Identification of C1q as a Binding Protein for Advanced Glycation End

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Identification of C1q as a binding protein for advanced glycation end products Miho Chikazawa, Takahiro Shibata, Yukinori Hatasa, Sayumi Hirose, Natsuki Otaki, Fumie Nakashima, Mika Ito, Sachiko Machida, Shoichi Maruyama, and Koji Uchida Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00777 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 8, 2016

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Biochemistry

Identification of C1q as a binding protein for advanced glycation end products †

Miho Chikazawa , Takahiro Shibata †

†, §

†

†

†

, Yukinori Hatasa , Sayumi Hirose , Natsuki Otaki ,

†

‡

¶

Fumie Nakashima , Mika Ito , Sachiko Machida , Shoichi Maruyama , Koji Uchida

*,†

†

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

§

PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012,

Japan §

¶

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Department of Nephrology, Graduate School of Medical Sciences, Nagoya University,

Nagoya 464-8601, Japan

To whom correspondence should be addressed: Koji Uchida, Ph.D. Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Tel: 81-52-789-4127, Fax: 81-52-789-5296, E-mail: [email protected]

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FOOTNOTES ABBREVIATIONS: Ab, antibody; AGEs, advanced glycation end products; BSA, bovine serum albumin; CLR, collagen-like region; CML, Nε-carboxymethyllysine; DHA, dehydroascorbic acid; dsDNA, double-stranded DNA; ELISA, enzyme-linked immunosorbent assay; GR, globular region; HRP, horseradish peroxidase; ITC, isothermal titration calorimetry; MALDI-TOF/TOF-MS, matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry; OPD, 1,2-phenylenediamine; PLL, poly-L-lysine; RAGE, the receptor for AGEs


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Biochemistry

ABSTRACT

Advanced glycation end products (AGEs) are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum protein(s) that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGEs-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including collagen-like domain, as the AGEs-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 (C5) to release C5a. These findings provide the first evidence for AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.


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Nonenzymatic glycation is a complex series of reactions between reducing sugars, such as glucose, and the amino groups of protein, resulting in the formation of a heterogeneous group of molecules called advanced glycation end products (AGEs) 1. The AGEs can also be produced from reactive carbonyl compounds derived from the autoxidation of carbohydrates and other metabolic pathways. It is generally accepted that early glycation processes result in the formation of Schiff bases followed by rearrangement to relatively stable Amadori products. Hemoglobin A1c is a well known example of glycated proteins containing Amadori products. Amadori products further undergo a series of reactions through dicarbonyl intermediates and cause molecular rearrangements, leading to the generation of a variety of AGEs. Several products, including Nε-carboxymethyllysine (CML) and pentosidine, have been chemically characterized as AGEs. The formation and accumulation of these AGEs has been implicated in many pathophysiologies associated with aging and the long-term complications of diabetes 2. Dehydroascorbate (DHA), an oxidized form of vitamin C, can serve as an excellent source of AGEs. DHA can be degraded to generate several oxidized products, such as 2,3diketogulonic acid, 3-deoxythreosone, xylosone, and threosone 3, that can covalently react with the positively charged amino acid side-chains of proteins to generate the AGEs 4-6. DHA and its oxidized products could form adduct species structurally similar to those obtained upon the incubation of proteins with other reducing sugars 7-9. Due to the high vitamin C levels in tissues, DHA and its degradation products could be causally involved in the formation of AGEs in vivo. Monnier and his colleagues have indeed shown that there is a similarity between modifications of proteins by ascorbate and those in aged human lenses and cataracts in vivo 8. In addition, they have also shown that the overexpression of a vitamin C transporter leads to the significant accumulation of AGEs 10.


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AGEs are some of the damage-associated molecular patterns that trigger a range of cellular responses, including immune and inflammatory responses. They exert their cellular effects through interactions with specific receptors called pattern recognition receptors, such as the receptor for AGEs (RAGE) 2. The activation of RAGE triggers the cellular activation and proliferation, leading to inflammation and tissue destruction 5. CML has been shown to bind RAGE and modulate the cellular response 6. These molecules, possessing an exposed epitope, are also recognized by the soluble receptors, such as antibodies and regulatory proteins 11-13. We have previously shown that AGEs, including the DHA-modified proteins, are commonly recognized by the natural IgM antibodies 14. Due to the formation and accumulation of AGEs not only in the physiological organism during aging, but also in a variety of pathophysiology and clinical implications, additional mechanisms for the recognition of these molecules were anticipated. Hence, in the present study, we examined the presence of serum protein(s) that are capable of binding AGEs and identified a complement protein as a hitherto unrecognized innate regulatory protein for the AGEs in human serum. Moreover, we characterized the structural and chemical criteria governing the interaction between this complement factor and AGEs.


