the amount of covalently stabilized complex, BS³-treated and vehicle-treated samples were subjected to SDS-PAGE. ¹²⁵I-A that was covalently cross-linked to ₂M-MA (bound) and free ¹²⁵I-A (free), which includes free A and A that was bound to ₂M-MA but not cross-linked, were quantitated by PhosphorImager analysis. Results were analyzed according to the following equation (12),

(Eq. 1)

The cross-linking efficiency, z, is a constant, derived from the y intercept, for each set of proteins and conditions (12). z is referred to as the BS³-cross-linking efficiency but may also be affected if a fraction of the radioiodinated protein is incapable of binding to the ₂M. The apparent K_D was determined from the slope when free/bound was plotted against 1/[₂M-MA]. This value is based on the assumption that there is a single binding site for A in ₂M. Assuming one A-binding site/₂M subunit, as suggested by our data, then the K_D must be corrected by multiplying the apparent K_D by a factor of four.

Ligand Blotting-- This method has been previously used to demonstrate specific and saturable binding of growth factors to denatured ₂M subunits, ₂M fragments, and GST-₂M-peptide fusion proteins (17-19). Protein preparations were denatured in 2% SDS or treated with 1 mM DTT in 2% SDS and then with 5 mM iodoacetamide for 2 h, as previously described (17). Samples were then subjected to SDS-PAGE and electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% milk in 20 mM sodium phosphate, 150 mM NaCl, 0.1% Tween 20, pH 7.4, and probed for 2 h with ¹²⁵I-A or biotinylated-A. ¹²⁵I-A ligand blots were washed and subjected to PhosphorImager analysis. Biotinylated A ligand blots were probed with horseradish peroxidase-conjugated avidin (1:5000 dilution). The membranes were then subjected to enhanced chemiluminescence (ECL) and densitometry. Equivalent loading and transfer of proteins were demonstrated by Coomassie Blue staining or, when applicable, by immunoblot analysis with GST-specific antibody (17).

A-peptide Immunoblotting-- Denatured ₂M subunits and BSA were treated with 1 mM DTT and then with 5 mM iodoacetamide to block free sulfhydryl groups, subjected to SDS-PAGE, and electrotransferred to PVDF. The membranes were blocked with 5% milk. Unlabeled A_(1-40) was incubated with the immobilized ₂M and BSA in PBS-T at 37 °C for 2 h. The membranes were then washed extensively and probed with rabbit A-specific IgG (1:4000) in PBS-T and 0.1% milk (v/v), followed by anti-rabbit IgG-horseradish peroxidase conjugate (1:10,000). Membranes were analyzed by ECL and densitometry.

Plasma Clearance Experiments in Mice-- ¹²⁵I-₂M-MA (20 nM) was incubated with 20 µM A or with vehicle for 2 h at 37 °C. The ¹²⁵I-₂M-MA (0.3 µCi) was then injected, in the presence and absence of GST-RAP (40 or 80 µg), into the lateral tail veins of CD-1 female mice (30 g). Blood samples (40 µl) were withdrawn from the retro-orbital venous plexus, using heparinized capillary tubes, at the designated times (0.5-30 min). The radioactivity in each sample was determined using a gamma counter and expressed as a fraction of that present in the 0.5-min time point.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A Binding to ₂M Is ₂M Conformation-dependent-- A_(1-40) and A_(1-42) function differently in the initiation and progression of AD; however, both forms of A bind to ₂M equivalently (33). Thus, we conducted our analysis of A binding to ₂M and its derivatives using one form of A (A_(1-40)). To determine whether A binding to ₂M is ₂M conformation-specific, as has been demonstrated with growth factors (12, 13), ¹²⁵I-A (2.5 nM) was incubated with native ₂M or ₂M-MA (each at 0.3 µM) in solution. The products were analyzed by non-denaturing PAGE, which preserves non-covalent interactions. As shown in Fig. 2A, ¹²⁵I-A bound to ₂M-MA, whereas binding was not detected with native ₂M. Free ¹²⁵I-A migrated near the dye front. These results suggest that A binds selectively to ₂M that has undergone conformational change.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   2M conformation dependence of A binding. A, 125I-A was incubated in buffer alone, with native 2M, or with 2M-MA (0.3 µM) for 2 h at 37 °C and then subjected to non-denaturing PAGE. The gel was dried and stained. 2M-associated 125I-A was detected by PhosphorImager analysis. Free 125I-A migrated near the dye front (not shown). B, increasing concentrations of 2M-MA were incubated with 25 nM 125I-A for 2 h at 37 °C. Samples were treated with BS3 and subjected to SDS-PAGE. BS3-stabilized 125I-A·2M-MA complex (bound) and 125I-A that was not cross-linked to 2M-MA (free) were determined and plotted against the reciprocal of the 2M-MA concentration.

