Comprehensive interaction of dicalcin with annexins in frog olfactory and respiratory cilia Tatsuya Uebi 1 , Naofumi Miwa 1,2, * and Satoru Kawamura 1,2 1 Department of Biology, Graduate School of Science, Osaka University, Japan 2 Graduate School of Frontier Biosciences, Osaka University, Japan Calcium ions are known to modulate signal transduc- tion in various cells. This effect is usually mediated by Ca 2+ -binding proteins. For example, in olfactory receptor cells, odorant stimuli induce Ca 2+ influx through a cyclic nucleotide gated channel [1]. The increase in the Ca 2+ concentration is detected by calmodulin, a well-known Ca 2+ -binding protein. The Ca 2+ -bound form of calmodulin has essential roles in olfactory adaptation [2,3]. In photoreceptor cells, sev- eral Ca 2+ -binding proteins are known to be present and to modulate phototransduction signals [4]. We previously found a Ca 2+ -binding protein, dical- cin (renamed from p26olf [5]), in frog olfactory epithe- lium, and reported that dicalcin is expressed in the olfactory epithelium, lung, and spleen [6,7]. In the olfactory epithelium and lung, dicalcin localizes in the cilia. Dicalcin has partial homology to S100 proteins, a family of EF-hand Ca 2+ -binding proteins, and consists of two S100A11-like regions aligned in sequence. The amino acid sequences in the N-terminal and the C-ter- minal halves show 58% and 45% identity, respectively, to chick S100A11 [7]. The predicted structure of dical- cin is similar to that of an S100 dimer [8]. S100 proteins are known to be involved in various cellular functions, such as cell cycle progression and cell survival [9–11]. S100 proteins show no enzymatic activities by themselves and, instead, modulate the function of other proteins through direct binding to Keywords annexin; dicalcin; olfactory cilia; respiratory cilia; S100 Correspondence S. Kawamura, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 1–3, Suita, Osaka 565-0871, Japan Fax: +81 6 6879 4614 Tel: +81 6 6879 4610 E-mail: kawamura@fbs.osaka-u.ac.jp *Present address Department of Physiology, School of Medicine, Toho University, Tokyo, Japan Database Amino acid sequences have been submitted to DDBJ under the following accession numbers: frog annexin A1, AB286845; frog annexin A2, AB286846; frog annexin A4, AB286848; frog annexin A5, AB286847 (Received 8 May 2007, revised 20 July 2007, accepted 24 July 2007) doi:10.1111/j.1742-4658.2007.06007.x Dicalcin (renamed from p26olf) is a dimer form of S100 proteins found in frog olfactory epithelium. S100 proteins form a group of EF-hand Ca 2+ -binding proteins, and are known to interact with many kinds of tar- get protein to modify their activities. To determine the role of dicalcin in the olfactory epithelium, we identified its binding proteins. Several proteins in frog olfactory epithelium were found to bind to dicalcin in a Ca 2+ -dependent manner. Among them, 38 kDa and 35 kDa proteins were most abundant. Our analysis showed that these were a mixture of annex- in A1, annexin A2 and annexin A5. Immunohistochemical analysis showed that dicalcin and all of these three subtypes of annexin colocalize in the olfactory cilia. Dicalcin was found to be present in a quantity almost suffi- cient to bind all of these annexins. Colocalization of dicalcin and the three subtypes of annexin was also observed in the frog respiratory cilia. Dicalcin facilitated Ca 2+ -dependent liposome aggregation caused by annexin A1 or annexin A2, and this facilitation was additive when both annexin A1 and annexin A2 were present. In this facilitation effect, the effective Ca 2+ con- centrations were different between annexin A1 and annexin A2, and there- fore the dicalcin–annexin system in frog olfactory and respiratory cilia can cover a wide range of Ca 2+ concentrations. These results suggested that this system is associated with abnormal increases in the Ca 2+ concentration in the olfactory and other motile cilia. FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4863 these proteins. p53, RAGE and annexins are known to be binding proteins of S100 proteins. S100 proteins are known to form dimers, and the dimer form binds to the binding protein to exert the effect. Because dicalcin consists of two S100-like domains aligned in sequence, the function of dicalcin is probably similar to that of an S100 dimer. Although the Ca 2+ -binding property has been inves- tigated in detail in dicalcin [12], little is known about its physiologic function. To investigate this, in the present study we first tried to determine the binding proteins of dicalcin. We found that several of the pro- teins in frog olfactory epithelium bind to dicalcin in a Ca 2+ -dependent manner. Among them, 38 kDa and 35 kDa proteins were the major proteins. We identified them as annexin A1, annexin A2 and annexin A5. We further examined their localizations and the effect of dicalcin on the activities of these annexins by measur- ing liposome aggregation. Results Purification of binding proteins of dicalcin Binding proteins of dicalcin were searched for among the soluble and membrane-associated proteins of frog olfactory cilia. Because dicalcin is an S100-like EF-hand Ca 2+ -binding protein, we expected that the binding proteins would bind to dicalcin in a Ca 2+ - dependent manner. The Chaps-solubilized fraction (see Experimental procedures) containing the mem- brane-associated proteins in frog olfactory cilia (Fig. 1, cilia) was loaded onto a dicalcin-Sepharose column at 1 mm Ca 2+ . Most of the proteins were found in the pass-through fraction (Fig. 1, elution peak A and lane A), but some of the proteins were retained, and eluted by reducing the Ca 2+ concentra- tion (Fig. 1, elution peak B and lane B). Several pro- teins were found in lane B, but the major proteins were 38 kDa and 35 kDa proteins. The latter could be one of the binding proteins detected in our previ- ous dicalcin-overlay analysis [13]. In control studies, we did not see the binding of these proteins when dicalcin was not attached to the Sepharose beads (Fig. 1C). Although the amount of each of the eluted proteins varied among preparations, 38 kDa and 35 kDa proteins were always the major constituents. We therefore focused on these proteins in the follow- ing study. Essentially similar binding proteins were detected when we used the soluble protein fraction, but the amounts of the proteins were greater in the Chaps-solubilized fraction. For this reason, we used this fraction in the following studies. Amino acid sequence analysis of 38 kDa and 35 kDa proteins During the course of this study, we realized that 35 kDa proteins contained proteolytic fragments of 38 kDa proteins: in the presence of protease inhibi- tors, the amount of 38 kDa proteins was larger than that in the absence of the inhibitors. However, we could not inhibit the proteolysis completely: even in the presence of a cocktail of inhibitors, our immuno- logic study detected signals of 38 kDa proteins at the 35 kDa position (see Fig. 3A below). In addition, the degree of inhibition was variable, depending on each preparation. Nevertheless, the binding proteins of di- calcin, mainly the 38 kDa and the 35 kDa proteins, were fragmented by a protease. The resultant proteo- lytic fragments were isolated by RP-HPLC, and their amino acid sequences were determined. The result suggested that the 38 kDa and the 35 kDa proteins are the annexin family proteins. The result, however, was complex: the amino acid sequences of the frag- ments did not match the sequence of a single annexin family protein. Instead, the sequence of a fragment showed some similarity to the sequence of annex- in A1, annexin A2, annexin A4 or annexin A5 of Fig. 1. Purification of binding proteins of dicalcin by affinity column chromatography. The Chaps-solubilized protein fraction of the cilia of frog olfactory epithelium was loaded to a dicalcin-Sepharose col- umn at 1 m M Ca 2+ . Most of the proteins passed through the col- umn (A in the elution profile) at a high (1 m M)Ca 2+ concentration, but some proteins remained in the column and came out only after addition of 5 m M EGTA (B in the profile). Inset: SDS ⁄ PAGE patterns of the Chaps-solubilized cilia protein fraction (cilia), the pass-through fraction (A) and the eluate in the presence of 5 m M EGTA (B). As a control, an eluate was obtained similarly as in (B), but with the use of Sepharose beads without dicalcin conjugated (C). Proteins were stained with silver. Role of dicalcin in frog olfactory cilia T. Uebi et al. 4864 FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS other animal species, which suggested that the 38 kDa and the 35 kDa proteins were a mixture of these annexins. We therefore tried to isolate cDNAs of annexin A1, annexin A2, annexin A4 and annex- in A5 to identify which annexins were in the fraction of the 38 kDa