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MUSCLE CELL BIOLOGY AND CELL MOTILITY
Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
Submitted 16 August 2006 ; accepted in final form 23 December 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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smooth muscle; protein-protein interaction
22 kDa with two EF hands per monomer, function as Ca2+ sensors that respond to an increase in cytosolic free [Ca2+] with a conformational change that exposes a hydrophobic site of interaction with a variety of target proteins and thereby regulate their subcellular localization and/or activity (32). They exhibit unique patterns of tissue- and cell type-specific expression and have been implicated in the Ca2+-dependent regulation of diverse physiological processes, including cell cycle regulation, differentiation, growth, and metabolic control (12). S100 proteins have also been associated with a variety of pathological events, including neoplastic transformation and neurodegenerative diseases such as Alzheimer's, usually via overexpression of the protein (24). S100A11, originally discovered in this laboratory (10), is a member of the S100 protein family. It is also known as calgizzarin (38) and S100C (26). S100A11 exhibits broad tissue distribution, is homodimeric with two Ca2+-binding sites per 11,282-Da monomer, and exposes a hydrophobic surface on binding Ca2+ (1, 34). The three-dimensional structure of apo-S100A11 was determined by multidimensional NMR spectroscopy (9). Comparison with the structure of Ca2+-saturated S100A11 in complex with a peptide corresponding to the binding site on annexin A1 (annexin I) (30) revealed the conformational change occurring upon Ca2+ binding and confirmed the exposure of a hydrophobic site of interaction with the target peptide. In addition to binding to annexin A1, S100A11 has been shown to interact with actin (42) and transglutaminase (31), and is capable of forming a heterodimer with S100B through subunit exchange (8).
The objective of this study was to identify additional molecular targets of S100A11, specifically in smooth muscle.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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80% based on protein concentration measurements before and after coupling. Control columns were prepared in identical fashion but without inclusion of S100A11 or annexin A6. Polyclonal antibodies to full-length chicken gizzard S100A11 were raised in rabbits and the IgG fraction purified as previously described (1). Polyclonal antibodies to annexin A6 were produced in rabbits by Global Peptide Services (Ft. Collins, CO) by injection of a synthetic peptide corresponding to the 12 NH2-terminal residues with a COOH-terminal Cys (AKIAQGAMYRGSC) of rat annexin A6 (NCBI accession no. NP_077070) coupled to keyhole limpet hemocyanin. The IgG fraction was purified from the antiserum by HiTrap Protein A HP chromatography. A monoclonal antibody to rat S100A11 was raised against the purified full-length protein expressed in Escherichia coli and purified by HiTrap Protein G HP chromatography. Purification of chicken gizzard S100A11. S100A11 was purified from chicken gizzard as previously described (10) and stored at –80°C.
Affinity chromatography. Frozen chicken gizzards (1 g for small-scale analysis and 25 g for large-scale analysis) were thawed, minced, homogenized with a Polytron (setting 7) for 3 x 10 s in 4 volumes of 30 mM Tris·HCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 1 mM dithiothreitol (DTT), 0.3% (vol/vol) Triton X-100, 0.1 mg/ml leupeptin, 10 µg/ml pepstatin A, and centrifuged at 11,400 g for 15 min. The supernatant was diluted 1:1 with 30 mM Tris·HCl, pH 7.5, 150 mM NaCl, 2.4 mM CaCl2, 1 mM DTT, 0.3% Triton X-100, 0.1 mg/ml leupeptin, 10 µg/ml pepstatin A to give a final free [Ca2+] of 0.2 mM and applied to a column (1 ml for small-scale and 5 ml for large-scale analysis) of S100A11-Sepharose Fast Flow previously equilibrated with 30 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.2 mM CaCl2, and 1 mM DTT (buffer A). Unbound proteins were washed from the column with buffer A and bound proteins eluted with (in mM) 30 Tris·HCl, pH 7.5, 150 NaCl, 4 EGTA, and 1 DTT (buffer B). Fractions (0.125 ml for small-scale and 2 ml for large-scale analysis) were collected at a flow rate of 0.5 ml/min for small-scale and 10 ml/h for large-scale analysis.
Purified annexin A6, 
(1–20) annexin A6, COOH-terminal half-annexin A6 (M340-D671), and NH2 terminal half-annexin A6 (G5-R339) were dialyzed against buffer A and loaded on the S100A11 affinity column. The column was washed with buffer A until A280 returned to baseline. Bound proteins were eluted with buffer B, and fractions (1 ml) were collected at a flow rate of 10 ml/h and subjected to SDS-PAGE.
Purified chicken S100A11 (0.2 mg) was dialysed against buffer A and applied to an annexin A6-Sepharose Fast Flow affinity column previously equilibrated with buffer A. Unbound protein was washed from the column until A280 returned to baseline. Bound protein was then eluted with buffer B. Fractions (0.5 ml) were collected at a flow rate of 15 ml/h and subjected to SDS-PAGE.
RT-PCR.