To estimate the K_D for A binding to ₂M-MA, we used the BS³-rapid cross-linking method, which has been used extensively to determine binding affinities for ₂M and growth factors (12, 13, 50). A major advantage of this method is that it is not necessary to resolve free and bound A, which typically involves the use of steps, such as chromatography or PAGE, that promote dissociation of non-covalent protein complexes. A representative study in which ¹²⁵I-A was incubated with increasing concentrations of ₂M-MA is shown in Fig. 2B. The exact fraction of the non-covalent ₂M·A complex, which was cross-linked by BS³ (z), was determined from the y intercept, as previously described (12). In three separate experiments, z = 0.06-0.14, compared with z values that are typically in the range of 0.15-0.40 for the binding of growth factors to ₂M (12). z may be decreased if a fraction of the ¹²⁵I-A was incapable of binding to ₂M-MA or if the A, which bound to ₂M-MA, multimerized so that individual A monomers could not be cross-linked to the ₂M-MA. Neither of these effects would be expected to influence the calculated apparent K_D.

The apparent K_D for the binding of A to ₂M-MA was 0.29 ± 0.02 µM (n = 3). This value is based on the assumption that each molecule of ₂M has one binding site for A. If each ₂M subunit has a distinct A-binding site, as the evidence to be presented will suggest, then the K_D for the binding of A to the individual binding site is 1.2 µM. Although this is a low affinity interaction, due to the homotetrameric structure of ₂M, the plasma concentration of ₂M subunits is 12-20 µM (11).

Binding of ¹²⁵I-A to Denatured ₂M Subunits-- Native ₂M was denatured in SDS and DTT and treated with iodoacetamide. A similar protocol was executed with ₂M-MA and with BSA. The preparations were then subjected to SDS-PAGE. Coomassie Blue staining revealed the 180-kDa ₂M subunit as the major band in both the native ₂M and ₂M-MA preparations, as anticipated (Fig. 3). Faint bands with apparent masses of 120 and 60 kDa were observed in the native ₂M lane. These bands correspond to the ₂M heat fragmentation products that result from an internal reaction involving the thiol ester bonds at 100 °C, as previously described (48). ₂M-MA does not undergo heat fragmentation, because the thiol esters have already undergone aminolysis (46, 47).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Ligand blot analysis of A binding to 2M. Native 2M, 2M-MA, and BSA were denatured in the presence of reductant and treated with iodoacetamide. The samples were then subjected to SDS-PAGE and electrotransferred to PVDF membranes. Some membranes were stained with Coomassie Blue. Other membranes were blocked with 5% milk and then probed with 125I-A, unlabeled A, or biotinylated A. 125I-A was detected by PhosphorImager analysis. Unlabeled A was detected by immunoblot analysis. Biotinylated A was detected with horseradish peroxidase-conjugated avidin and ECL.

¹²⁵I-A bound to denatured ₂M subunits that were electrotransferred to PVDF membranes. No difference in ¹²⁵I-A binding was observed with native ₂M and ₂M-MA, as was anticipated, because the difference in structure between these two forms of ₂M is mainly conformational. ¹²⁵I-A did not bind to BSA, suggesting that the interaction with ₂M is specific. The interaction of ¹²⁵I-A with ₂M, in the ligand blotting system, suggests that the individual ₂M subunit binds A and that ₂M tertiary and quaternary structure are not necessary. In this respect, A binding to ₂M resembles the interaction observed with growth factors (17) but not with proteinases (11, 15).

To confirm that the interaction of A with ₂M was not dependent on an unanticipated modification occurring during A radioiodination, we developed alternative methods for detecting A binding to PVDF-immobilized ₂M subunits. In the first protocol, unlabeled A was used to probe the PVDF membranes. ₂M-associated A was then detected by immunoblot analysis. In the second protocol, biotinylated A was substituted for ¹²⁵I-A. In both cases, binding of A to PVDF-immobilized native ₂M and ₂M-MA was detected whereas A binding to BSA was not.

