Báo cáo khoa học: Identification of intracellular target proteins of the calcium-signaling protein S100A12 pdf

Identification of intracellular target proteins of the calcium-signaling protein S100A12 Takashi Hatakeyama 1 , Miki Okada 1 , Seiko Shimamoto 1 , Yasuo Kubota 2 and Ryoji Kobayashi 1 Departments of 1 Signal Transduction Sciences and 2 Dermatology, Kagawa University Faculty of Medicine, Japan In this report, we have focused our attention on i dentifying intracellular mammalian proteins that bind S100A12 in a Ca 2+ -dependent manner. Using S100A12 affinity chroma- tography, we have identified cytosolic NADP + -dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehy- drogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins . Immunoprecipitation experiments indicated th e formation o f stable c omplexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S10 0A9 in vivo. Surface plasmon resonance analysis showed that the b inding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca 2+ -dependent, whereas that of GAPDH and IDH was only weakly Ca 2+ -dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal t runcation mutants to the S100A12-immobilized sensor chip. The results indicated t hat the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87–92). However, cross-linking experiments with the truncation mutants in dicated that residues 87–92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca 2+ -dependent affinity chromatography revealed that C-terminal residues 75–92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. T he thermal agg regation of IDH or GAPDH was facilitated by S100A12 in the a bsence of Ca 2+ , whereas in the presence of Ca 2+ the protein sup- pressed the aggregation of aldolase t o less t han 50%. T hese results suggest that S100A12 may have a chaperone/anti- chaperone-like f unction which is Ca 2+ -dependent. Keywords: annexins; affinity chromatography; Ca 2+ -bind- ing p roteins; molecular chaperone; surface plasmon reson- ance ana lysis. Ca 2+ , a second messenger, is involved in regulating various cellular responses through a class of Ca 2+ -binding proteins, including calmodulin (CaM), troponin C and S100 pro- teins. These proteins exhibit a general structural principle o f so-called EF-hand. They undergo a conformational c hange through C a 2+ binding and consequently interact with their target proteins. S100 proteins were first considered to exist mainly in the nervous system, but subsequently new members of the S100 family were identified in other tissues [1,2]. Based on amino acid sequence similarity and other molecular properties, 20 different proteins have been assigne d to the family [2]. The y have been found to interact in a Ca 2+ -dependent manner with a diverse group of proteins, i ncluding those involved i n cell proliferation and differentiation, cellular architecture, signal transduction, and intracellular metabolism. For example, S100A1 interacts with guanylate cyclase [3] and glycogen phosphorylase [4]; S100A4 interacts with nonmus- cle m yosin and tropomyosin [5,6]; S100B, S100A1, S100A8 and S100A9 interact with intermediate filaments [7–10]; S100A1 and S100B interact with microtubules [11–13], twitchin kinase [14] and fruct ose-1,6-bisphosphate aldolase [15]; S100B and S100A1 interact with N dr-kinase [ 16] and phosphoglucomutase [17]; a nd S100A1, S100A6, S100A10, S100A11 and S100B inteact with annexins [18–22]. S100A12 (also known as calgranulin C) has been detec- ted in large amounts in neutrophils [23] and lung [24]. Recently, S100A12 from bovine lung was identified as a ligand for the receptor for advanced glycation end-products (RAGE), and anti-S100A12 immunogloblin was found to substantially block leucocyte recruitment in delayed hypersensitivity and colit is [25]. M ore recently, Yang et al. reported the chemotactic properties of S100A12 for neu- trophils and monocytes [26]. These reports indicate that extracellular S 100A12 may contribute to the pathogenesis of inflammatory responses. The X-ray structure of S100A12 hasbeensolvedintwocrystalforms:R 3 and P2 1 [27]. In the R 3 crystal form, S100A12 is a dimer, and in the P2 1 crystal form the dimers a re arranged as a h examer. Ca 2+ -depend- ent hexamer formation could facilitate the binding of S100A12 t o i ts rece ptor (RAGE) and r esult i n r eceptor oligomerization [28]. The protein has been found to show Correspondence to R. Kob ayashi, Department of S ignal Transduction Sciences, Kagawa University Faculty of Medicine, 1750–1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761–0793, Japan. Fax/Tel.: +81 87 8912249; E-mail: ryoji@kms.ac.jp Abbreviations: Aldolase, fructose-1,6-bisphosphate aldolase A; BS 3 , bis-(sulfosuccinimidyl) suberate; CaM, calmodulin; GAPDH, glycer- aldehyde-3-phosphate dehydrogenase; Hsp, heat shock protein; IDH, NADP + -dependent isocitrate dehydrogenase; NF-jB, nuclear factor- jB; NHS, N-hydroxysuccinamide; RAGE, receptor for advanced glycation end-products; SPR, surface plasmon resonance. (Received 1 8 March 2004, revised 21 July 2004, accepted 2 A ugust 2004) Eur. J. Biochem. 