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MATERIALS AND METHODS Materials Dehydroascorbic acid (DHA) and calf thymus double-stranded DNA (dsDNA) were obtained from Sigma-Aldrich. Complement C1q (human) was obtained from Calbiochem. Bovine serum albumin (BSA) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Hemoglobin A0 and hemoglobin A1c isolated from human blood were obtained from Sigma-Aldrich and BBI Solutions, respectively. Dynabeads M-270 carboxylic acid was obtained from Invitrogen. Horseradish peroxidase (HRP)-NeutrAvidin and ECL Western blotting detection reagents were obtained from GE Healthcare. The HRP-linked anti-mouse IgM (for ELISA analysis) were obtained from Cappel Laboratories. The normal mouse IgM (MM-30) was purchased from Abcam (Cambridge, UK). EZ-link biotin-LChydrazide was obtained from Pierce. C1qA peptides were synthesized by Medical & Biological

Laboratories

AGRPGRRGRPGLK

Co., (C1qA

Ltd.,

Japan.

14-26);

The

following

peptides

KGGAPRRGGLPRR

were

(C1qA

made:

14-26/S);

GIKGTKGSPGNIKDQPR (C1qA 76-92); KPIGTGKSGGNIDQKPR (C1qA 76-92/S); AGRAGRRGRAGLK

(C1qA

14-26/P-A);

AGGPGGGGGPGLG

(C1qA

14-26/0+);

AGGPGRGGRPGLG (C1qA 14-26/2+). All of the other reagents used in the study were of analytical grade and obtained from commercial sources. Human serum Normal human serum was stored at -70°C. MgEGTA-treated serum contained 10 mM magnesium and 10 mM EGTA. Complement C1q-depleted serum from a human was purchased from Calbiochem. Preparation of AGEs in vitro


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AGEs were prepared by incubating BSA (1.0 mg/ml) with DHA (25.0 mM) in 0.2 M phosphate buffer (pH 7.4) at 37 °C under atmospheric oxygen. After 7 days, aliquots were collected and dialyzed against PBS. The DHA-modified BSA (AGEs-BSA) was characterized by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (AB SCIEX 5800) analysis (Supporting Information Fig. S1). The acetylated, succinylated, and maleylated proteins were prepared according to published procedures 15-17. Purification and identification of AGEs-binding proteins BSA coupled to the Dynabeads was incubated with 25 mM DHA in 0.2 M phosphate buffer (pH 7.4) for 7 days to obtain the AGEs-coupled beads. The beads (2 x 107) were then added to microcentrifuge tubes and incubated with 150 µl of x2 dilution human serum in PBS/Tween for 1 h at room temperature. After washing three times with PBS/Tween, binding protein was eluted by adding sample buffer and heating (80 ˚C, 10 min). Protein separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) performed under reducing conditions was processed for tryptic digestion and matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) (AB SCIEX 5800). Immunoblot analysis and ligand blotting The samples were run on 15% SDS-polyacrylamide gels. After electrophoresis, the gel was transblotted onto a PVDF membrane (GE Healthcare), incubated with skim milk for blocking, washed, and then incubated with an anti-human C1qA antibody (Ab) (ab76425, abcam), anti-human C1qB Ab (Santa Cruz Biotechnology.), anti-human C1qC Ab (ab75756, Abcam), or biotinylated AGEs-BSA (100 µg) in TTBS overnight at 4˚C. After washing, the membrane was incubated with x1000 HRP-linked secondary Ab or HRP-NeutrAvidin for 1 h


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at room temperature. This procedure was followed by the addition of ECL reagents. The bands were visualized using a Cool Saver AE-6955 (ATTO, Tokyo, Japan). Preparation of biotinylated proteins in vitro Biotinylation of the protein was performed by incubating protein (1 mg/ml) with a 20-fold molar excess of EZ-Link Biotin-LC-Hydrazide in PBS for 30min at RT. After incubation, aliquots were collected and dialyzed against PBS. To prepare the biotinylated-AGEs-BSA, biotinylated BSA (1.0 mg/ml) was incubated with DHA (25.0 mM) in 0.2 M phosphate buffer (pH 7.4) for 7 days at 37 °C. After the incubation, aliquots were collected and dialyzed against PBS. ELISA (enzyme-linked immunosorbent assay) ELISA was performed as previously reported 14. Co-immunoprecipitation studies Diabetic db/db (BKS.Cg-+Leprdb/+Leprdb/Jcl), and their control m/m (BKS.Cg-m+/m+/Jcl) mice were purchased from Japan Clea (Tokyo, Japan). Twelve-week-old male mice (n=5) were humanely killed and intracardially perfused with ice-cold PBS. After perfusion, the kidney was isolated, frozen in liquid nitrogen and stored at -80 ˚C. The removed kidneys were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X100, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor mixture, and phosphatase inhibitor mixtures (Sigma). Serum was separated by centrifugation and stored at -80 ˚C until analysis. All animal protocols were approved by the Animal Experiment Committee in the Graduate School of Bioagricultural Sciences, Nagoya University.