A Binding to ₂M Heat Fragments-- When ₂M is heated in the presence of denaturant, the thiol ester bonds, which are formed from the side chains of Cys-949 and Gln-952, react internally with the ₂M polypeptide backbone, causing scission of the ₂M subunit at residue 952 (46-48). The N-terminal 120-kDa fragment includes the bait region and the growth factor binding sequence. The C-terminal 60-kDa fragment includes the LRP recognition sequence (38-42) and the region identified by Hughes et al. (37) as a candidate A-binding site. To determine whether A binding activity is localized to either or both of these denatured ₂M fragments, ₂M was subjected to heat fragmentation and analyzed by ¹²⁵I-A-ligand blotting. Only the 60-kDa ₂M heat fragment bound ¹²⁵I-A (Fig. 4). The 120-kDa ₂M heat fragment was without activity. This result provides evidence that a specific sequence is responsible for the interaction of ₂M with A. Furthermore, this result suggests that the ₂M growth factor-binding site and the A-binding site are distinct.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Binding of 125I-A to 2M heat fragments. Native 2M was heated at 100 °C, in the presence of SDS, for 5 or 30 min and subjected to ligand blot analysis with 125I-A (bottom panel). As a control, unheated native 2M and BSA were studied simultaneously. Equivalent PVDF membranes were stained with Coomassie Blue (top panel).

The 18-kDa RBF Competes with ₂M-MA for A Binding-- A second method for defined fragmentation of ₂M involves papain treatment of the activated conformation under mildly acidic conditions. An 18-kDa fragment, which retains LRP binding activity, is dissociated from the C terminus of each ₂M subunit (aa 1314-1451) (38-40). The residual 600-kDa fragment retains the major structural characteristics of ₂M-MA, as determined by electron microscopy (45). The 18- and 600-kDa ₂M fragments were purified and assessed for their ability to bind A without prior denaturation. When ¹²⁵I-A was incubated with the 600-kDa fragment, in solution, binding was not detected by non-denaturing PAGE (Fig. 5A). Under equivalent conditions, binding was readily detected with intact ₂M-MA.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   A binding to 2M fragments derived by papain treatment. The 600- and 18-kDa papain fragments of 2M-MA were prepared and purified. A, 125I-A was incubated with 2M-MA or the 600-kDa fragment (1.0 µM), under non-denaturing conditions, for 2 h at 37 °C. The samples were subjected to non-denaturing PAGE. 125I-A was detected by PhosphorImager analysis. B, 125I-A was incubated with 2M-MA (0.3 µM) and increasing concentrations of the 18-kDa RBF for 2 h at 37 °C. The samples were then subjected to non-denaturing PAGE and PhosphorImager analysis. The figure shows the fraction of the 125I-A that was associated with 2M-MA, compared with the amount observed in the absence of 18-kDa RBF.

In separate experiments, 2.5 nM ¹²⁵I-A was incubated with 0.3 µM ₂M-MA and increasing concentrations of purified 18-kDa RBF in solution. ¹²⁵I-A binding to ₂M-MA was decreased in the presence of the 18-kDa RBF, and the magnitude of the effect was dependent on the RBF concentration (Fig. 5B). The IC₅₀ was 0.7 µM. Because of the relatively high concentration of ₂M-MA, the K_D for A binding to the 18-kDa RBF was ~2-fold lower (0.3-0.4 µM) than the IC₅₀. These results provide further evidence that an A-binding site is localized near the C terminus of the ₂M subunit and that this site is distinct from the growth factor-binding sequence.

A Binding to GST-₂M-peptide Fusion Proteins-- To comprehensively analyze the ₂M sequence with regard to A binding, we utilized ligand blotting to screen a series of six previously described ₂M-peptide-GST fusion proteins (FP1-FP6) (18). In the intact ₂M subunit, the bait region and growth factor-binding site are located in FP3. The residues that comprise the thiol ester bond are located in FP4, and the LRP recognition sequence is in FP6. To assess A binding, the fusion proteins were treated with iodoacetamide, without prior reduction, and subjected to SDS-PAGE. After electrotransfer to PVDF, only FP6 bound ¹²⁵I-A (Fig. 6A).