271, 3765–3775 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04318.x the highest homologies to S100A8 (40%) and to S100A9 (46%) [29]. Upon Ca 2+ binding, S100A12 undergoes a conformational c hange, supporting the idea t hat this p rotein is involved in intracellular C a 2+ signal transduction [30]. In the present study, we have focused our attention on identifying i ntracellular m ammalian proteins that bind S100A12 in a Ca 2+ -dependent manner. Utilizing S100A12-coupled Sepharose in affinity chromatography, we have identified cytosolic NADP + -dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), annexin V, S100A9 and S100A12 itself as S100A12-binding proteins. Experimental procedures Materials Recombinant rat S100A1 and S100B were prepared as described previously [31]. Recombinant human annexin V was a gift from A. Iwasaki 1 (Kowa C o., L td, N agoya, Japan) and recombinant human S100A9 a gift from K. Fukuda (Asahi Kasei Corp., Osaka, Japan). Aldolase was obtained from Sigma-Aldrich, IDH and GAPDH from Roche Diagnostics, N-hydroxysuccinimide (NHS) 2 -activated Seph- arose 4 Fast Flow and Protein-G Sepharose from Amer- sham Biosciences, and bis-(sulfosuccinimidyl) suberate (BS 3 ) from Pierce Biotechnology, Inc. Anti-aldolas e i mmu- nogloblulin was purchased from Roche Diagnostics, anti- IDH a nd anti-GAPDH immunoglobulin from Nordic Immunological Laboratories, and anti-annexin V and anti-S100A9 immunoglobulin from Santa C ruz Biotechno- logy, Inc. All other r eagents were at l east of analytical grade. Construction and purification of S100A12 and its truncation mutants The bovine lung S100A12 coding sequence was obtained b y PCR using the corresponding full-length cDNA, w hich was cloned from a bovine lung Uni-Zap XR library [18], and mutations were also induced by PCR. The oligonucleotides employed as PCR primers installed an Nde Irestriction enzyme site 5 ¢ to the ATG start c odon (sense primer), and different stop codons and an adjacent BamHI restriction enzyme site at the 3¢ end of the cDNA (antisense primers). The P CR products were gel-purified and cloned into pT7Blue T-Vector (Novagen). The nucleotide sequence of each mutant was confirmed by automated DNA sequencing (model 377; Applied Biosystems). An NdeI–BamHI frag- ment of wild-type and all mutant cDNAs was ligated into the same restriction en zyme site of pET-11a (Nov agen) and then the plasmid was introduced into competent Escheri- chia coli BL 21(DE3) (Stratagene). Bacterial expression and purification was carried out as described previously [32]. All proteins were judged to be greater than 9 5% pure, based on 1 2% Tricine/SDS/PAGE [33]. Affinity chromatography The affinity matrix was prepared as follows: 2 mg of recombinant wild-type S 100A12 and its t runcation mutants was suspended in 2 mL of NaCl/P i (PBS) ( pH 7.0) and mixed with a slurry (2 mL) of NHS-activated Sepharose. Coupling w as allowed to proceed for 5 h at r oom tempera- ture on a rocking table. The gel was then washed once with NaCl/P i and incubated for 4 h at room temperature with 50 mL of 1 M Tris/HCl ( pH 7.5). Af finity chromatography was performed as described previously [24]. Briefly, bovine lung (5 g) was homogenized with 6 volumes of 20 m M Tris/ HCl, 0.1 m M EGTA (pH 7.5) a nd centrifuged at 1 8 000 g for 2 0 min at 4 °C. The s upernatant w as adjust ed to a Ca 2+ concentration of 1 m M with 1 M CaCl 2 , followed by paper filtration. The extract was applied to the affinity column (2 mL of bed v olume), equilibrated in, and w ashed with, 20 volumes of the same buffer as used for homogenization and adjusted to the same Ca 2+ concentration a s for the supernatant. After washing the column, bound proteins were eluted first with Tris buf fer containing 5 m M EGTA alone, and then with the addition of 1 M NaCl. The eluted proteins were analyzed by SDS/PAGE. Protein sequencing Protein samples were subjected to SDS/PAGE and trans- ferred to poly(vinylidene difluoride). After staining with Ponceau S [34], the protein bands were cut out and digested with lysylendopeptidase [35]. After overnight incubation at 37 °C, the proteolytic fragments were separated by HPLC (model L C-10A; Shimadzu) in a C18 reverse-phase column (TSK-gel ODS-80; Tosoh) with a linear gradient o f 0–80% (v/v) a cetonitrile in the presence of 0 .1% (v/v) trifluoroacetic acid at a flow rate of 1 mLÆmin )1 . The amino acid sequence of each proteolytic fragment was determined with an automated protein sequencer (model PPSQ-21; Shimadzu). Antibody production Antibody to bovine S100A12 was produced by intramus- cular injection of 1 mg of recombinant S100A12 protein (emulsified in Freund’s complete adjuvant) into rabbits. Booster shots of t he antigen in Freund’s i ncomplete adjuvant were given three times at 2-week intervals. The rabbits were bled 10 days after the last injection and the specificity of antibody was check ed by Western blotting. Immunoprecipitation and