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Co-immunoprecipitation of C1q-AGEs complexes was carried out using an anti-C1q-B Ab (T-20) (Santa Cruz, sc-27664). Following pre-clearing with Dynabeads Protein G, serum or kidney homogenate proteins (1 mg/ml) from diabetic db/db mice and their nondiabetic controls were incubated with the anti-C1q-B Ab (2 µg) coupled to protein G beads in 1 ml of PBS/Tween for 1 h at room temperature under rotation. Afterward, beads were washed twice with PBS/Tween, and 200 µl of PBS was added to each tube. Samples were vortex-mixed, heated for 5 min at 80 ˚C. Dynabeads were then pulled down using magnet, and the supernatant was collected. A 100 µl aliquot of the supernatant solution was added to each well of a microtiter plate. After overnight incubation, the wells were blocked with blockace in PBS/Tween then washed with PBS/Tween. A biotinylated anti-AGEs IgG ADL13 prepared from the MRL-lpr mouse 14 was added to the plates and incubated for 2 h at 37 ˚C. The plates were washed again, and the bound IgG was detected using HRP-NeutrAvidin followed by the addition of 1,2-phenylenediamine. Measurement of zeta potential The zeta potential of the modified proteins were measured by the Zetasizer (Nano-ZS) from Malvern Instruments and its software, Dispersion Technology Software (DTS). A 1 ml aliquot of 1 mg/ml native or modified BSA solution in 10 mM phosphate buffer (pH 7.4) was injected into a cuvette and the electrophoretic mobility was measured at room temperature. C3 activation assay To measure the C3 activation, we used the method of Ali et al.

18

with the following

modifications. Nunc MaxiSorp microtiter plates were coated with 100 µl of 50 µg/ml of antigens in PBS. After overnight incubation, the wells were blocked with blockace in PBS/Tween then washed with PBS. Serum samples were diluted in BBS (4 mM barbital, 145


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mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), added to the plates and incubated for 1 h at 37˚C. The plates were washed again, and the bound C3b was detected using rabbit antihuman C3c (Dako) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG followed by the colorimetric substrate, 1,2-phenylenediamine (OPD). C4b deposition assay Nunc MaxiSorp microtiter plates were coated with 100 µl of 50 µg/ml of antigens (BSA or AGEs-BSA) in PBS. After overnight incubation, the wells were blocked with blockace in PBS/Tween then washed with PBS/Tween. Serum (normal human serum or C1q-depleted serum) were diluted in BBS (4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) with or without C1q (50 µg/ml), added to the plates and incubated for 1 h at 37˚C. The plates were washed, and the bound C4b was detected using goat anti-human C4 (ab47788, Abcam) followed by HRP-conjugated anti-goat IgG followed by the colorimetric substrate, 1,2-phenylenediamine. C5a release assay For C5a ELISA, microtiter plates were coated with BSA or AGEs-BSA and blocked as described above for the complement activation assay. The wells were then incubated with serum dilutions in BBS buffer for 15 min at 37 °C. The samples were immediately assayed using a C5a-ELISA kit (Abcam) according to the instructions of the manufacturer. Isothermal titration calorimetry (ITC) ITC experiments were performed using a MicroCal ITC titration calorimeter (ITC200, GE Healthcare). Titrations were performed at a temperature of 25°C. The AGEs-BSA used for the ITC experiments was enriched using a Microcon concentrator (Millipore). The raw data


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(buffer titration) corresponding to the dilution curve were shown in Supporting Information Figs. S2-S3. Each experiment was performed at least twice. Binding of C1q to human hemoglobin A1c. Proteins (BSA, AGEs-BSA, hemoglobin A0, hemoglobin A1c) (10 µg/well) were immobilized on a plate and incubated with biotinylated C1q (10 µg/well). Binding was detected using streptavidin-peroxidase. Collagenase-digested C1q. Human C1q (0.5 mg/ml) was incubated overnight at 37 °C with 0.1 mg/ml collagenase (Type VII, purified from Clostridium histolyticum, Sigma) in 50 mM Tris-HCl buffer, pH 7.4, containing 10 mM CaCl2 and 250 mM NaCl. Statistical analysis The data represent the mean ± s.d. where indicated. Statistical significance was evaluated by using the unpaired two-tailed Student's t-test or, when appropriate, Dunnett’s or Tukey’s HSD test.