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Ligand blot analysis of A binding to GST-2M fusion proteins. A, PVDF membranes with FP1-FP6, which had been denatured in the absence of reductant and treated with iodoacetamide, were probed with 125I-A. B, a representative study in which FP3 and FP6 were denatured in the presence of DTT, treated with iodoacetamide, and subjected to ligand blot analysis with 125I-A. C, FP3, FP4, and FP6 were denatured in 2% SDS and DTT, treated with iodoacetamide, subjected to SDS-PAGE, and electrotransferred to PVDF membranes. The membranes were probed with biotinylated A or subjected to immunoblot analysis with GST-specific antibody.

Because intact disulfide bonds may allow partial restoration of non-denatured structure following protein electrotransfer to PVDF membranes, ligand-blotting experiments were also performed using FP3 and FP6 that were reduced with DTT and then alkylated with iodoacetamide. In these experiments, ¹²⁵I-A binding was still detected only with FP6 and not with FP3 (Fig. 6B). Equivalent results were obtained when biotinylated A was substituted for ¹²⁵I-A (Fig. 6C). Based on these results, a model emerges in which the structure of ₂M includes at least two distinct protein interaction sites with differing specificity. A site located near the center of the ₂M subunit is responsible for the binding of growth factors whereas a separate site near the C terminus is exclusively responsible for the binding of A.

Resolution of the LRP- and A-binding Sites in ₂M-- In intact human ₂M, aa 1370-1377 constitute an  helix that is the center of the LRP recognition site (41, 42). The  helix is anchored in position by a  sandwich so that the side chains of two critical Lys residues (aa 1370 and 1374) protrude at 45° angles and are surrounded by hydrophobic surface residues (41). This complex secondary and tertiary structure may explain why the 18-kDa RBF is recognized by LRP, whereas tryptic peptides corresponding to the same region and partially denatured forms of the 18-kDa RBF are not (38, 41).

Because our ligand blotting results demonstrated that tertiary structure is not necessary for A binding to FP6, we generated a new set of fusion proteins to explore the relationship between the LRP- and A-binding sites in ₂M. FP6c included all of the amino acids that form the LRP recognition  helix, five amino acids N-terminal to the  helix and the entire sequence C-terminal to the  helix; however, FP6c did not bind A (Fig. 7). FP6a, which included the N-terminal segment of FP6 but terminated five amino acids before the start of the  helix, bound A, albeit at lower levels than FP6. These results suggest that the A-binding site is located N-terminal to the LRP binding  helix. FP6b, which was equivalent to FP6a but extended through the  helix to aa 1400, bound slightly increased levels of A, suggesting that amino acids 1365-1400 may impact positively on this interaction; however, ligand blotting results provide only an approximation of differences in binding affinity.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Resolution of the A- and LRP-binding sites in the 2M subunit. A, fusion proteins containing amino acids that constitute the LRP recognition site and other fusion proteins that do not were subjected to ligand blot analysis with 125I-A. As a control for load, blots were stained with Coomassie Blue or subjected to immunoblot analysis with GST-specific antibody. B, the results of at least eight separate experiments with each fusion protein were pooled. In each case, 125I-A binding to a fusion protein was standardized against that observed with FP6 (mean ± S.D.).

Fusion proteins, which correspond exactly to the sequence of the 18-kDa RBF, were generated and mutated to essentially eliminate the LRP-binding site (FP6d-AA) (39) or substantially reduce binding to LRP while eliminating binding to the previously described ₂M signaling receptor (FP6d-AR) (40). By ligand blotting, both forms of FP6d retained A binding activity, supporting our hypothesis that the A and ₂M receptor recognition sequences in ₂M are non-identical. A slight decrease in the binding of A to FP6-AA and FP6-AR, compared with FP6, may indicate that the mutated Lys residues, although non-essential, impact positively on the interaction.

Determination of the K_D for A Binding to FP6-- In the ligand blotting experiments, the concentration of ¹²⁵I-A, used as probe, was substantially lower than the likely K_D value for ¹²⁵I-A binding to any of the fusion proteins. Thus, assuming equivalent load and insignificant contributions from "low affinity" or "nonspecific" binding sites, the amount of binding observed is inversely proportional to the K_D for each interaction. To more accurately assess the binding affinity of A for FP6 and FP6d-AA, specific binding experiments were performed.