Western blotting Bovine lung (1 g) was homogenized in five volumes of 20 m M Tris/HCl, p H 7.6, 0.1 M KCl, 0.2 m M phenyl- methanesulfonyl fluoride with 1 m M CaCl 2 and t hen centrifuged at 15 000 g for 30 min. The extract (100 lL) was incubated for 1 h at 25 °Cwith1lg of anti-S100A12 immunoglobulin. Protein G–Sepharose (20 lL) was added to the sample, which was then rotated for 3 h. Sepharose beads were washed four times and processed for SDS/ PAGE. Immunoprecipitates were analyzed by Western blotting with antibodies specific for IDH, aldolase, GAPDH, annexin V, S100A12 or S100A9. Surface plasmon resonance (SPR) analysis SPR analysis of real-time protein–protein interactions was carried out using BIAcore X (Biacore, Inc.). All steps were 3766 T. Hatakeyama et al. (Eur. J. Biochem. 271) Ó FEBS 2004 performed at 25 °C. Recombinant S100A12 was covalently linked to carboxymethylated dextran on the surface of the s ensor chip , CM5, by amine coupling, according to the manufacturer’s instructions. R ecombinant S100A12 (50 lgÆmL )1 ,60lL) was immobilized in 20 m M sodium acetate, 0.15 M NaCl, pH 6.0, until 13 000 response units (1.45 p mol) were bound and a st able baseline was obtained. As a control for nonspecific binding, a surface without S100A12 (untreated surface) was tested in all experiments. Samples of S100A12 and its mutants (27 n M ,54n M , 108 n M , 216 n M , 436 n M and 873 n M ), S100A9 (0.23 l M , 0.46 l M ,0.93l M ,1.85l M and 3.70 l M ), annexin V (0.35 l M ,0.70 l M ,1.39 l M ,2.78 l M and 5.56 l M ), GAPDH (1.50 l M ,3.0l M ,4.5l M ,6.00l M ,9.00l M and 12.00 l M ) and IDH (1 .31 l M ,2.63l M ,5.26l M ,7.89l M and 10.52 l M ), were prepared in a running buffer containing 10 m M HEPES, pH 7.0, 150 m M NaCl, and 0.005% (v/v) Tween-20 in the p resence of 1 m M CaCl 2 or 1 m M EGTA at a flow rate of 20 lLÆmin )1 . The S100A12-coupled sensor chip was regenerated between protein injections with a brief (60s)washin5m M EGTA, 2 M NaCl until the baseline returned to its preinjection level. The equilibrium dissoci- ation constant, K D , was deduced from the kinetics of the binding from the on (k a ) and off (k d )rates(K D ¼ k d /k a ). The rate constants were determined using BIAEVALUATION 3.0 software using numerical integration algorithms. Dimer formation and cross-linking of S100A12 and its mutants Purified wild-type S100A12 and its mutant proteins were preincubated each at a concentration of 0.5 mgÆmL )1 in 20 m M HEPES (pH 7.0) in the presence of 1 m M CaCl 2 or 2m M EGTA for 20 min at 25 °C. Subsequently, aliquots were incubated with 5 m M BS 3 crosslinker for 30 min at 25 °C. Reactions were quenched by adding Tris to a final concentration of 50 m M . Proteins were visualized by 12% Tricine/SDS/PAGE. Gel filtration Gel filtration was carried out on a Superdex 75 column (HR 10/30; Amersham Biosciences)-connected FPLC chroma- tography system (Amersham Biosciences). The hydro- dynamic analysis was carried out with 50 lgofthe respective protein. The column buffer w as 20 m M HEPES, pH 7 .0, 150 m M NaCl and 1 m M CaCl 2 or 1 m M EGTA. The column w as operated at a flow rate of 0.5 mLÆmin )1 and a p rotein was detected using th e Bradford a ssay ( Bio-Rad). The column was calibrated using transferrin (81 kDa), ovalbumin (43 k Da), myoglobin (17.6 kDa), ribonuclease A (13.7 kDa) and aprot inin (6.5 kDa). Chaperone-like activity assay The thermal-induced aggregation of IDH, aldolase and GAPDH was performed according to established protocols [36]. Protein solutions with or without S100A12 were mixed at room temperature and then heated at 65 °C for 5 min in a thermal cycler. The light scattering of the solution a t 488 n m was measured in a spectrophotometer (Model UV - 1600; Shimadzu). Results Identification of target proteins for S100A12 in bovine lung Affinity chromatography with recombinant S100A12 cou- pled to Sepharose was used to determine whether cytosolic proteins of bovine lung were able to bind S100A12 in a Ca 2+ -dependent manner (Fig. 1). Lung extract with 1 m M CaCl 2 added was applied to the S100A12 affinity column. When Ca 2+ -dependent binding proteins were released following application of a buffer c ontaining 5 m M EGTA, a distinct protein peak was eluted f rom the column (Fig. 1 A). The proteins found in the peak fraction migrated at 38, 36, 35, 34, 23 and 6.5 kDa on SDS/PAGE ( Fig. 1B). To identify these S100A12-binding proteins, a mino acid sequencing o f the proteins was performed. The proteins were digested with lysylendopeptidase, and the proteolytic products were separated by reverse-phase HPLC on a C18 column. Amino acid sequence analysis was performed on several major peptides. Referring to the NBRF protein sequence database and the SwissProt database, the sequences of 38, 36, 35, 34, 23 and 6.5 kDa proteins matched IDH, aldolase, GAPDH, annexin V, S 100A9 and S100A12, respectively (Fig. 1C). Of the 12 determined amino acid sequences of the 23 kDa protein, 11 residues were found to be identical t o those of bovine S 100A9, except