Total RNAs were prepared from fresh tissues using the RNeasy Mini Kit (Qiagen), and oligo(dT)-primed first-strand cDNAs were prepared from the purified RNAs using either Omniscript (Qiagen) or Sensiscript (Qiagen) reverse transcriptases, according to protocols supplied by the manufacturer. All PCR reactions involved in cloning were performed using high-fidelity ProofStart DNA polymerase (Qiagen). Oligonucleotides were obtained from the University of Calgary Core DNA and Protein Services Facility. All constructs generated in this study were verified by complete sequence analysis at the same facility. Restriction endonucleases and DNA modifying enzymes were from either New England Biolabs or Invitrogen. Vectors used for cloning were dephosphorylated prior to ligation. All clones were first transformed into DH5
(Invitrogen) or Top 10 cells (Invitrogen) for purification of plasmid DNAs, which were then transformed into BL21 (DE3) cells (Invitrogen) for isopropyl-
-D-thiogalactopyranoside (IPTG)-induced expression of recombinant proteins.
Chicken S100A11 cDNA (NCBI accession no. P24479) was amplified and subcloned into pGEX-6P-1 (Amersham Biosciences) via BamHI and SmaI sites, from a first-strand cDNA prepared from chicken gizzard total RNA. The forward primer introduced a BamHI cleavage site (underlined) upstream of the ATG start codon (5'-CGGGATCCatgTCCAAGGTTTCCCCCACT-3') and the reverse primer introduced a SmaI cleavage site (underlined) downstream of the stop codon (5'-TCCCCCGGGctaAGGATGAGGAGGCTGAAC-3').
Rat S100A11 (NCBI accession no. AY688465) was amplified from a rat cerebral vessel first-strand cDNA using oligonucleotides designed against the mouse S100A11 cDNA (NCBI accession no. BC086903) using a KpnI site-containing forward primer (5'-GGGGTACCatgCCTACAGAGACTGAGAG-3') and a XbaI site-containing reverse primer (5'-GCTCTAGAttaGATTCGCTTCTGGGAAGT-3'), followed by cloning into the same sites of pcDNA3.0. For protein expression in bacteria, the coding region was amplified using the forward NdeI primer (5'-CACACATatgCCTACAGAGACTGAGAGATG-3') and reverse EcoRI primer (5'-GTGTGAATTCttaGATACGCTTCTGGGAAG-3') and cloned into the same sites in the expression vector pAED4, a pET-derived expression vector generously provided by Dr. J.-P. Jin (Northwestern University, Chicago, IL).
Chicken annexin A6 (NCBI accession no. Q6B344) was amplified from a first-strand cDNA prepared from chicken gizzard total RNA, and subcloned into pGEX-6P-1 (Amersham Biosciences) via the EcoRI and XhoI sites. The forward primer introduced an EcoRI cleavage site (5'-CGGAATTCatgGCACCCAAAGGAAAGGTT-3') and the reverse primer introduced a XhoI cleavage site (5'-CCGCTCGAGctaGTCGTCCCCCCCGCACAG-3'). The pGEX-6P-1/(1–20) annexin A6 mutant was prepared by amplifying the above clone using the forward EcoRI site-containing primer (5'-CGGAATTCAGCCAGGATGCAGACGCCTTG-3') and the same reverse primer listed above for the full-length chicken annexin A6. pGEX-6P-1/annexin A6 (G5-R339) was amplified and cloned using the forward EcoRI site-containing primer (5'-GAGAGAATTCGGAAAGGTTTACAGGGGCTCGGTGAAGGAC-3') and the reverse XhoI site-containing primer (5'-TCTCTCTCGAGtcaCCGATAGGCCACCTGCGCTGCCTCG-3'). pGEX-6P-1/annexin A6 (M340-D672) construct was generated from the pGEX-6P-1/annexin A6 clone by digestion with EcoRI and BlpI and replacement of the COOH-terminal fragment released with the annealed oligonucleotides EcoBpuUpper (5'-AATTCATGTGGGAGC-3') and EcoBpuLower (5'-TAAGCTCCCACATG-3'), annealed as described above.