FP6 and FP6-AA were purified to homogeneity; however, unlike intact ₂M-MA and the 18-kDa RBF, the fusion proteins did not bind A in solution, even when refolding protocols were executed. Possible explanations for this observation are provided under "Discussion." As an alternative approach, we immobilized purified FP6, FP6d-AA, and FP3 on PVDF after exposure to SDS and probed the membranes with ¹²⁵I-A (0.1 µM) and increasing concentrations of unlabeled A. Nonspecific binding was defined by the level of ¹²⁵I-A binding observed in the presence of 30 µM unlabeled A. As shown in Fig. 8, specific binding of ¹²⁵I-A to both FP6 and FP6d-AA was detected. The K_D values were 2.4 ± 0.8 and 5.2 ± 0.6 µM, respectively (mean ± S.E., n = 3 with internal triplicate replicates). The B_max, which is not an informative value when this method is used, was 20-25% higher with FP6. Importantly, the binding of ¹²⁵I-A to FP3 was entirely nonspecific.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Specific binding isotherms for the interaction of A with FP6 and FP6d-AA. Multiple samples, containing the equivalent amount of FP6, FP6d-AA, or FP3, were subjected to SDS-PAGE and electrotransferred to PVDF membranes. The membranes were incubated with 125I-A and increasing concentrations of unlabeled A. A binding was determined by PhosphorImager analysis. The results of three separate experiments with triplicate replicates were averaged to generate the curves that are shown. Specific binding to FP6 () or FP6d-AA () is plotted as a function of the total A concentration. A did not demonstrate specific binding to FP3.

Effects of A on ₂M Recognition by LRP-- Others have demonstrated that LRP mediates cellular uptake and degradation of A by binding A·₂M complexes (33-35). Because of the close proximity of the A and LRP recognition sequences in ₂M, we considered the possibility that A might inhibit binding of ₂M to LRP even though the binding sites are non-identical. Each ₂M tetramer has four independent LRP recognition sequences (38). Thus, even if some of the LRP-binding sites were blocked by A, receptor recognition may still occur. To address this question, we examined the plasma clearance of ₂M-MA in mice. In this well characterized system, conformationally modified forms of ₂M, such as ₂M-MA, clear from the plasma as a first-order process with a t_1/2 of 3-5 min, and clearance competition is observed when ¹²⁵I-₂M-MA is injected in the presence of excess unlabeled ₂M-MA (51). The rapid plasma clearance of ₂M-MA represented a clear advantage for our experiments with A, compared with binding or endocytosis experiments performed in vitro, because of the opportunity to minimize the time period during which dissociation of A·₂M-MA complex may occur.

Fig. 9 shows the plasma clearance of ¹²⁵I-₂M-MA (n = 4) in the absence of competing ligand and in the presence of 40 or 80 µg of GST-RAP. The GST-RAP inhibited the clearance of ¹²⁵I-₂M-MA from the plasma, as anticipated due to competition for plasma-accessible LRP, which is mainly located in the liver (51). To determine whether A inhibits ₂M-MA binding to LRP, ¹²⁵I-₂M-MA was preincubated with a nearly saturating concentration of A (20 µM, 16-fold the K_D) for 2 h at 37 °C and then injected intravenously in mice. The rate of ¹²⁵I-₂M-MA clearance was completely unchanged. We cannot rule out the possibility that A dissociated from the ¹²⁵I-₂M-MA after the preparation was injected intravascularly, due to dilution in the bloodstream; however, given the rapid timeframe of the plasma clearance experiments, our results demonstrate that ₂M-associated A either does not interfere with LRP recognition or rapidly dissociates from some sites to free up LRP recognition sequences and allow uptake of the remaining A.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Plasma clearance of 125I-2M-MA after incubation with A. 125I-2M-MA (20 nM) was incubated with 20 µM A () or with vehicle () for 2 h and then injected intravascularly in mice (n = 4). 125I-2M-MA that was not preincubated with A was injected in the presence of 40 µg of GST-RAP () or 80 µg of GST-RAP () (average of duplicate determinations with each concentration of GST-RAP).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

LRP and its ligands, ₂M, apolipoprotein E4, and Kunitz-proteinase inhibitor domain-containing isoforms of APP, form an intriguing functional family, the members of which have been implicated in familial or late-onset AD (52). Deciphering the mechanisms whereby these proteins affect AD may be difficult because of their multifunctional nature. For example, LRP may mediate the clearance of A in association with ₂M and may be essential for A transport across the blood-brain barrier (53). However, LRP may also mediate the transfer of APP into intracellular compartments where there is increased access to amyloidogenic proteinases (54).