for one residue at position 41. The sequences of th e other proteins were completely identical to those of each protein compared. To i dentify, more strongly, binding proteins of S100A12, we also eluted the S100A12-Sepharose column with a 1 M NaCl-containing buffer, after elution with the EGTA-containing buffer. As shown in lane 2 of Fig. 1B, trace amounts of h igh molecular mass proteins (ranging from 45 to 100 kDa) were detected. S100A12 interacts with IDH, aldolase, GAPDH, annexin V and S100A9 in vivo We performed co-immunoprecipitation experim ents to demonstrate that in vitro S100A12 and target protein associations also occur under physiological conditions in mammalian cells. A physical interaction between S100A12 and IDH, aldolase, GAPDH, annexin V or S100A9 was demonstrated by the observation that I DH, aldolase, GAPDH, annexin V or S100A9 immunoprecipitate with the a nti-S100A12 immunoglob ulin from bovine lung extracts (Fig. 2). S100A12, IDH, aldolase, GAPDH, ann- exin V and S100A9 were not detected after immunopreci- pitation with rabbit preimmune serum or other nonspecific antibodies (data not shown). These experiments indicate the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V or S100A9 under physiolo- gical c onditions in mammalian cells, although t he possibility that the complexes were formed after lysis cannot be excluded. Affinity determination of the interaction between S100A12 and its target proteins To study t he real-time binding kinetics of S100A 12, S100A9, annexin V, GAPDH and IDH to S100 A12, Ó FEBS 2004 S100A12 target proteins (Eur. J. Biochem. 271) 3767 recombinant S100A12 was immobilized on a biosensor chip surface and the protein complex formation was analyzed by SPR. The sensorgrams of the interactions are shown i n F ig. 3 . The binding of S100A12, S100A9 and annexin V to immobilized S100A12 was strictly Ca 2+ dependent (Fig. 3A–C). These observations are consistent with the results of affinity chromatograp hy experiments as described above. In contrast, the binding of GAPDH and IDH showed weak Ca 2+ dependency (Fig. 3D,E). The discrepancy between the Ca 2+ -sensitive elution of GAPDH and IDH from the S100A12-Sepharose and the relative Ca 2+ insensitivity in the SPR a nalyses, remains t o be explained. The immobilization of S 100A12 to a CM-sensor chip via its primary amines could have modified the Ca 2+ -dependent conformational change of the protein. The binding curves of S100A12, S100A9, annexin V, GAPDH, and IDH were fitted to the bivalent binding model. When the b inding curves were fitted t o the bivalent model, the Ôgoodness of fitÕ was indicated by a v 2 value of < 1.0. All other models had a v 2 of > 1.0, indicating higher nonrandom deviation from the fitted curve. The binding affinities determined for these target proteins to S100A12 are summarized in Table 1. S100A12 bound to immobilized S100A12 approximately 100-fold more strongly than it bound to S100A9 or annexin V, and about 10-fold more strongly than it bound to GAPDH or IDH. There is a discrepancy between the relatively low- affinity binding of S100A9 and annexin V to S100A12 and the complex formation of these proteins observed by the affinity chromatography (Fig. 1) and the coimmunopre- cipitation experiments (Fig. 2). T his discrepancy appears t o be explained by the high levels of S100A9 and annexin V expressed in bovine lung [24,37]. As aldolase has a s trong net positive charge (pI ¼ 8.9), it bound nonspecifically to the CM5 sensor chip, presumably owing to electrostatic interaction to the surface (data not shown). Proteins Sequences a : NADP -dependent isocitrate dehydrogenase (IDH) b : fructose-1,6-bisphosphate aldolase A (aldolase) c : glyceraldehyde 3-phosphate dehydrogenase (GAPDH) d : annexin V e : S100A9 f : S100A12 ISGGSVVEM 5 ISGGSVVEM 13 IFPYVELDLHSYD 31 IFPYVELDLHSYD 43 ADDGRPFPQVIK 87 ADDGRPFPQVIK 98 RVIISAPSADAPMFV 116 RVIISAPSADAPMFV 130 FNGTVK 53 FNGTVK 58 QVYEEEYGSSLEDD 127 QVYEEEYGSSLEDD 140 QLVQK 35 QLVQK 39 EQPNFLK 40 ELPNFLK 46 IFQDLDADK 56 IFQDLDADK 64 TAHIDIHK 84 TAHIDIHK 91 0.6 0.3 0 Fraction number 5 mM EGTA 5 mM EGTA/1 M NaCl MW 97.4 66.2 45.0 31.0 21.5 6.5 a b c d e f 0 10 20 30 40 EGTA EGTA/NaCl 14.4 A C B Protein concentration (A 595 ) (kDa) + Fig. 1. Identification o f S100A12-binding proteins fro m bovine lung. (A) Elution p rofile obtained f rom bovine lung extract a fter S100A12 affinity chromatography. (B) Tricine/SDS/PAGE (12%) of E GTA and EGTA/NaCl eluates. The 38, 36, 35, 3 4, 23 and 6.5 kDa proteins were tentatively nameda,b,c,d,eandf,respectively.(C)Partialaminoacidsequencesof a–f. The amin o acid se qu ence(s) o f protein a w as (were) compared with those of NADP + -dependent isoc itrate dehydro genase (IDH), p rotein b with fructose-1,6-bisphosphate aldolase A (aldolase), p rotein c with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein d with annexin V, protein e with S100A9, and protein f with S100A12. The numbers indicate residue numbers for each protein. 