Smooth muscle cells were enzymatically isolated from rat cerebral arteries as previously described (39) and mRNA purified using the RNeasy Mini Kit (Qiagen) with on-column DNase treatment, as described by the manufacturer. Reverse transcription was performed using the Sensiscript RT Kit (Qiagen) with 12 µl of isolated RNA as described by the manufacturer. To ensure the presence of RNA and successful transcription of cDNA, a positive control PCR was carried out using primers for RhoA [forward primer: 5'-CGGGATCCCGATGGCTCCCATC(C/A)GGAAG-3'; reverse primer: 5'-GGAATTCCTCACAAGA(T/C)(G/A)AGGCA(A/C)-3'] with 4 µl of cDNA and recombinant Taq polymerase (Invitrogen) as follows: 94°C for 3 min, 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, and extension at 72°C for 10 min. Endothelin-1 and ERG-3 were selected as markers to screen for contamination of the isolated smooth muscle cells with endothelial and neuronal cells, respectively. In each case, nested PCR was performed with 4 µl of cDNA, recombinant Taq polymerase and 35 cycles with outer primers (forward primer for endothelin-1: 5'-GAGCTGAGAAGGAAGTGCAGAG-3'; reverse primer for endothelin-1: 5'-GGTCTTGATGCTGTTGCTGATG-3'; forward primer for ERG-3: 5'-CCCAAGGTTAAAGAGAGGACACA-3'; reverse primer for ERG-3: 5'-AGCGGCACCATATTCTGAGTATC-3'). A 4-µl aliquot of this reaction was used as template with nested primers (forward primer for endothelin-1: 5'-TGTGTCTACTTCTGCCACCTG-3'; reverse primer for endothelin-1: 5'-GCCTCCAACCTTCTTAGTTTTCTT-3'; forward primer for ERG-3: 5'-GCAGACGCCACGCATCAAC-3'; reverse primer for ERG-3: 5'-ACGCACAAGACGCAGGAGAC-3') under the following conditions: 94°C for 3 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and extension at 72°C for 10 min. To screen for S100A11 and annexin A6 mRNAs, PCR was carried out on 4 µl of cDNA using gene-specific primers based on the mouse (NCBI accession no. 086903) and rat (NCBI accession number NM024156) sequences, respectively [forward primer for S100A11: 5'-CCTACAGAGACTGAGCG(G/C/A)TGCAT-3'; reverse primer for S100A11: 5'-CCTTCTGGTTCTTTGTGAAGGCA-3'; forward primer for annexin A6: 5'-AGGAGGATTATCACAAGTC-3'; reverse primer for annexin A6: 5'-ATGTTGAGCAGGTCTATC-3'] under the following conditions: 94°C for 3 min, 40 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 45 s, and extension at 72°C for 10 min for S100A11, and 94°C for 3 min, 40 cycles of 94°C for 45 s, 55°C for 60 s and 72°C for 135 s, and extension at 72°C for 10 min for annexin A6. PCR was carried out on a GeneAmp PCR System 2400 (Perkin Elmer) or MyCycler (Bio-Rad) Thermal Cycler.
Quantitative PCR.
mRNA was purified from freshly isolated cerebral arteries pooled from four rats with the RNeasy Mini Kit (Qiagen) with additional on-column digestion of DNA with the RNase-free DNase Set (Qiagen) according to the manufacturer's protocol. Purified RNA was used to synthesize cDNA by reverse transcription using the Sensiscript RT Kit (Qiagen), RNaseOUT ribonuclease inhibitor and oligo(dT)12–18 primer (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions in an iCycler Thermal cycler (Bio-Rad) with the following forward and reverse primers, respectively: for -actin, 5'-TATGAGGGTTACGCGCTCCC-3' and 5'-ACGCTCGGTCAGGATCTTCA-3'; for calmodulin, 5'-GCTGACCAACTGACTGAAGAG-3' and 5'-CCCAGAGACCGCATCACC-3'; for S100A11, 5'-GTCCCTGATTGCTGTTTTC-3' and 5'-GGTCCTTCTGGTTCTTCG-3'; for S100A10, 5'-TGCTTACATTTCACAGGTTTGC-3' and 5'-GTCCACAGCCAGAGGGTC-3'; and for annexin A6, 5'-GCCGCTTGCCTATTGTGAC-3' and 5'-GCTGGTGTATCTGCTCATTGG-3'. Reaction efficiencies were >90% for all primers and were within 5% of each other. Relative quantification was performed by normalizing threshold cycles (Ct) values to Ct values of -actin (Ct). Results represent the means of triplicate samples from two independent experiments.
Expression and purification of recombinant S100A11. E. coli BL21 (DE3) cells were transformed with the plasmid (pGEX-6P-1/S100A11) and grown at 37°C until the OD600 reached 0.6–0.8. Protein expression was induced by the addition of 0.1 mM IPTG. After 4 h of incubation, the cells were harvested by centrifugation at 8,000 g. The pellet was resuspended in phosphate-buffered saline (PBS; 25 ml) and frozen at –80°C. The cell pellet was thawed and sonicated (6 x 30 s) on ice. Triton X-100 (1%) was added and mixed gently for 30 min. The solution was centrifuged for 20 min at 15,000 g at 4°C. The supernatant was collected, glutathione-Sepharose 4 Fast Flow beads added and the mixture rotated gently at room temperature for 30 min. Following centrifugation (3,000 g for 1 min at 4°C), the supernatant was discarded and the beads were washed three times with PBS and poured into a column (1.6 x 10 cm). The bound glutathione S-transferase (GST)-S100A11 was eluted with 10 mM reduced glutathione in 50 mM Tris·HCl, pH 8.0. The eluate containing GST-S100A11 was collected and dialyzed against (in mM) 50 Tris·HCl, pH 7.0, 150 NaCl, 1 EDTA, and 1 DTT. PreScission protease (0.34 U/100 µg GST-fusion protein) was added to the dialysate and incubated at 4°C for 16 h. The bulk of the cleaved GST was removed on a glutathione-Sepharose 4 Fast Flow column and further purification of S100A11 was achieved by Ca2+-dependent hydrophobic interaction chromatography (10).
Expression and purification of full-length and truncated annexin A6 species.