and the 35 kDa proteins. Cloning of annexin cDNAs On the basis of the partial amino acid sequences of the proteolytic fragments as determined above, we synthe- sized oligonucleotide degenerate primers and used them to search for the cDNA fragments of the corres- ponding annexins. Partial cDNA fragments of annex- in A1, annexin A2, annexin A4 and annexin A5 were amplified, and the frog olfactory cDNA library was screened with these fragments. The full-length sequences of frog annexin cDNAs were obtained, and the amino acid sequences were deduced (supplemen- tary Fig. S1). The amino acid sequences detected in the proteolytic fragments were found in the deduced amino acid sequences of frog annexin A1, annexin A2, and annexin A5, but not in the sequence of frog an- nexin A4. This result indicated that annexin A4 was not present, or the content of annexin A4 was small in the fraction of the 38 kDa and 35 kDa proteins. Among our recombinant annexins (see below), the apparent molecular mass of annexin A4 was slightly lower than 35 kDa on our SDS ⁄ PAGE gel. Because the density of the corresponding position on the SDS ⁄ PAGE gel of the binding proteins of dicalcin was faint, this result also suggested that the content of annexin A4 in the 35 kDa proteins was small even if it was present. For these reasons, we did not study annexin A4 further. Identification of annexin A1, annexin A2 and annexin A5 as the binding proteins of dicalcin Our results were so far consistent with the notion that the 38 kDa and the 35 kDa proteins are annexin A1, annexin A2, and annexin A5. However, we were not totally sure of this at this stage. Therefore, we first tried to confirm that annexin A1, annexin A2 and an- nexin A5 show Ca 2+ -dependent binding to dicalcin, as the 38 kDa and the 35 kDa proteins do. For this, we obtained recombinant annexin A1, annexin A2 and annexin A5 expressed in Escherichia coli. The apparent molecular masses of recombinant annexin A1 and ann- exin A2 were both 38 kDa, and that of annexin A5 was 35 kDa (Fig. 2), and all of them bound to the di- calcin-Sepharose beads in a Ca 2+ -dependent manner (Fig. 2), as the native 38 kDa and 35 kDa proteins do. Second, we identified the 38 kDa and the 35 kDa pro- teins as annexin A1, annexin A2 and annexin A5 immu- nologically. We raised specific antiserum against annexin A1, annexin A2 or annexin A5 in mouse and rabbit using recombinant annexins (supplementary Fig. S2). Antiserum against annexin A1 recognized both the 38 kDa and the 35 kDa proteins (Fig. 3A, low Ca 2+ eluate), and antiserum against annexin A2 also detected the 38 kDa and the 35 kDa proteins. Antiserum against annexin A5 detected only the 35 kDa proteins. From the above results, it became evident that the 38 kDa proteins contained both full-length annexin A1 and annexin A2, and the 35 kDa proteins contained full-length annexin A5 together with proteolytic fragments of annexin A1 and annexin A2. Our two- dimensional electrophoresis confirmed this (Fig. 3B). This two-dimensional analysis also indicated that pro- teins other than annexin A1, annexin A2 and annex- in A5 were not present in significant amounts in the 38 kDa and 35 kDa proteins (Fig. 3B). In Fig. 3B, there are weak signals of annexin A1 at around pH 5.1. They are probably the signals of annexin A1 that was not focused in our two-dimensional electro- phoresis. Fig. 2. Ca 2+ -dependent binding of recombinant annexins to dicalcin. The cell lysate of E. coli (lysate) expressing recombinant annex- in A1, annexin A2 or annexin A5 was mixed with dicalcin-Sepha- rose beads at 1 m M Ca 2+ . The beads were washed 10 times by centrifugation with K-gluc buffer supplemented with 1 m M Ca 2+ , and the 1st and the 10th extracts were subjected to SDS ⁄ PAGE (high-Ca 2+ wash 1 and high-Ca 2+ wash 10). The beads were finally washed with K-gluc buffer supplemented with 5 m M EGTA, and the extract was subjected to SDS ⁄ PAGE (low-Ca 2+ wash). T. Uebi et al. Role of dicalcin in frog olfactory cilia FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4865 Colocalization of annexins and dicalcin in frog olfactory and respiratory epithelium Dicalcin has been reported to localize in the cilia of frog olfactory and respiratory epithelium [7]. To understand the possible association of annexin A1, annexin A2 and annexin A5 with the function of dical- cin, we examined the colocalization of each annexin with dicalcin, using specific