3768 T. Hatakeyama et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Mapping of the S100A12 binding site on S100A12 itself To localize the site of S100A12 interaction on immobilized S100A12, we examined the binding of a series o f C-terminal truncation mutants to wild-type S100A12 coupled to the sensor chip in the presence o f C a 2+ . A schematic r epre sen - tation of the amino acid sequences of the four mutants is given in F ig. 4A. Figure 4B and Table 1 show the kinetics of the interactions between the t runcation mutants and wild-type S100A12. C89 bound readily to the immobilized wild-type S100A12 in the presence of Ca 2+ , while further truncation resulted in a significant reduction of the binding. C74 did not bind at all t o the immobilized S100A12. Owing to a faster dissociation rate, the C89 fragment bound to the immobilized S100A12 about 10-fold more weakly than full- length S100A12. C86 and C79 had much lower affinities for the immobilized S100A12 t han the full-length S100A12. C74 did not bind to the immobilized S100A12. The results of these experiments suggest that the S100A12-binding site on S100A12 itself is located at the extreme C terminus (residues 87–92) of the protein. Dimerization of S100A12 mutants To determine whether the C-terminal e xtension (residue 87–92) truncation mutant of S100A12 affects the ability to dimerize, chemical cross-linking experiments were employed using BS 3 . The results clearly indicated that wild-type S100A12 and all of the S100A12 mutants, except C74, form homodimers (Fig. 5). The formation of homodimers was found to occur both in the presence and absence of Ca 2+ , suggesting that Ca 2+ is not essential for S100A12 dimeri- zation. Furthermore, these data indicate that the C-terminal residues 87–92 are not critical for dimerization. In cross-linking experiments, C74 did not migrate into t he gel because of possible aggregation. The total absence of C-terminal extension, including helix IV, m ight cause instability of the molecule. Gel filtration To circumvent the use of a chemical cross-linking reagent, which could stabilize rather weak and nonspecific protein interactions, we next employed analytical gel filtration to analyze the hydrodynamic p arameters of S 100A12 and its truncation mutants in solution. Results f or wild-type S100A12, and the C89, C86 and C74 mutants, are summarized in Fig. 6. When compared with elution posi- tions of marker proteins, the elution position of wild-type S100A12 corresponds to  30.3 kDa. This differs slightly from the 21.3 kDa position expected for a dimeric S100A12 of perfectly globular shape, and indicates that dimeric S100A12 s hows s ome deviations from a globular shape. The dimeric nature of wild-type S100A12 in the 30.3 kDa peak was verified by gel filtration of covale ntly cross-linked wild- type S100A12 dimers, which showed an elution position indistinguishable from that of the noncross-linked protein (data not shown). When compared with the wild-type S100A12, C89 and C86 mutants show almost identical elution profiles, i.e. they elute in a s ymmetrical peak at the dimer position. When analyzed by analytical gel filtration, the elution position of the C74 mutant corresponds to  129 kDa, in dicating possible aggregation. Interaction of C-terminal truncation mutants of S100A12 and the S100A12 target proteins To further determine the role of S100A12 C-terminal residues i n S100A12 target protein interactions, we prepared affinity matrices coupledwith C-terminal truncation mutants andusedthemfortheCa 2+ -dependent affinity chromato- graphy o f bovine l ung extracts (Fig. 7). Immobilized C89, as well as immobilized wild-type S100A12, were able to interact with all t he target proteins such as IDH, aldolase, GAPDH, annexin V, S100A9, and S100A12. However, immobilized C86 and C74 mutants readily bound to IDH, aldolase, GAPDH and annexin V, b ut not at all to S100A9 or S100A12. These results demonstrate that C-terminal residues 75–92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH a nd annexin V. Effects of S100A12 on the heat-induced aggregation of IDH, aldolase, and GAPDH To analyze the functional properties of S100A12, we studied its action on protein-folding reactions in vitro (Fig. 8). The thermal unfolding and aggregation of IDH, aldolase, and GAPDH was used as a typical assay system. Heat ing 3 l M IDH, aldolase o r G APDH at 65 °Ccausedprotein aggregations in less than 5 min. In t he absence of S100A12, 1 m M Ca 2+ had essentially no effect on the unbound bound IP anti-S100A12 IDH aldolase GAPDH annexin V S100A9 S100A12 WB: anti-IDH WB: anti-aldolase WB: anti-GAPDH WB: anti-annexin V WB: anti-S100A9 WB: anti-S100A12 41.6 - kDa 28.2 - 14.8 - 41.6 - 28.2 - 41.6 - 41.6 - 41.6 - Fig. 2. S100A12 interaction with NADP + -dependent IDH, aldolase, GAPDH, annexin V, S 100A9 and S100A12 in vivo. Bovine lung extracts were immunoprecipitated with an anti-S100A12 immuno- globulin in the presence o f 1 m M CaCl 2 . The immunoprecipitates were analyzed by Western blotting with anti-IDH, anti-aldolase, anti- GAPDH, anti-annexin V, anti-S100A9 and anti-S100A12 immuno - globulins. IP, i mmunoprecipitation, WB, Western blotting. Molecular mass standards a re shown at t he left. Ó FEBS 2004 S100A12 target proteins (Eur. J. Biochem. 