Chicken annexin A6 (NCBI accession no. Q6B344) was subcloned into pGEX-6P-1 via the EcoRI and XhoI sites. The forward primer introduced an EcoRI cleavage site (underlined) upstream of the ATG start codon (5'-CGGAATTCatgGCACCCAAAGGAAAGGTT-3') and the reverse primer introduced an XhoI cleavage site (underlined) downstream of the stop codon (5'-CCGCTCGAGctaGTCGTCCCCCCCGCACAG-3'). The pGEX-6P-1/(1–20) annexin A6, pGEX-6P-1/annexin A6 (G5-R339), and pGEX-6P-1/annexin A6 (M340-D672) constructs were generated from the pGEX-6P-1/annexin A6 construct. E. coli BL21 (DE3) cells were transformed with the different annexin A6 plasmids and grown at 37°C until the OD600 reached 0.8. Protein expression was induced by the addition of 0.1 mM IPTG. After 3 h of incubation, the cells were harvested by centrifugation at 8,000 g for 10 min. The expressed GST-fusion proteins were purified and the GST moiety removed as described above for recombinant S100A11. The bulk of the cleaved GST was removed on a glutathione-Sepharose 4 Fast Flow column and further purification of annexins was achieved by anion-exchange chromatography. The solution was dialyzed against 20 mM Tris·HCl, pH 7.5, and applied to a Mono Q HR 16/10 column previously equilibrated with 20 mM Tris·HCl, pH 7.5. The column was washed with 20 mM Tris·HCl, pH 7.5, until A280 returned to baseline and bound proteins were eluted with a linear (0–0.5 M) NaCl gradient in 20 mM Tris·HCl, pH 7.5. Fractions were collected at a flow rate of 0.5 ml/min. In the case of annexin A6 (M340-D672), the protein was eluted from the FPLC column by stepping to 0.18 M NaCl in 20 mM Tris·HCl, pH 7.5. All cDNA sequences were verified by the University of Calgary Core DNA and Protein Services Facility. The purity of all recombinant proteins was confirmed by SDS-PAGE.
Cross-linking of S100A11 and annexin A6. Purified recombinant chicken annexin A6 (0.2 mg/ml) was incubated for 10 min at room temperature with purified recombinant chicken S100A11 (0.2 mg/ml) in 0.2 M triethanolamine, pH 8.0, in the presence of 4 mM EGTA or 1 mM CaCl2. A 50-fold molar excess of dimethylsuberimidate (DMS) relative to annexin A6 was added. Samples were withdrawn 5 h following DMS addition and made 50 mM in Tris to quench the cross-linking reaction. In separate experiments, 5-h incubation with DMS was found to consistently give maximal cross-linking. Controls contained annexin A6 alone or S100A11 alone. Samples containing 10 µg of annexin A6 and/or S100A11 were subjected to SDS-PAGE and Western blot analysis with anti-S100A11.
Ca2+-dependent binding of trypsin-digested annexin A6 to immobilized S100A11. Purified recombinant annexin A6 was dialysed against 25 mM Tris, pH 7.5, diluted to a final concentration of 0.2 mg/ml (10 ml total) and partially digested with 40 µg [1:50 (wt/wt) ratio of trypsin:annexin A6] of sequencing grade modified TPCK-trypsin at 30°C for 15 min. Soybean trypsin inhibitor (STI; 1 mg) was added to stop the digestion. Buffer composition was adjusted to 30 mM Tris·HCl, pH 7.5, 0.15 M NaCl, 0.2 mM CaCl2, and 1 mM DTT, and loaded on an S100A11-Sepharose affinity column. The column was washed with 30 mM Tris·HCl, pH 7.5, 0.15 M NaCl, 0.2 mM CaCl2, 1 mM DTT, and 0.1 mg/ml STI until A280 returned to baseline. Bound proteins were eluted with 30 mM Tris·HCl, pH 7.5, 0.15 M NaCl, 4 mM EGTA, 1 mM DTT, 0.1 mg/ml STI, and fractions (1 ml) were collected at a flow rate of 10 ml/h.
Gel electrophoresis and Western blot analysis. Protein was extracted from rat caudal arterial smooth muscle strips devoid of endothelium with 0.2 ml of 50 mM Tris·HCl, pH 6.8, 1% SDS, 0.1 mM diisopropylfluorophosphate, heated to 95°C for 5 min and mixed at room temperature for 60 min before addition of 0.1 ml of 2x sample buffer and SDS-PAGE at 35 mA for 3.5 h. Proteins were transferred to 0.2-µm nitrocellulose membranes for 3 h at 80 V and 4°C in 25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol. Membranes were blocked with 5% nonfat dried milk or 0.5% (wt/vol) I-Block in Tris-buffered saline (TBS; 20 mM Tris·HCl, pH 7.5, 0.5 M NaCl) containing 0.05% Tween (TBST) for 1 h and incubated with primary antibody (polyclonal anti-[rat annexin A6] at 1:10,000 dilution, polyclonal anti-[chicken S100A11] at 1:10,000 dilution or monoclonal anti-[rat S100A11] at 1:100 dilution in TBST containing 1% nonfat dried milk or 0.1% I-Block) for 1 h. Membranes were washed with TBST and incubated with anti-[rabbit IgG]-horseradish peroxidase-conjugated secondary antibody (Chemicon; 1:10,000 dilution) or anti-[mouse IgG]-horseradish peroxidase-conjugated secondary antibody (Chemicon; 1:10,000 dilution) before chemiluminescence detection. SDS-PAGE using 7.5–20% acrylamide gradient gels and Western blot analysis were performed as previously described (40).