Like LRP, ₂M expresses multiple activities that may be involved in AD progression. Because ₂M is a broad-spectrum proteinase inhibitor, which reacts with proteinases from all four major mechanistic classes (15, 55), various proteinases that are involved in A catabolism, such as neprilysin, insulysin, and plasmin (56-59), may be ₂M targets. The possibility that ₂M regulates the activity of proteinases involved in APP processing has also been considered; however, De Strooper et al. (60) demonstrated that this does not occur. The lack of an effect of ₂M on APP processing is consistent with other studies demonstrating that ₂M is poorly reactive with proteinases functioning at or near the cell surface (61).

The ability of ₂M to function as a carrier and deliver proteins to LRP for catabolism was first demonstrated with TGF- and PDGF-BB (62, 63). Only activated ₂M is functional in this capacity, because native ₂M is not recognized by LRP (51). Growth factors, such as TGF-, bind to native ₂M, as well as activated ₂M, and the resulting effects on growth factor activity are complicated. When bound to native ₂M, growth factors are typically inactive; however, because this interaction is reversible, ₂M-associated growth factors may provide a reservoir, buffering against changes in the free growth factor concentration (13, 64).

In our experiments, A bound selectively to the LRP-recognized or -activated form of ₂M, confirming the work of Narita et al. (33). To activate ₂M, we reacted the protein with methylamine. This reaction induces a conformational change in ₂M that is equivalent to the structural rearrangement induced by proteinases (11, 15, 44). In the ligand blotting system, denatured ₂M subunits retained A binding activity. This result suggests that ₂M tertiary and quaternary structures are not necessary for A binding. Instead, we hypothesize that ₂M activation reveals an otherwise cryptic linear sequence of amino acids that constitute a binding site for A. ₂M conformational change is also necessary for recognition by LRP; however, this interaction apparently requires retention of secondary and tertiary structure in the ₂M RBF (38, 41).

Although the primary sequence in ₂M that is responsible for the binding of growth factors is fairly promiscuous, this sequence did not interact with A and apparently did not contribute to the A binding activity of intact ₂M. Instead, a distinct protein-interaction site was identified, and our analysis of GST fusion proteins suggests that the center of this site is located between amino acids 1314 and 1365. The A binding sequence identified in our experiments may be equivalent to the candidate A-binding site identified by yeast-two hybrid screen (37). Based on these results, we now propose that ₂M contains at least two distinct protein-interaction sequences that are functional even when higher order ₂M structure is eliminated. These two binding sites demonstrate distinct ligand binding specificities, because the growth factor-binding site in FP3 does not bind A, and the A-binding site in FP6 does not bind TGF-, PDGF-BB, or NGF- (18).

The LRP recognition sequence includes two Lys residues within an  helix that includes amino acids 1370-1377 (41, 42). In the intact three-dimensional structure of the ₂M RBF, the two Lys residues are oriented so that the side chains are readily available for interaction with LRP. Furthermore, the Lys residues are surrounded by a high density of hydrophobic surface residues. All of our evidence indicates that the A-binding site and the LRP recognition sequence are adjacent but distinct. FP6c, which includes the entire  helix, did not bind A, whereas FP6a, which lacks the  helix, did. Mutants of the RBF, which have been previously shown to not bind LRP (39, 40), still bound A. Furthermore, a saturating concentration of A did not inhibit the plasma clearance of ₂M-MA, which is mediated by LRP (51). We propose that the center of the A binding sequence is located on the N-terminal side of the LRP recognition  helix. Comparison of the structure of RBFs from various -macroglobulins has demonstrated that one surface of the RBF is highly conserved (41). Jenner et al. (41) proposed that the conserved surface is divided into two patches, one of which constitutes the LRP recognition site. Only speculation was offered regarding the function of the second patch, which includes amino acids from our fusion proteins that bind A. The possibility that this second conserved surface patch represents an A-binding site merits consideration.

Determining the binding affinity of A for ₂M and its derivatives i