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RESULTS Identification of an AGEs-binding protein in serum To examine the presence of serum protein(s) which are capable of binding AGEs, normal human serum was incubated with beads coupled to either unmodified BSA or AGEs-BSA (DHA-modified BSA), then the bound proteins were eluted and separated by SDS-PAGE under reducing conditions (Fig. 1A). Some unique bands including the protein with a molecular mass of about 26 kDa (indicated by the arrow) were exclusively found in the AGEs-BSA pull-downs. To identify the 26-kDa protein, the band resolved by Coomassie Brilliant Blue (CBB) staining was excised and analyzed by MALDI-TOF MS (Supporting Information Table S1). From three independent proteomic experiments, the protein was identified as the complement component C1q subcomponent subunit A (C1qA). We also detected another protein band with similar molecular mass in the SDS-PAGE, but were unable to identify it. This may be due to the fact that the C1q components contains either hydroxylated or glycosylated lysine residues

19

. Immunoblot analysis revealed the presence

of C1qA in the elutions from normal human serum on AGEs-BSA beads, but not on the unmodified BSA beads (Fig. 1B, left). C1qA was also detected in the poly-L-lysine (PLL)bound AGEs (AGEs-PLL) pull-downs (Fig. 1B, right). The ligand blot and solid phase binding assays showed the binding of the biotinylated AGEs-BSA to the purified C1q (Figs. 1C, D). C1q is comprised of six copies of 3 similar but distinct polypeptide chains, A, B, and C 20. It is therefore reasonable to speculate that full-length serum C1q assembled from the A, B and C chains might bind to the AGEs-BSA beads, and the three chains should be eluted and detected. Indeed, all three chains were detected in the AGEs (AGEs-BSA) pull-downs (Fig. 1E). However, the ligand blot analysis showed that the AGEs-BSA selectively binds to the


12

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Biochemistry

C1qA (Fig. 1F), suggesting that both B and C chains might be pulled downed with the A chain bound to the AGEs-BSA beads. We also performed a pull-down experiment using C1qdepleted human serum and observed the disappearance of the bands, corresponding to the C1q components (Supporting Information Fig. S4). The data also support the idea that the main target of AGEs-BSA is C1qA. Involvement of C1q in the recognition of AGEs in vivo Because AGEs are formed upon reaction of protein with the short-chain glycolytic intermediates, such as glyoxal, methylglyoxal, glycolaldehyde, glyceraldehyde, and dihydroxyacetone 21, we tested the binding of the C1q to various AGEs. The pull-down assay indeed showed the binding of C1q to all of the tested lysine-bound AGEs (Fig. 2A). In addition, C1q showed significant binding to human glycated hemoglobin (hemoglobin A1c) as compared to non-glycated hemoglobin (hemoglobin A0) (Fig. 2B). Furthermore, coimmunoprecipitation studies with the serum and kidney homogenates revealed that the C1qAGEs complexes were accumulated more in the diabetic (db/db) mice as compared to the control mice (Fig. 2C). These data suggest the involvement of C1q in the recognition of AGEs in vivo. Characterization of AGEs-bind regions of C1q Each of three chains (A, B, and C) of C1q has collagen-like N-terminal “stalk” (collagenlike region, CLR) and a C-terminal, globular “head” (globular region, GR) 22, which are involved in the binding with a variety of ligands, such as DNA and aggregated IgG (AggIgG)

23-25

. The biotinylated C1q indeed recognized DNA and Agg-IgG (Fig. 3A). To gain

insight into the region(s) of C1q responsible for its binding to the AGEs-BSA, we performed a competition assay, in which the biotinylated C1q was allowed to bind to microtiter plates coated with AGEs-BSA in the presence or absence of the C1q ligands. We tested DNA and


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Agg-IgG as the C1q ligands and observed that only DNA competed in a concentrationdependent manner for the binding of C1q to the coated AGEs-BSA (Fig. 3B). Even though Agg-IgG preferentially binds to the GR of C1q 26, it did not block binding of the C1q to the AGEs-BSA. The binding of the biotinylated C1q to the immobilized DNA was also inhibited by the AGEs-BSA (Fig. 3C). These data suggest that AGEs-BSA and DNA might share a binding region on the A chain of C1q. However, the observations that DNA only partially inhibited the binding of AGEs-BSA to C1q (Fig. 3B) whereas the AGEs-BSA completely inhibited the binding of DNA to C1q (Fig. 3C) also implicated that C1qA might have multiple binding sites, including CLR and GR, for the AGEs-BSA. Indeed, the ligand blot analysis of the collagenase-digested human C1q showed that the AGEs-BSA recognized a 15 kDa protein, corresponding to GR (Supporting Information Fig. S5). Identification of an AGEs-binding site on the CLR of C1qA The previous findings that DNA bound preferentially to the A chain of C1q and that the binding sites for DNA were localized by using synthetic C1qA chain peptides to two cationic regions within residues 14-26 and 76-92, respectively (Fig. 4A), 27 suggest that the AGEsbinding sites in C1qA may at least include CLR, containing either hydroxylated or glycosylated lysine residues 19. To identify the binding site for the AGEs-BSA on the CLR of C1qA, we performed binding studies using recombinant CLR fragments. We tested C1qA 14-26 and C1qA 76-92 peptides for their ability to compete with the AGEs-BSA binding to C1q in the solid-phase binding assay. The biotinylated AGEs-BSA were preincubated with C1qA 14-26 and C1qA 76-92 and their scrambled peptides C1qA 14-26/S and C1qA 76-92/S (Fig. 4B), containing identical amino acids but different sequences, and their inhibitory effects on the binding of the AGEs-BSA to the immobilized C1q were examined. The C1qA 14-26 and C1qA 14-26/S peptides showed about 60% inhibition whereas no significant