Gel overlay (Far Western analysis).
Purified S100A11 (positive control; 1 µg) and purified full-length annexin A6, (1–20) annexin A6, annexin A6 (G5-R339), or annexin A6 (M340-D672) (1 µg each) were subjected to SDS-PAGE. The proteins were transferred to 0.2-µm nitrocellulose membranes as described above and the membranes dried overnight. Membranes were wet with TBS in the presence of 0.2 mM CaCl2 or 4 mM EGTA and blocked by treatment with 0.5% (wt/vol) I-Block in TBS for 1 h at room temperature in the presence of 0.2 mM CaCl2 or 4 mM EGTA. Membranes were incubated with S100A11 (0.1 mg/ml in 30 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.2 mM CaCl2, 1 mM DTT or 30 mM Tris·HCl, pH 7.5, 150 mM NaCl, 4 mM EGTA, 1 mM DTT) for 4 h at room temperature. Membranes were washed (4 x 5 min) with TBS in the presence of 0.2 mM CaCl2 or 4 mM EGTA and incubated with primary antibody (polyclonal rabbit anti-[chicken S100A11]) at 1:10,000 dilution in TBS containing 0.1% I-Block in the presence of 0.2 mM CaCl2 or 4 mM EGTA. Membranes were washed (4 x 5 min) with TBS in the presence of 0.2 mM CaCl2 or 4 mM EGTA and incubated for 1 h with goat anti-(rabbit IgG)-horseradish peroxidase conjugate. Finally, membranes were washed (4 x 5 min) with TBS in the presence of 0.2 mM CaCl2 or 4 mM EGTA and immunoreactive bands visualized by enhanced chemiluminescence. Duplicate gels were stained overnight in 45% (vol/vol) ethanol, 10% (vol/vol) acetic acid containing 0.1% (wt/vol) Coomassie brilliant blue, and destained in 10% (vol/vol) acetic acid.
Preparation of native liposomes from vascular smooth muscle membrane lipids. Rat caudal arterial smooth muscle (ten 6 x 1.5 mm strips) was ground under liquid N2 in 15 volumes of 50 mM Tris·HCl, pH 7.5, 0.15 M NaCl, 1 mM DTT, 1 mM CaCl2, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), rotated end-over-end for 30 min at room temperature and centrifuged at 100,000 g for 30 min. The supernatant was discarded. The pellet was resuspended in 15 volumes of 50 mM Tris·HCl, pH 7.5, 0.15 M NaCl, 1 mM DTT, 4 mM EGTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM PMSF (buffer C), and homogenized with a Pyrex homogenizer. The homogenate was rotated end-over-end for 15 min and centrifuged at 100,000 g for 30 min. The supernatant was discarded. The pellet was resuspended in 15 volumes of buffer C, homogenized, rotated end-over-end for 15 min, and centrifuged at 100,000 g for 30 min. The supernatant was discarded. The pellet was resuspended in 15 volumes of EGTA buffer, homogenized, rotated end-over-end for 15 min, 1 ml of CHCl3/methanol (1:1, vol/vol) was added and the mixture rotated end-over-end for 5 min. Following low-speed centrifugation (3,000 rpm for 10 s), the lower (CHCl3) layer was recovered, dried in a stream of N2 and 50 mM imidazole-HCl, pH 7.4, 0.15 M NaCl added. Following incubation at 30°C for 30 min, the sample was vortexed and sonicated (model 1510 sonic probe, Braun) on ice for 10 x 30 s.
Binding of annexin A6 and S100A11 to native liposomes. Binding studies were carried out in a total volume of 0.2 ml of 50 mM imidazole-HCl, pH 7.4, 0.15 M NaCl, 40 µl liposomes, 5 µg purified recombinant chicken S100A11, and annexin A6 in the presence of 0.2 mM CaCl2 or 4 mM EGTA at room temperature for 20 min. Samples were centrifuged at 100,000 g for 30 min. The pellets were resuspended in 50 µl of 80 mM imidazole, pH 7.4 containing either 4 mM EGTA or 0.2 mM CaCl2, an equal volume of SDS gel sample buffer was added and boiled for 5 min before SDS-PAGE of supernatant and pellet.