antisera (supplementary Fig. S2). In addition, we also examined whether differ- ent subtypes of the annexins colocalize in the same cilia. Figures 4 and 5 show the immunohistochemical studies of dicalcin and annexin A1, annexin A2 and annexin A5. In Fig. 4, the olfactory cilia, which were identified immunohistochemically with olfactory cilia- specific G olf antibody (Fig. 4M), were found to be reactive to antiserum against dicalcin (Fig. 4A,D,G). The cilia were also positively stained with antiserum against annexin A1 (Fig. 4B), annexin A2 (Fig. 4E), and annexin A5 (Fig. 4H). The merged image clearly showed colocalization of dicalcin with each of the an- nexins (Fig. 4C,F,I). In this study, the conditions for obtaining immunofluorescence were kept constant in each of the observations with rabbit antiserum (Texas Red) or mouse antiserum (fluorescein isothiocyanate), and therefore the color in the merged picture was dependent on the relative intensities of red and green fluorescence, namely, the titers of antisera against di- calcin and annexins. Preabsorption of the specific anti- bodies by recombinant proteins significantly reduced the signals (Miwa et al. [13] for anti-dicalcin serum and Fig. 4N for anti-annexin A2 serum). Because all the annexins examined in this study co- localized with dicalcin, we then examined whether ann- exin A1, annexin A2 and annexin A5 all colocalize in the same cell. Figure 5 shows the immunohistochemi- cal study of colocalization of annexin A1, annexin A2, and annexin A5. For any combination of these three subtypes of annexin, colocalization was demonstrated (Fig. 5). Therefore, it was evident that all three sub- types of annexin are present in the same olfactory cilium. From the results in Figs 4 and 5, it became evident that dicalcin, annexin A1, annexin A2 and annexin A5 all colocalize in the same olfactory cilium. In the respiratory epithelium, similar colocalization was observed (supplementary Fig. S3), although the signal of G olf , a marker protein of olfactory cells, was not seen. Estimation of the relative molecular abundance of dicalcin and annexins in frog olfactory cilia The above immunohistochemical study showed that all subtypes of the annexins studied here colocalize with AB Fig. 3. Identification of annexin A1, annexin A2 and annexin A5 by western blot analysis. (A) Determination that the 38 kDa proteins are a mixture of annexin A1 and A2 and that the 35 kDa proteins are a mixture of annexin A5 and proteolytic fragments of annexin A1 and annex- in A2. Purified recombinant annexin A1, annexin A2 and annexin A5 (A1, A2 and A5), together with the binding proteins of dicalcin (low-Ca 2+ eluate), were electrophoresed on an SDS ⁄ PAGE gel, and the proteins were stained with silver (silver stain). The proteins were probed with specific antisera against annexins (anti-A1, anti-A2 and anti-A5) by western blot. The 38 kDa proteins contained both annexin A1 and annex- in A2, and the 35 kDa proteins contained annexin A5 together with annexin A1 and annexin A2, possibly fragmented by proteolysis during preparation. (B) Two-dimensional electrophoretic identification of the 38 kDa and the 35 kDa proteins as annexin A1, annexin A2, and annex- in A5. A similar analysis as in (A) was performed by two-dimensional electrophoresis. Annexins were identified at the apparent molecular mass of 38 kDa with pI values of 6.2–7.1 (annexin A1), and of c. 8 (annexin A2), and a single spot at 35 kDa with pI ¼ 5.6 (annexin A5). Each annexin subtype is indicated by a circle. Role of dicalcin in frog olfactory cilia T. Uebi et al. 4866 FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS dicalcin in frog olfactory cilia. To understand the sig- nificance of this colocalization, we tried to estimate the relative molecular abundance of dicalcin and annexins. In this quantification, we used both the soluble and the membrane fraction after detachment of the cilia (see Experimental procedures). They were solubilized with the SDS ⁄ PAGE sample buffer, and were directly electrophoresed with known amounts of recombinant dicalcin and annexins. The contents of annexins and dicalcin in the cilia were estimated by western blot, and their ratio determined in three frogs was annex- in A1 ⁄ annexin A2 ⁄ annexin A5 ⁄ dicalcin ¼ 1.0 : 0.42 ± 0.09 : 0.54 ± 0.15 : 1.9 ± 0.6. Dicalcin is a soluble protein, and annexins were mostly present in the Chaps-solubilized fraction. Dicalcin