271) 3769 thermal aggregation of IDH (Fig. 8A), aldolase (Fig. 8B) and GAPDH (Fig. 8C). However, Ca 2+ had a dramatic effect on the chaperone/antichaperone behavior of S100A12. The thermal aggregation of IDH or GAPDH was greatly facilitated by S100A12 in the absence of Ca 2+ (Fig. 8 A,C), whereas S 100A12 suppressed the aggregation of aldolase to less than 50% in the presence of Ca 2+ (Fig. 8B). S100A12 alone caused slight optical changes in the light-scattering analysis (Fig. 8). These behaviors of S100A12 are similar to those of some of the well-studied molecular chaperones or antichaperones [36]. Discussion Recently, S100A12 was demonstrated to trigger a signal transduction cascade by binding to the cell-surface recepto r RAGE in endothelium, lymphocytes, and mononuclear phagocytes, resulting in activation of the transcription factor nuclear factor-jB(NF-jB) [25]. Based on these findings, a molecular model for the involvement of extra- cellular S100 p roteins in i nflammatory processes w as proposed [25]. I dentification of the in vivo S100A12 target proteins, a nd characterization of their m ode of interaction with S100A12, is essential to d etermine the exact contribu- tion of the S 100A12-dependent signaling pathways in cellular function. S100 proteins have been implicated in intracellular and extracellular regulatory activities. Mem- bers of this protein family have been shown to interact with several target proteins w ithin cells, t hereby regu lating enzyme activities, cell growth and differentiation, and Ca 2+ homeostasis. In the present study, we have focused our attention on identifying intracellular S100A12-binding proteins and presented evidence showing that IDH, aldo- lase, G APDH, annexin V, S100A9 a nd S100A12 a re putative S100A12 target proteins. An overlap between t he specificities of different S100 proteins and CaM for inter- acting target proteins, peptides, and drugs is often observed [14,16,38]. Hence, aldolase, GADPH and annexin V in 0 50 100 Time (sec) Responses (RU) 450 350 250 150 50 0 0 50 100 0 50 100 0 50 100 0 50 100 250 200 150 100 50 0 Responses (RU) Time (sec) 250 200 150 100 50 0 Responses (RU) 250 200 150 100 50 0 Responses (RU) 250 200 150 100 50 0 Responses (RU) (S100A12) (S100A9) (annexin V) (GAPDH) (IDH) Time (sec) Time (sec) Time (sec) Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ EGTA EGTA EGTA EGTA EGTA AB D C E Fig. 3. The b inding of S100A12 (A), S100A9 (B), annexin V (C), GAPDH (D), and IDH (E) to immobilized S100A12. Overlayplotofthereal-time binding data r ecord ed upon interaction of i mmobiliz ed S100A12 with its target proteins. S10 0A12 was cova lently attached to the surface of th e CM5 senso r ch ip, as described i n t he Exp erime ntal pro cedures. T he trac e s re flect the amount of p rote in c omplex formed at the surface over time. Each sensorgram consists of an association phase and a dissociation phase during the sample flow, reflecting the decay of the complex when the target protein was removed from the flow cell. S100A12 (873 n M ), S100A9 (3.70 l M ), annexin V (5.56 l M ), GAPDH (12.0 l M )andIDH (10.52 l M ) were injected at a flow rate of 20 lLÆmin )1 in the presence of 1 m M Ca 2+ or 2 m M EGTA. Table 1. The affinity kinetics of interaction between S100A12 and its target proteins in the presence of 1 m M CaCl 2 . Therateconstants(k d and k a ) were derived from analysis of the dissociation and association phases, respectively, of the s ensorgrams. K D was calculated from the ratio o f k d and k a . G APDH, g lyceraldehyde-3-phosphate dehydroge- nase;IDH,NADP + -dependent isoc itrate dehydro genase. WT , wild- type S100A12; C89, C86, C79 and C74, truncated mutants of S100A12. Analyte K D ( M ) k a (1/Ms) k d (1/s) S100A12 4.09 · 10 )9 1.32 · 10 5 5.40 · 10 )4 S100A9 1.52 · 10 )7 2.03 · 10 4 3.08 · 10 )3 Annexin V 6.21 · 10 )7 6.30 · 10 3 3.91 · 10 )3 GAPDH 8.27 · 10 )8 9.43 · 10 3 7.80 · 10 )4 IDH – 8.51 · 10 3 – S100A12 (WT) 2.18 · 10 )9 6.23 · 10 4 1.36 · 10 )4 C89 6.62 · 10 )8 7.27 · 10 4 4.81 · 10 )3 C86 1.86 · 10 )7 1.23 · 10 4 2.29 · 10 )3 C79 – – – C74 – – – 3770 T. Hatakeyama et al. (Eur. J. Biochem. 