Binding of S100A11 to phosphatidylserine liposomes. Porcine brain phosphatidylserine (4 mg) was dried in a stream of N2, 50 mM imidazole-HCl, pH 7.4, 0.15 M NaCl (1.5 ml) added, and incubated at 30°C for 30 min. After vortexing, the mixture was sonicated on ice (10 x 30 s). Binding studies were carried out in a total volume of 0.2 ml by incubation for 20 min of purified recombinant chicken S100A11 (0.5–10 µg) with liposomes (0.1 mg) in 50 mM imidazole-HCl, pH 7.4, 0.15 M NaCl and either 4 mM EGTA or 0.2 mM CaCl2. Samples were then centrifuged at 100,000 g for 30 min. Separated supernatants and pellets were boiled in SDS-gel sample buffer for 5 min before SDS-PAGE.
Protein concentration. Protein concentrations were determined using the bicinchoninic acid method (Pierce) or by amino acid analysis at the Alberta Peptide Institute (University of Alberta, Edmonton, AB, Canada).
Mass spectrometry. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry (MS) was carried out in the Southern Alberta Mass Spectrometry Centre at the University of Calgary.
Immunocytochemistry. Rat caudal arterial smooth muscle cells were isolated as follows. Male Sprague-Dawley rats (300–350 g) were killed by halothane inhalation and decapitation as approved by the Animal Care Committee (Faculty of Medicine, University of Calgary). Excess adventitia and adipose tissue were dissected free from the caudal artery in Ca2+-free SMDS buffer, freshly prepared daily with ultrapure distilled water, containing (in mM) 120 NaCl, 25 NaHCO3, 4.2 KCl, 0.6 KH2PO4, 1.2 MgCl2, 11 glucose, and 0.01 CaCl2. Tissue segments were placed over a 0.31-mm needle and moved back and forth 40 times to remove the endothelium and then cut into 1 x 1 mm pieces. Tissues were then incubated in SMDS containing collagenase type 1A (7 mg in 5 ml; Sigma) and Protease type XXVII (0.2 mg in 5 ml; Sigma) for 18–24 min at 35–37°C with bubbling with 95% O2-5% CO2. Enzymatic digestion was quenched by gentle washing with cold (4°C) SMDS and placed on ice for 1 h. Single isolated myocytes were obtained by gentle trituration of the digested arteries with a fire-polished Pasteur pipette. Cell suspensions were dropped onto coverslips, left to stand for at least 30 min at room temperature (20°C), fixed with 2% paraformaldehyde in PBS and washed 3 x 5 min with PBS. The cells were permeabilized and blocked with quench solution (0.1 M Trizma base, pH 7.4, 1.8% NaCl, 2% BSA, 0.2% Triton X-100). Labeling was performed in a humidified box overnight at 4°C using a 1:1,000 dilution of a polyclonal antibody raised in rabbits against an NH2-terminal peptide of rat annexin A6 or an undiluted mouse monoclonal antibody directed against full-length rat S100A11. Cells were washed 3 x 5 min with PBS and then treated for 1 h at room temperature with secondary antibody: Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) or Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch), each diluted 1:2,000 in quench solution. Hoechst 33342 nucleic acid stain (Molecular Probes) was applied at 1:10,000 dilution to visualize the nuclei. Cells were washed 3 x 5 min with PBS before visualization of immunofluorescently labeled cells with a laser-scanning cytometry microscope (model BX50-FLA, CompuCyte) equipped with a cooled scientific charge-coupled device (CCD) camera (Diagnostic Instruments) and CCD detector with Sony XC-003/003P camera.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In gel overlay assays, full-length annexin A6 bound S100A11 in the presence of Ca2+ but not EGTA following SDS-PAGE of annexin A6, transfer to nitrocellulose and overlay with S100A11 (Fig. 4).
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81 and 93 kDa, corresponding to complexes of 1 S100A11:1 annexin A6 and 2 S100A11:1 annexin A6. S100A11 in the absence of annexin A6 was cross-linked to dimers and higher-order multimers in a Ca2+-independent manner (Fig. 5, lanes 1 and 5).
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Identification of S100A11-binding domains in annexin A6.
The NH2-terminal head regions of annexin A1 and annexin A2 mediate their binding to S100A11 and S100A10, respectively (29, 30). To determine whether the unique NH2-terminal head region of annexin A6 is involved in binding to S100A11, a deletion mutant lacking this domain [(1–20) annexin A6] was expressed and purified. Figure 7 shows that this deletion mutant of annexin A6 binds to immobilized S100A11 in a Ca2+-dependent manner. The NH2-terminal head region of annexin A6 is, therefore, not required for interaction with S100A11. The Ca2+-dependent interaction of S100A11 with annexin A6 lacking the NH2 terminal head region was confirmed in gel overlay assays (Fig. 4).