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Biochemistry

inhibitory effect was observed with the C1qA 76-92 and C1qA 76-92/S peptides (Fig. 4C). In addition, we tested the binding of the biotinylated AGEs-BSA to these peptides and observed that the AGEs-BSA were bound to the C1qA 14-26 and C1qA 14-26/S peptides, but not to the C1qA 76-92 and C1qA 76-92/S peptides (Fig. 4D). Thus, C1q binds to the AGEs-BSA at the region within the C1qA 14-26 peptide independent of its peptide sequence and conformational space. We also examined the stoichiometry of the AGEs-BSA and these C1q peptides by ITC. When the peptides were titrated into the ITC cell containing the AGEsBSA, stoichiometry values of 7.74 molecules of the C1qA 14-26 peptide and 7.89 molecules of the C1qA 14-26/S peptide bound per molecule of the AGEs-BSA were obtained (Fig. 4E). Consistent with the solid-phase binding assay, the C1qA 76-92 and C1qA 76-92/S peptides did not show any specific binding to the AGEs-BSA (Fig. 4F). We further tested the charge control peptides, C1qA 14-26/0+ in which all cationic residues were replaced with glycine residues and C1qA 14-26/2+ in which three cationic residues were replaced with glycine residues, for their ability to compete with C1q during the solidphase binding assay (Figs. 5A-C). As expected, both charge control peptides did not inhibit the C1q-AGEs binding, indicating that more than two cationic residues were required to interact with the AGEs. In addition, these peptides did not show measurable interactions by ITC (Figs. 5D-F). We also tested C1qA 14-26/P-A, in which both proline residues were replaced with alanine residues, and observed that the peptide still retained its ability to bind the AGEs-BSA (Fig. 5). These data suggest that the binding capacity of these cationic peptides had no sequence or conformation specificity, rather, the amount of positive charge on the peptides was the determining factor. Involvement of electronegative potential of AGEs during the recognition by C1q


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The C1qA peptide 14-26 is characterized by the presence of a number of charged side chains 27. This region is distinct from the remainder of the C1q CLR due to the presence of a cluster of five residues that are positively charged at physiological pH (one lysine and four arginines). Based on the observations that the AGEs-BSA-binding to C1q was inhibited by both the C1qA peptide 14-26 and its scrambled peptide 14-26/S (Fig. 4), but not by the charge control peptides (C1qA 14-26/0+ and 14-26/2+) (Fig. 5), we speculated that the specificity of C1q directed against the AGEs-BSA might arise through an electric interaction between the AGEs-BSA and C1q. To prove this hypothesis, we tested whether C1q could recognize electronegative molecules, such as acetylated BSA (AcBSA), succinylated BSA (ScBSA), and maleylated BSA (MaBSA) (Fig. 6A). These modifications neutralize the positive charge of the lysine residues, thereby increasing the negative charge on the protein surface. In addition to the neutralization, the succinylation and maleylation of lysine further introduce a negative charge. As expected, the solid-phase C1q binding assay showed that the C1q recognized these negatively-charged proteins (Fig. 6B). We further examined the correlation between the electronegativity and C1q-binding activity by measuring the zeta potential, an electrochemical property determined by the net electrical charge of the molecules. The zeta potential of the protein decreased upon glycation, in which a zeta-potential as low as -40 mV was observed (Fig. 6C). In addition, the decrease in the zeta potential was well correlated with the increase in the C1q-binding potential. Thus, the reduction in the zeta potential was consistent with the enhanced electronegativity of the AGEs-BSA. Moreover, the involvement of the electric interaction between C1q and the AGEs-BSA was further evaluated by manipulation of the ionic strength. As shown in Fig. 6D, NaCl significantly inhibited the binding of the biotinylated-C1q to the AGEs-BSA in a dose-dependent manner. The data also support our hypothesis that the electronegative potential of the AGEs might be involved, at least in part, in the recognition by the C1q.