might have been lost during isolation of the olfactory epithelium, and therefore the content of dicalcin could be higher than the value determined above. Because the number and the volume of the cilia in the sample were not known, it was not possible to determine the actual concentra- tions of these proteins. Effect of dicalcin on the activity of annexins As has been reported previously, annexins are known to induce membrane aggregation in a Ca 2+ -dependent manner [14], and it is also known that this activity of annexins is enhanced by binding of S100 proteins [15]. We therefore examined the effect of dicalcin on the membrane aggregation activity of annexins. The ABC DEF GHI JKL MNO Fig. 4. Colocalization of dicalcin with annexin A1, annexin A2 or annexin A5 in frog olfactory epithelium. (A–I) Immunofluorescence double- staining of dicalcin and annexins. A section was treated with rabbit anti-dicalcin serum (red; A, D and G) and mouse antiserum raised against one subtype of annexin (green: B, annexin A1; E, annexin A2; H, annexin A5). The corresponding images were merged (merged; C, F and I). (J–L) Controls. A section for controls was treated with normal serum of rabbit (J) and mouse (K), and the images were merged (L). (M) A representative section treated with antibody to G olf . All positive signals against dicalcin, annexins and G olf were observed in the cilia layer (arrowheads). (N) A control. Antiserum against annexin A2 was preabsorbed with recombinant annexin A2. (O) Frog olfactory epithelium stained with toluidine blue. Bars indicate 20 lm in (L) (applicable to A–N) and 50 lm in (O). T. Uebi et al. Role of dicalcin in frog olfactory cilia FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4867 activity was measured as the increase in the absorbance due to aggregation of phosphatidylserine liposomes (see inset in Fig. 6E, for example). The dose effect of each of the annexins in the presence or absence of di- calcin was examined (Fig. 6A). Annexin A1 and annex- in A2 alone increased liposome aggregation similarly in a dose-dependent manner (filled rectangles and filled circles, respectively). Dicalcin increased their activities, and the effect was higher on annexin A2 (open circles) than on annexin A1 (open rectangles). Annexin A5 did not show liposome aggregation activity (open and filled triangles). Although the effect of dicalcin was obvious at annexin concentrations above 40 nm, the increase in the absorbance was often too rapid for reliable data to be obtained. For this reason, we used annexins at low concentrations. The concentrations of annexins were kept at 12.5 nm (annexin A1), 5 nm (annexin A2) and 7.5 nm (annexin A5) throughout the measurement, based on the relative molecular abundance of annex- ins in the cilia, i.e. annexin A1 : annexin A2 : annex- in A5 ¼ 1.0 : 0.42 : 0.54 (see above). Dicalcin was added in excess. The effect of dicalcin on liposome aggregation induced by annexins was measured at various Ca 2+ concentrations, and the initial rate of increase was plotted as a function of Ca 2+ concentrations. As shown in Fig. 6B, no significant aggregation was observed in the absence of annexins (filled triangles) or dicalcin (open triangles). In the absence of liposomes, no significant increase in absorbance was detected (not shown). In the presence of annexins alone, slight aggregation was observed, but the effect was not so large (filled circles in Fig. 6B–E) at the annexin con- centrations used (see above). When dicalcin was present (open circles), the liposome aggregation activi- ties of annexin A1 or annexin A2 were facilitated ABC DEF GHI JKL Fig. 5. Colocalization of annexin A1, annexin A2 and annexin A5 in frog olfactory epithelium. (A–I) Immunofluorescence double-staining of one subtype of annexin with the other subtype of annexin. A section was treated with rabbit antiserum raised against one subtype of annex- in (red: A, annexin A1; D, annexin A5; G, annexin A5) and mouse antiserum raised against the other subtype of annexin (green: B, annex- in A2; E, annexin A1; H, annexin A2). The corresponding images were merged (C, F, I). (J–L) Controls. A section for controls was treated with normal serum of rabbit (J) and mouse (K), and the images were merged (L). Positive signals were observed only in the cilia layer (arrowhead). Role of dicalcin in frog olfactory cilia T. Uebi et al. 4868 FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS greatly when the Ca 2+ concentration was increased (Fig. 6B,C). Essentially, the effect of dicalcin was not seen with annexin A5 (Fig. 6D). The effective Ca 2+ concentrations depended on the subtype of annexin: annexin A2 was more sensitive to Ca 2+ than annexin A1. The half-maximal dicalcin effect was observed at < 5 lm Ca 2+ with annexin A2, but at about 30 lm with annexin A1. Although the ini- tial rate of aggregation increased to a similar level for both annexin A1 and annexin A2 at high Ca 2+ con- centrations (Fig. 6B,C), this was partly because of the difference in the concentrations used (12.5 nm annex- in A1 vs. 5 nm annexin A2; see above). When the con- centration of annexin A2 was increased to the same level as that of annexin A1, the effect of dicalcin was at least two times larger for annexin A2 than for annexin A1 (Fig. 6A). To simulate the effect of dicalcin in a cell, dicalcin was added to the mixture of annexin A1, annexin A2 and annexin A5 according to their ratios of the con- centrations in the cilia (see above). The observed acti- vity (Fig. 6E, filled lines) was equal to the calculated sum of each of the activities of annexin A1, annex- in A2 and annexin A5 (Fig. 6E, thick dotted lines). Binding of truncated forms of annexins to dicalcin In the present study, we found that dicalcin binds to annexin A1, annexin A2 and annexin A5, and that it facilitates the membrane aggregation activities of ann- exin A1 and annexin A2. In mammal S100 proteins and annexins, an S100–annexin complex is formed in a subtype-specific manner: S100A10 binds to annex- in A2 [16], and S100A11 binds to annexin A1 [17]. In the case of mammal annexin A1 and annexin A2, the specificity has been reported to arise in part at their N-terminal 1–13 amino acids [18,19]. Because dicalcin binds to both annexin A1 and annexin A2, in addi- tion to annexin A5, as shown in this study, the bind- ing sites of dicalcin and those of frog annexins could be different from those known previously. To test this possibility, we examined the binding to dicalcin of Fig. 6. Effect of dicalcin on liposome aggregation induced by an- nexins. Time courses of annexin-induced liposome aggregation were measured as the increase in the absorbance at 350 nm [see inset in (E)]. In (A), the time course was measured at various con- centrations of annexin in the presence (open symbols) and absence (filled symbols) of 200 n M dicalcin at 100 lM Ca 2+ . The initial rate of the absorbance increase was plotted against the annexin concen- tration. In (B)–(E), liposome aggregation was measured at various Ca 2+ concentrations in the presence (open circles) and absence (filled circles) of dicalcin (DC). The initial rate of the absorbance increase was plotted against the Ca 2+ concentration [annexin A1 in (B), annexin A2 in (C), annexin A5 in (D), annexin A1 + annex- in A2 + annexin A5 in (E)]. Data points represent mean ± standard error determined in two different preparations (n ¼ 3 in each prepa- ration). For controls, the result with dicalcin but no annexins pres- ent (open triangles) and that with neither dicalcin nor annexins (filled triangles) are shown in (B). These two controls are shown as thin dotted lines in (C) and (D). The result obtained in the presence of dicalcin and all of the annexins (E) was compared with the calcu- lated sum of each of the initial rates obtained in (B)–(D) (thick dot- ted lines). T. Uebi et al. Role of dicalcin in frog olfactory cilia FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4869 N-terminal-truncated forms of frog annexin A1 and annexin A2. The result showed that, indeed, dicalcin binds to these truncated forms (Fig. 7A), which indi- cated that the N-terminal region is not essential for the interaction of frog annexin A1 and annexin A2 with dicalcin. Consistently, we observed that the 35 kDa forms of annexin A1 and annexin A2 found in the fraction of the binding proteins of dicalcin (Fig. 1) were the N-terminal-truncated annexins (Fig. 7B). Discussion In the present study, we showed that the major bind- ing proteins of dicalcin in frog olfactory epithelium are annexin A1, annexin A2 and annexin A5 (Figs 1–3 and supplementary Fig. S1). The binding does not require the N-terminal region of annexins (Fig. 7). Di- calcin and all these annexins colocalize in the olfactory and respiratory cilia (Figs 4 and 5 and supplementary Fig. S3). Dicalcin was found to increase