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68 and 34 kDa, suggesting that cleavage occurred near one end of the molecule to generate the 68-kDa fragment and near the middle of the molecule to generate two halves of 34 kDa each (Fig. 8, lane 4). The digest was subjected to affinity chromatography on immobilized S100A11 and all fragments bound in a Ca2+-dependent manner (Fig. 8, lanes 13-18). The 34-kDa bands were cut out of the gel, completely digested with trypsin and subjected to MALDI-TOF MS. Several annexin A6 peptides were detected, which were evenly distributed throughout the length of the molecule, although no peptides at the extreme NH2- or COOH-termini were detected (Table 2). The 68- and 34-kDa tryptic fragments of annexin A6 were transblotted from separate gels to polyvinylidenedifluoride (PVDF) membrane and their NH2-terminal sequences determined by Edman degradation (Table 3). MALDI-TOF MS analysis of the tryptic digest generated four principal peaks (Fig. 9A), one of which (19,984.72 Da) is attributable to the soybean trypsin inhibitor used to terminate the trypsin digestion. Table 4 summarizes these MS data, which support the results of Edman degradation and provide additional information about the COOH-terminal ends of the peptide fragments. Thus the 68-kDa fragment corresponds to G5-D672, and the 34-kDa fragments include M340-D672 and V349-D672. An additional peak is evident (Fig. 9B) of 37,883 Da, corresponding to G5-R339 (Table 4). Full-length annexin A6 gave a molecular mass by MS of 76,087.18 Da (Fig. 9C), which contains a short NH2-terminal extension due to the cleavage by PreScission protease (Table 4). From the data of Figs. 8 and 9 and Tables 2–4, we conclude that annexin A6 is cleaved by trypsin at Lys4 within the NH2-terminal head region and at Arg339 and Lys348 in the linker region connecting the two halves of the molecule (each containing 4 annexin repeats). All of these tryptic fragments bound to immobilized S100A11 in a Ca2+-dependent manner (Fig. 8), indicating that annexin A6 contains binding sites for S100A11 in each half of the molecule.
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Coexpression of S100A11 and annexin A6 in smooth muscle tissue and in isolated smooth muscle cells.
Western blot analysis has revealed the expression of S100A11 and annexin A6 in smooth muscle tissues (1, 29). We confirmed this conclusion by Western blot analysis of rat caudal arterial smooth muscle (see online supplementary Fig. S5). However, smooth muscle tissues contain many different cell types, including endothelial cells, neurons, and fibroblasts, in addition to smooth muscle cells, and, if the S100A11-annexin A6 interaction is of physiological relevance, the two proteins must be coexpressed in the same cell. RT-PCR was therefore performed on mRNA prepared from isolated vascular smooth muscle cells of the rat basilar artery and indicated that both S100A11 and annexin A6 messages are indeed expressed in smooth muscle cells: PCR products of the expected sizes (148 and 478 bp, respectively) were detected (Fig. 10). Real-time PCR on mRNA isolated from rat cerebral arteries allowed the quantification of message levels in the tissue relative to the -actin housekeeping gene (Table 5). S100A11 mRNA was found to be 20% and annexin A6 mRNA 8% of the -actin content. For comparison, the levels of two other EF-hand Ca2+-binding proteins, calmodulin and S100A10, were similar to the S100A11 message level (19% and 17%, respectively of the -actin content). Dual-labeling immunocytochemistry confirmed at the protein level that both annexin A6 and S100A11 are expressed in isolated vascular smooth muscle cells (Fig. 11, left and middle panels, respectively). The nucleus, identified by Hoechst nucleic acid stain (Fig. 11, right panel), was not stained with either antibody. The specificity of antibody staining was confirmed by loss of the immunofluorescent signal following preadsorption of the primary antibodies with the annexin A6 peptide to which the antibody to annexin A6 was raised (Fig. 11) or full-length S100A11 (Fig. 11, bottom left), respectively, and the absence of an immunofluorescence signal following treatment with secondary antibody alone (Fig. 11, bottom right).
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70 amino acids. Most annexins contain four such repeat motifs packed into a highly -helical disk, which forms the membrane-binding module. Annexins differ primarily in the NH2-terminal head region, which varies in length. For example, annexin A1 has an NH2-terminal head region composed of 40 amino acids, the first 10–14 residues of which constitute the S100A11-binding site (30). Other S100-annexin partnerships have also been identified. Annexin A2 forms a tetramer composed of two subunits of annexin A2 and two of S100A10, which is Ca2+-independent (20). Annexins A2, A6, and A11 interact with S100A6 (calcyclin) in a Ca2+-dependent manner (16, 36, 41). S100B binds to annexins A2 (6), A5, and A6 (17). Finally, S100A1 interacts with annexins A5 and A6 (17). Annexins have been implicated in diverse physiological functions, including regulation of membrane organization, membrane trafficking, membrane-cytoskeleton linkage, ion conductance across membranes, anti-inflammatory and anti-coagulant effects, and as mediators or regulators of certain cell-cell and cell-matrix interactions (18). Annexin A6 specifically has been implicated in modulation of Ca2+ and K+ conductance (25), Ca2+ homeostasis (21) and cell growth and proliferation (37, 15). The formation of tetramers composed of two annexin subunits and two S100 subunits could link two distinct membranes in a Ca2+-dependent manner. Annexins have also been shown to bind to actin-based cytoskeletal elements (e.g., 11, 20, 28), which could explain their possible involvement in Ca2+-dependent regulation of membrane-cytoskeleton dynamics.