271) Ó FEBS 2004 crude cell extracts that bind t o S100A12–Sepharose beads also bind to other S100 proteins or CaM [15,18–22,39]. IDH, S100A9 and S100A12 are the only proteins identified, to date, that demonstrate strict interactions with S100A12 and a bsolutely n o binding to CaM (data not shown). The results of specific binding proteins of S100A12, examined in the present study, are briefly d iscussed below. S100A12 and S100A9 Complex formation is regarded to be an essential pre- requisite for the biological function of S100 proteins [1]. A characteristic property of S100 proteins is their tendency to dimerize. The question raised by the interaction between S100A12 and S100A12 itself o r S100A9 c oncerns homo- or A B Fig. 4. Binding of the C-terminal trun cation mutants of S100A12 to immobilized S100A12. (A) Amino ac id sequences of the wild-type S100A 12 (WT; 92 amino acids) a nd the C -terminal trun cation mut ants (C89, C 86, C79 a nd C74). S haded boxe s indicate fi rst and s econd Ca 2+ -binding loops o f S100A12. (B) Overlay plot of sensorgrams recorded on injection of WT, C89, C86, C79 and C74 (873 n M each) over surfaces containing immobilized S100A12 in the presence of Ca 2+ . CaCl 2 − + − − + − − + − − + − EGTA − − + − − + − − + − − + MW 97.4 66.2 45.0 31.0 21.5 (kDa ) dimer monomer WT C89 C86 C74 6.5 14.4 a b c a b c a b c a b c Fig. 5. Dimerization of wild-type S100A12 (WT) and its C -terminal truncation mutants. S100A12 dimerization, as revealed by chem- ical cross-linking. WT, C89, C86 and C74 (5 mg) were cross-linked using 2 m M bis- (sulfosuccinimidyl) suberate (BS 3 ) cross- linking agent. D etails of the assays are described in the Experimental procedures. Lane a, noncross-linked proteins; lane b, proteins cross-linked in the p re sence of 1 m M CaCl 2 , and lane c, proteins cross-linked in the presence of 2 m M EGTA. The symbols Ô+Õ and Ô–Õ indicate the presence and absence, respectively, o f the pertinent reactant in the reaction mixture. B lank arrows indicate possible aggregation. Ó FEBS 2004 S100A12 target proteins (Eur. J. Biochem. 271) 3771 hetero-dimerization processes. Deletion of the C-terminus (residues 87–92) o f S100A12 was found to be sufficient to abrogate interaction with S100A12 (Fig. 4). To study the influence of this moiety on the dimerization, a set of C-terminal deletion experiments were also carried out. T he deletion of the C-terminal six residues did not prevent S100A12 homodimerization (Figs 5 and 6). It is therefore possible to state that S100A12 interaction with S100A12 itself or S100A9 has a similarity with the i nteraction between S100B (or S 100A1) and its target proteins [40,41]. Recently, Ost erloh et al. reported that the C-terminal domain (residues 88–90) of S100A1 is essential for TRTK peptide bind ing, but dispensable for dimerization [40]. More recently, Deloulme et al. s howed that deletion of the extreme C-terminal residues (84–91) of S100B had no effects on its dimerization but significantly decreased the interaction between S100B and S100A6 or S100A11 [41]. Further d eletions which affect he lix IV completely abolished both S100A12 and S100A9 interactions, and S100A12 dimerization, con firming the importance of t his r egion fo r S100A12 complex formation. The observed Ca 2+ depend- ency of the interaction of S100A12 with S100A 12 itself or S100A9, b ut not of the S100A12 dimerization, also supports such a model. Annexin V Members of the annexin family have been shown to bind proteins of the S100 family. Annexin I has been shown to bind S100 A11 [ 22], annexin II binds S100A10 [ 42], S100A6 [43] and S100B [19], annexin XI binds S100A6 [20], and annexin V and VI bind S100A1 and S100B [18]. The annexin II–S100A10 interaction is Ca 2+ independent, while other annexin–S100 protein interactions are shown to be Ca 2+ -dependent [44]. The mode of interaction o f annexin V with S100A12 differs from the mod e of a nnexin II– S100A10 i nteraction. In the c ase o f t he latter, the C-terminal domain of S100A10 was proven to be critical for the recognition of annexin [45]. However, in the case of the annexin XI–S100A6 interaction, helix I of S100A6 was found to be e ssential for the recognition o f annexin XI [46]. Similarly, helix I o f S100A11 was shown to be critical in the recognition of annexin I [22]. Thus, there are a number o f modes of interaction between members of the annexin family and those of t he S100 family. Although t he precise physiological significance of these interactions is not known, Garbuglia et al. reported that binding of annexin VI, but not annexin V, to S100A1 and S100B blocks the ability of these two S100 proteins to inhibit glial fibrillary acidic protein and desmin assemblies [18]. Enzymes Previous biochemical studies have identified many potential S100 target enzymes, such as glycogen phosphorylase [4], twitchin kinase [14], aldolase [15], Ndr kinase [16], phospho- glucomutase [17] and G APDH [ 47], some e xhibiting Ca 2+ -dependent modulation while others show Ca 2+ - independent modulation. Both the affinity chromatography and the BIAcore experiments showed a high-affinity inter- action between IDH, aldolase and GAPDH, and immobi- lized S100A12. These observations prompted us to identify Fraction (ml) Fraction (ml) Fraction (ml) Fraction (ml) V 0 81kDa 43kDa 17.6kDa 13.7kDa 6.5kDa WT C89 C86 C74 0 0.05 0.1 0.15 0.2 51015 0 0.05 0.1 0.15 0.2 51015 0 0.05 0.1 0.15 0.2 51015 0 0.05 0.1 0.15 0.2 51015 V 0 81kDa 43kDa 17.6kDa 13.7kDa 6.5kDa V 0 81kDa 43kDa 17.6kDa 13.7kDa 6.5kDa V 0 81kDa 43kDa 17.6kDa 13.7kDa 6.5kDa Absorbance at 595nmAbsorbance at 595nmAbsorbance at 595nmAbsorbance at 595nm Fig. 6. Analytical gel filtration of wild-type S100A12 (WT) and its C-terminal truncation mutants. Gel filtration of the purified proteins (WT, C89, C86 and C74) was carried out on a Superdex 75 c olum n i n the absen ce of Ca 2+ . The respective protein peaks are given as a function of volume. E lution volumes of the marker proteins tranferrin (81 kDa), ovalu min (43 k Da), myoglob in (17.6 kDa), ribonuclease A (13.7 kDa) and ap rotinin (6.5 kDa) are indicated for comparison. 