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On the other hand, natural IgM Abs are also known to recognize multiple exogenous and endogenous ligands, including DNA and the AGEs 14. We indeed observed that the binding of IgM and C1q to AGEs-BSA showed linear correlations (Figs. 6E, Supporting Information Fig. S6). In addition, the anti-AGEs IgM competed with C1q for the binding to the AGEsBSA (Fig. 6F). No significant binding of the IgM to C1q was observed (Supporting Information Fig. S7). These results suggest that, as a charge pattern recognition molecule of the innate immunity, C1q may share similar ligands with IgM (Fig. 6G). Activation of C1q-dependent classical complement pathway by the AGEs-BSA Based on the finding that C1q bind the AGEs as a ligand, we investigated the ability of AGEs-BSA to activate the complement pathway using an ELISA-based plate assay, in which the AGEs-BSA coated on a microtiter plate were incubated with normal human serum followed by detection of the complement activation product C3b. AGEs-BSA, but not the unmodified BSA, mediated the deposition of C3b in a serum-dependent manner (Fig. 7A). However, the C3b deposition was significantly diminished when the AGEs-BSA were incubated with the heat (56 ˚C)-treated normal human serum at 37 ˚C or with the normal human serum at 4˚C (data not shown). The complement system participates in host immune reactions as a result of activation of the classical, lectin, and alternative complement pathways, among which C1q is involved in the classical complement pathway. The observation (Fig. 7B) that the serum-dependent activation of C3 deposition by the AGEsBSA was totally inhibited by the addition of MgCl2-EGTA, a chelating agent which blocks the activation of both the classical and lectin pathways, suggests that the binding of C1q to the AGEs-BSA might lead to the activation of the classical complement pathway. This hypothesis was supported by the observation that the AGEs-BSA-induced deposition of C3b was significantly reduced in the C1q-depleted serum, whereas the reconstitution of the serum


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with C1q restored the ability of the depleted serum to enhance the C3b deposition (Figs. 7C, D). Similar results for the involvement of C1q was observed in the AGEs-BSA-induced deposition of complement component C4b (Figs. 7E, F). Furthermore, the observation that the AGEs-BSA induced the proteolytic cleavage of complement protein 5 (C5) to release C5a (Fig. 7G) further supports the idea that the AGEs-BSA stimulates the classical complement pathway.


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DISCUSSION High levels of circulating AGEs occur in diabetes and end-stage renal disease 28, 29. AGEs are recognized and taken up by innate receptors called pattern recognition receptors that have been identified to be present on diverse cell types. In the present study, to identify serum protein(s), which are capable of binding AGEs, we analyzed human serum using proteomics approaches and identified C1q subcomponent A as an AGEs-binding protein (Fig. 1). C1q is the target recognition protein of the classical complement pathway and a major connecting link between the innate and acquired immunity. C1q also binds Frizzled receptors and modulates mammalian aging-related phenotypes via activation of Wnt signaling 30. Based on the results that (i) DNA, which binds to collagen-like region CLR, GR, or to both regions of C1qA, markedly inhibited the binding of C1q to the AGEs, and (ii) Agg-IgG, which binds to GR, did not block the binding of C1q to the AGEs-BSA, we speculated that the AGEs binding capacity of C1qA is attributable to the residual CLR. The C1q CLR has been reported to have two binding sites for a variety of ligands within residues 14-26 and 76-92 26, 27. Indeed, when peptide 14-26 was preincubated with the AGEsBSA, it blocked the binding of the AGEs-BSA to the immobilized intact C1q. In addition, peptide 14-26 was found to directly interact with the AGEs-BSA (Fig. 4). However, peptide 76-92 lacked these properties. These data imply that the sequence of C1qA 14-26 represents a major AGEs-BSA-binding site. An important characteristic of this sequence is that it contains an arginine-rich motif. This highly positively charged peptide has been shown to specifically inhibit C1q binding to Aβ, DNA, C-reactive protein, and serum amyloid P

27, 31-33

. Many of

these antibody-independent activators are multimeric and some indeed contain a high density of negative charges. It has been suggested that, due to the polyanionic nature of C1qinteracting molecules, C1q may recognize a specific pattern and/or spacing of charged


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residues or groups 17. This mechanism appears to be responsible for the recognition of AGEs by C1q based on the finding that scrambling the order of the residues within peptide 14-26 did not result in the loss of the peptide binding activity. In addition, no binding to the AGEsBSA was detected with the neutral peptide (C1qA 14-26/0+) and the peptide with two cationic charges (C1qA 14-26/2+) (Fig. 5). Thus, the charge, but not an appropriate amino acid sequence and conformation, may be sufficient for the peptide to be optimally reactive with the AGEs. Taken together, it was speculated that the basis for the ability of peptide 1426 to bind with the AGEs-BSA might be attributable to its positively charged character. On the other hand, it has been shown that C1q GR is involved in the binding for CRP 34, βamyloid peptide