As noted above, S100A11 is known to bind to annexin A1 in a Ca2+-dependent manner. However, we did not observe annexin A1 in the EGTA eluate following affinity chromatography of an extract of chicken gizzard smooth muscle on immobilized S100A11. This could be due to lack of expression of annexin A1 in this tissue, or levels of expression that are below the limit of detection by Coomassie blue staining following SDS-PAGE of column fractions. We did, however, identify a novel S100-annexin partnership between S100A11 and annexin A6. The direct Ca2+-dependent interaction of S100A11 with annexin A6 was confirmed by affinity chromatography with purified recombinant proteins (binding of annexin A6 to S100A11-Sepharose and of S100A11 to annexin A6-Sepharose), gel overlay, chemical cross-linking and co-precipitation with liposomes. Deletion mutagenesis indicated that the small NH2-terminal head region of annexin A6 is not required for S100A11 binding. This is perhaps not surprising since the NH2-terminal head region of annexin A6 is small compared with other annexins known to interact with S100 proteins, e.g., annexins A1 and A2, and it lacks the consensus S100-binding motif, 
XXX (where denotes a hydrophobic amino acid and X is any amino acid). On the other hand, examination of the amino acid sequence of annexin A6 reveals three putative S100-binding consensus sequences: V157-A163, L241-V247, and V590-V596 (Fig. 2). Consistent with this prediction, we have shown that there are distinct S100A11-binding sites within each half of the annexin A6 molecule, as shown by the S100A11-binding properties of tryptic fragments and deletion mutants of annexin A6. Thus, tryptic fragments corresponding to G5-R339, M340-D672, and V349-D672 and deletion mutants G5-R339 and M340-D672 all bound to S100A11 in a Ca2+-dependent manner.
Quantification of the interaction of S100A11 with liposome-bound annexin A6 in the presence of Ca2+ indicated 1:1 stoichiometry. Since S100A11 is a stable dimer, this suggests the Ca2+-dependent complex consists of one S100A11 dimer and two annexin A6 molecules. This complex was confirmed by chemical cross-linking. S100A11, therefore, could cross-link two annexin A6 molecules and thereby connect two membranes through the phospholipid-binding surface on annexin A6. For example, such a mechanism could serve to bring the sarcoplasmic reticulum in close apposition to the plasma membrane. Since annexin A6 can also interact with cytoskeletal elements, the possibility arises that the S100A11-annexin A6 complex may connect the plasma membrane to the cytoskeleton. An agonist-induced increase in cytosolic free [Ca2+] could lead to the formation of a heteromeric complex composed of two annexin A6 polypeptides cross-linked by an S100A11 dimer, to form a physical connection between the plasma membrane and the cytoskeleton that is essential for transmission of mechanical signals leading to contraction. Ca2+ plays a dual role in this process since both S100A11 and annexin A6 are Ca2+-binding proteins. Ca2+ ions play a key role in association of annexin A6 with the membrane and Ca2+ binding to S100A11 inducing a conformational change with exposure of a hydrophobic surface that interacts with annexin A6.
The sarcolemma of smooth muscle cells is segregated into force-transmitting regions (adherens junctions), which are directly linked to the cytoskeleton, and flexible vesicular domains containing numerous caveolae and noncaveolar lipid raft domains (13, 35). Together, the caveolae and stabilized noncaveolar rafts form an extensive lipid superstructure, a macroraft (sites of localization of signaling complexes). Since smooth muscle cells must adapt rapidly and continuously to changes in length, sarcolemmal and cytoskeletal protein reorganization must be precise and flexible. Members of the annexin protein family, including annexin A6, have been implicated in smooth muscle membrane organization, constituting a reversible, Ca2+-dependent link between the sarcolemma and the cytoskeleton (2). Effective regulatory mechanisms for coordinated cytoskeletal and sarcolemmal rearrangement are required to protect smooth muscle cells from mechanical damage during contraction-relaxation cycles. Most interestingly, Draeger and coworkers provided evidence that annexin A6 translocates to the non-caveolar raft domains neighboring the caveolae to form a Ca2+-sensitive linker connecting this domain to the actin cytoskeleton (2–4, 14). They proposed that this Ca2+-regulated connection protects the sarcolemma from mechanical damage during contraction and relaxation and enhances force transduction between the contractile machinery and the extracellular matrix. Since we have shown that S100A11 interacts with annexin A6 in a Ca2+-dependent manner, the possibility arises that this Ca2+-dependent linkage of the plasma membrane to the cytoskeleton also requires S100A11.
Evidence from other cell types has implicated annexin A6 in Ca2+-dependent formation of signaling complexes. For example, annexin A6 has been shown to associate in a Ca2+-dependent manner with lipid rafts along with PKC and neurocalcin- (27) and direct interaction between annexin A6 and skeletal muscle PKC has been demonstrated (33). Annexin A6 has also been identified in a complex with the GTPase activating protein p120 Ras-GAP and the tyrosine kinases Fyn and Pyk2, annexin A6 interacting directly with p120 Ras-GAP and Fyn, but not Pyk2 (7). Future studies will focus on the association of S100A11 and annexin A6 with other proteins that will shed light on the physiological function of this Ca2+-dependent complex.
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ACKNOWLEDGMENTS |
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Present address for D. P. Wilson: Discipline of Physiology, University of Adelaide, Adelaide 5005, South Australia.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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