3772 T. Hatakeyama et al. (Eur. J. Biochem. 271) Ó FEBS 2004 residues on S 100A12 involved i n the int eraction of S 100A12 with the enzymes IDH, aldolase and GAPDH. While the C-terminal domain of S100A12 was found to be essential for the interaction of imm obilized S100A12 with S100A12 itself or S100A9, this domain was not n ecessary for the interaction of S100A12 with the three enzymes. Several S100 proteins have also been shown to regulate other enzyme activ- ities. For example, Zimmer et al. reported the activation of aldolase by S100A1 and S100B [15], the inhibition of glycogen phosphorylase by S100A1 [4], the activation of WT C89 C86 C74 MW S100A12 S100A9 IDH aldolase GAPDH annexin V (kDa ) 31.0 21.5 14.4 6.5 66.2 45.0 97.4 Fig. 7. Role of C-terminal residues of S100A12 in S100 A12 target p rotein inter actions. Affinity chromatographies with wild-type S100A12 (WT), C89, C86 and C74 coupled to NHS 4 - activated Sepharose were used to determin e whether c ytosolic proteins o f bovine lung were able to bind to S100A12 in a Ca 2+ -dependent manner. Lung extract (3 mL) with 1 m M CaCl 2 added was applied to the affinity columns (0.5 m L of the bed volume). After washing, the b ound proteins were e luted with SDS sample buffer. The individual fractions (5 lL) were then subjected to S DS/PAGE. 0 10 50 0.4 0.3 0.2 0.1 0 0 10 50 0 10 50 0.5 0.4 0.3 0.2 0.1 0 0.6 0.4 0.2 0 Absorbance at 488 nm Absorbance at 488 nm S100A12 (µg/ml) S100A12 (µg/ml) Absorbance at 488 nm S100A12 (µg/ml) (IDH) (aldolase) (GAPDH) AB C Fig. 8. Effects of S100A12 on th e thermal aggregation of NADP + -dependent IDH, aldolase and GAPDH. Light s cattering owing to t hermal (65 °C) aggregation was measured as a bsorbance at 488 nm (vertical axis) in a spectrophotometer. Thermal aggregation of IDH (A), aldolase (B), and GAPDH (C), 3 l M each, in the p resence (0.2 m M CaCl 2 , close d t riangles) or a bsen ce (2 m M EGTA, op en t rian gles) of C a 2+ , with the addition o f S100A12 a t various d ifferen t concentrations. O pen and c losed circles i ndicate the prese nce (1 m M CaCl 2 ) a nd ab sence ( 2 m M EGTA), respectively, of Ca 2+ without the addition of target p rotein (IDH, aldolase or GAPDH). Each value represents the m ean and SD of three e xperiments. Ó FEBS 2004 S100A12 target proteins (Eur. J. Biochem. 271) 3773 phosphoglucomutase by S100B [17], and the inhibition of phosphoglucomutase by S100A1 [17]. Filipek et al. showed that S100A6 had no influence on the V max and K m of GAPDH [47]. These reports suggest that S100 proteins might have a role in the regulation of energy metabolism. Further experiments are required to draw definitive conclu- sions about the physiological relevance o f these data. Molecular c haperones consist of several groups of proteins that suppress the aggregation of unstable inter- mediates of proteins and are implicated in protein f olding, protein targeting to membranes, protein renaturation, subcellular transport and degradation [48]. In contrast, some proteins, such as disulfide isomerase [ 49] and t protein [50], facilitate the misfolding and aggregation of their substrate proteins. These functions, termed Ôanti-chaperone activityÕ, may provide a common mechanism for aggregate formation in the cell [49]. Recently, we have identified S100A1, b ut not CaM, as a potent m olecular chaperone and a new member of the heat shock protein (Hsp)70/Hsp90 multichaperone complex [51]. In the present report, we investigated whether S100A12 could also have a c haperone- like o r an antichaperone-like a ctivity. We used IDH, aldolase, and G APDH as substrates to examine t he possible chaperone/antichaperone function of S100A12 in vitro.In the absence, but not in the presence, of Ca 2+ , S100A12 could facilitate the thermal aggregation o f IDH and GAPDH. In contrast, in the presence of Ca 2+ , S100A12 served as a chaperone and inhibited the thermal aggregation of aldolase. 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Interaction of C-terminal truncation mutants of S100A12 and the S100A12 target proteins To further determine the role of S100A12 C-terminal residues i n S100A12 target protein interactions, we prepared affinity. pre- requisite for the biological function of S100 proteins [1]. A characteristic property of S100 proteins is their tendency to dimerize. The question raised by the interaction between S100A12 and S100A12. sequences of th e other proteins were completely identical to those of each protein compared. To i dentify, more strongly, binding proteins of S100A12, we also eluted the S100A12- Sepharose column with