35

, RAGE

36

, and DNA

37

. Based on the ligand blot analysis of the

collagenase-digested human C1q, we indeed observed the binding of the AGEs-BSA to the 15 kDa protein, corresponding to GR (Supporting Information Fig. S5). The data strongly suggest that the AGEs-BSA could also bind GR. The involvement of multiple domains is therefore likely to participate in the binding of multiple AGEs-BSA molecules. In the present study, using a variety of carbonyl compounds, we evaluated the correlation between the zeta potential of the modified proteins and C1q-binding activity and observed that the decrease in the net electrical charge was well correlated with the increase in the C1qbinding potential (Fig. 6). We also observed that the C1q recognized a variety of electronegative proteins, such as acetylated, succinylated, and maleylated proteins. Moreover, the observation that NaCl significantly inhibited the binding of the C1q to AGEs-BSA also supports our hypothesis that the electronegative potential of the AGEs-BSA might be involved, at least in part, in the recognition by the C1q. Neutralizing the positive charges on the surface of proteins may therefore make the negatively charged side chains of proteins more exposed and therefore available for the electrostatic interaction with the C1q. The


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formation of electronegative molecules is also commonly seen in many physiological and pathophysiological processes. The introduction of electronegative groups, such as phosphate and sulfate groups, into proteins significantly changes the net electrical charge of the proteins, resulting in a significant conformational change in their structures. Therefore, the electrostatic interaction mechanism may ubiquitously constitute the fundamental event in molecular interactions and signal transductions. On the other hand, C1q is known to be an activation ligand for phagocytosis, rapidly triggering enhanced phagocytosis of a variety of targets, including apoptotic cells 38. In this study, we observed activation of the complement pathway by the AGEs-BSA as measured by C3b deposition (Fig. 7). The activation of the classical pathway for the AGEs-BSA-induced C3b deposition was also demonstrated by the experiments using the calcium-free sera. The deposition of C3b was significantly reduced in the C1q-depleted serum, whereas the reconstitution of the serum with C1q restored the ability of the depleted serum to enhance the C3b deposition. In addition, similar effects of C1q were observed on the AGEs-BSA-induced deposition of C4b (Figs. 7E, F). These findings and the observation that the AGEs-BSA induced the proteolytic cleavage of complement protein 5 (C5) to release C5a (Fig. 7G) suggest that the AGEs-BSA stimulates the classical complement pathway. We have also observed that, in our preliminary study, the phagocytosis of the AGEs-BSA by human THP-1 monocytes was enhanced in the normal human serum, whereas the phagocytosis was significantly abrogated when the cells were incubated with the AGEs-BSA in the heat-treated serum (Chikazawa, M., Shibata, T., and Uchida, K., unpublished data). In addition, the reconstitution of C1q-depleted serum with serum levels of C1q restored the serum-mediated phagocytosis of the AGEs-BSA. These results also support our idea that C1q may contribute to the AGEs clearance in the presence of other serum components, including down-stream complement components, and that the classical complement-dependent (C1-dependent)


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phagocytosis may be responsible for promoting the clearance. On the other hand, although classical complement-dependent (C1-dependent) phagocytosis has been demonstrated to play a major role in promoting apoptotic cell clearance 39, 40, accumulating evidence also suggests that C1q serves as a bridging molecule for the apoptotic cell clearance by binding to the apoptotic cell and the phagocyte and stimulating engulfment, independent of the classical complement pathway

41-44

. The finding that C1q directly binds AGEs as a ligand also

suggests the involvement of this mechanism in the phagocytosis of AGEs. In summary, we report the identification of C1q as a hitherto unrecognized innate regulatory protein for the AGEs, which are ubiquitously generated during aging and many degenerative diseases, such as diabetes and atherosclerosis 45-50. These data suggest a novel role for C1q in enhancing the AGEs uptake that leads to the beneficial metabolism of modified proteins. In addition, based on the findings that C1q is bound not only to DNA and AGEs, but also several other modified proteins, including the acylated proteins, we propose a mechanism in which the electronegative potential of AGEs might be involved, at least in part, in the recognition by C1q. Thus, our discovery of C1q as a major AGEs-binding protein demonstrates that the innate immunity may play a pivotal role in providing homeostatic responses against electronegative molecules ubiquitously generated in biological systems. Because mechanisms governing the removal of AGEs are critical to the suppression of agerelated diseases, including diabetic complications and atherosclerosis, further investigation of the molecular components of these C1q-mediated processes should lead to novel strategies or areas for therapeutic intervention in these diseases.

ACKNOWLEDGMENTS


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We thank Dr. Noritada Kaji (Nagoya University, Graduate School of Engineering) for technical assistance with Zeta potential analysis and Dr. Kazukiyo Yamamoto (Nagoya University, Graduate School of Bioagricultural Sciences) for his valuable discussion about statistical analysis. We also thank Ms Yuki Hondoh for her excellent editorial support.

Supporting Information Available Supplementary Table (Table S1) and Supplementary Figures (Figure S1-S7)

Funding Sources This work was supported in part by Grants-in-Aid for Scientific Research (A) (No. 21248016 and No. 26252018 to K.U.), (C) (24580177 to T.S.), and Challenging Exploratory Research (No. 24658122 to K.U.) and a Grant-in-Aid for Scientific Research on Innovative Areas "Signaling Functions of Reactive Oxygen Species" (No. 20117007 to K.U.) and "Oxygen Biology: a new criterion for integrated understanding of life" (No. 26111011 to K.U.) of The Ministry of Education, Culture, Sports, Science and Technology, Japan; a grant from the JST PRESTO program (T.S.); grants from the Ministry of Health, Labor and Welfare of Japan. Notes The authors declare no competing financial interest.


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(2007) Serum C3 is a stronger inflammatory marker of insulin resistance than Creactive protein, leukocyte count, and erythrocyte sedimentation rate. Comparison study in an elderly population, Diabetes Care 30, 2362-2368. 48.

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FIGURE LEGENDS Fig. 1. Identification of C1q as an AGEs-binding protein in human serum. (A) Pull-down assay for the detection of AGEs-binding proteins in human serum. The human serum was incubated with the BSA-coupled or DHA-treated BSA (AGEs)-coupled beads for 1 h at room temperature. The proteins bound to the beads were eluted by adding sample buffer and heating (80 ˚C, 10 min) and separated by SDS-PAGE. The protein indicated by the arrow represents C1qA identified by MALDI-TOF MS. (B) Immunoblot blot analysis of C1qA in the eluates from the BSA- or AGEs-coupled beads incubated with human serum (left). Immunoblot blot analysis of C1qA in the eluates from the polylysine (PLL) or DHA-treated polylysine (AGEs-PLL) incubated with human serum (right). (C) Ligand blot assay for the binding of the biotinylated AGEs to C1q. The purified human C1q (0.5 µg) was separated by 15% gel SDS-PAGE, transferred to PVDF, and probed with biotinylated AGEs or anti-C1qA Ab. (D) Binding of biotinylated AGEs to coated C1q. Purified human C1q (5 µg/ml) was immobilized on a plate and incubated with biotinylated AGEs (0-25 µg/well) at 4 ˚C for 12 h. Binding of the AGEs was assessed by ELISA using the OPD substrate with signal absorbance measured at 490 nm. (E) Immunoblot analysis of C1qA, C1qB, and C1qC in the eluates from the BSA- or AGEs-coupled beads incubated with human serum. Data are representative of three individual experiments in duplicates. (F) Ligand blot assay for the binding of the biotinylated AGEs to C1q. The purified human C1q was separated by 15% gel SDS-PAGE, transferred to PVDF, and probed with biotinylated AGEs (left) or anti-C1qA, C1qB, and C1qC Ab (right). Data are representative of three individual experiments. Fig. 2. Involvement of C1q in the recognition of AGEs in vivo


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Biochemistry

(A) Immunoblot for C1q using eluates from the poly-L-lysine (PLL) or modified polylysine (AGEs-PLL) incubated with human serum. The AGEs-PLL were prepared by incubating poly-L-lysine (PLL) (1.0 mg/ml) with 25 mM glycolytic aldehydes in 0.2 M PB (pH 7.4) at 37 ˚C for 7 days. (B) Binding of biotinylated C1q to the in vitro- and in vivo-derived AGEs. The ligands (BSA, AGEs-BSA, hemoglobin A0, and hemoglobin A1c) (50 µg/ml) were immobilized on a plate and incubated with biotinylated C1q (20 µg/ml) at 4 ˚C for 12 h. Data are from single experiments, performed in triplicate wells, representative of three individual experiments. ***, P < 0.001. Differences were analyzed by the unpaired two-tailed Student’s t test. (C) Co-immunoprecipitation of AGEs-C1q complexes. The serum or kidney homogenates from db/db mice and their nondiabetic controls were subjected to coimmunoprecipitation using anti-C1q Ab. ELISA analysis was performed using a biotinylated anti-AGEs Ab ADL-13. ***, P < 0.001. Differences were analyzed by the unpaired twotailed Student’s t test. Fig. 3. AGEs bind the DNA-binding region on the C1q collagen-like domain. (A) Binding of biotinylated C1q to the coated ligands. The ligands (BSA, AGEs-BSA, DNA, and Agg-IgG) (50 µg/ml) were immobilized on a plate and incubated with biotinylated C1q (20 µg/ml) at 4 ˚C for 12 h. Data are mean ± s.d. of three separate experiments, with each performed in duplicate. **P

Identification of C1q as a Binding Protein for Advanced Glycation End

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