the rate of liposome aggregation caused by annexins (Fig. 6). Specificity of the binding between dicalcin and annexins In the present study, we identified the 38 kDa and the 35 kDa proteins as annexin A1, annexin A2 and ann- exin A5. Annexins are known to bind to a dimer form of S100 proteins. In mammals, the binding between annexins and S100 dimer proteins has been shown to be subtype-specific. S100A11 binds to annex- in A1 [17] (but see [20] also), and S100A10 binds to annexin A2 [16]. Because dicalcin in frogs shows the highest amino acid sequence homology to S100A11 (45–58%), the binding of dicalcin to annexin A1 is not surprising. However, binding to all of annexin A1, annexin A2 and annexin A5 is a rather unique charac- teristic of dicalcin, although similar comprehensive binding has been suggested for some of the S100 pro- teins [11]. The comprehensive binding of dicalcin to various subtypes of annexin could be due to the char- acteristics of frog annexins and ⁄ or dicalcin (see below). Annexin consists of two domains, the N-terminal region and the C-terminal protein core. Although the N-terminal region has been suggested to be responsible for the binding to S100 proteins [21], the N-terminal truncated forms of annexin A1 and annexin A2 bind to dicalcin (Fig. 7). The binding of these forms sug- gests that these annexins bind to dicalcin not with the N-terminal regions but with the sites that have not yet been identified in their core domains. In S100A10 and S100A11, the amino acid residues contacting the corresponding annexins are known [22– 24]. In dicalcin, several of them are conserved (supple- mentary Fig. S4). The amino acids thought to give the subtype-specificity of S100 binding to annexin are also known [25]. However, these residues in dicalcin are dif- ferent from those in S100A10 or S100A11 (supplemen- tary Fig. S4), which suggests that the specificity of binding of dicalcin to annexins is not so strict. From the above considerations, we speculate that the binding between annexins and dicalcin occurs via the interaction between the conserved amino acids in dicalcin and the still unknown site in the core domain of annexin. Because annexin A5 lacks the correspond- ing N-terminal region of annexin A1 or annexin A2 (supplementary Fig. S1), it would not be surprising if frog annexin A5 bound to dicalcin. Recombinant frog annexin A4, which also lacks the corresponding N-ter- minal region, also showed Ca 2+ -dependent binding to dicalcin (data not shown). Similarly, as in the present study, it was reported recently that the N-terminus of A B Fig. 7. Ca 2+ -dependent binding to dicalcin of N-terminal region-trun- cated annexin A1 and annexin A2. (A) Recombinant annexin A1 and annexin A2 were truncated at their N-termini with elastase and chy- motrypsin, respectively, and mixed with dicalcin-Sepharose beads. The truncated annexin A1 and annexin A2 bound to the beads at a high Ca 2+ concentration, but they were eluted by reducing the Ca 2+ concentration (low-Ca 2+ wash). (B) Amino acid sequence analysis showed that the proteolytic fragments used in (A) lacked the N-ter- minal regions. Arrowheads show the sites cleaved and the mole- cular masses of the rest of the cleaved peptides. Arrows show the N-termini of the 35 kDa forms of annexin A1 and annexin A2. Role of dicalcin in frog olfactory cilia T. Uebi et al. 4870 FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS annexin 6 is not required for the interaction of annexin 6 with S100A11 [26]. Colocalization of dicalcin and annexins in the cilia We previously reported that dicalcin is present in the olfactory and the respiratory cilia [7]. Expression of S100 proteins has been reported in the olfactory epi- thelium in teleosts and rodents [27,28], and in the cilia of human bronchial epithelial cells [29]. Annexins have been detected in the tissues containing ciliated cells: the respiratory epithelium [30,31] and bronchial epithe- lial cells [29]. So far, however, localization of annexins in the olfactory cilia has not been reported, and there- fore, this is the first report that annexin A1, annex- in A2 and annexin A5 are expressed in the cilia of olfactory cells. In the present study, we showed that dicalcin, annexin A1, annexin A2 and annexin A5 co- localize in the olfactory cilia. Because ciliated cells seem to express both S100 proteins and annexins, our result could apply to cells that contain motile cilia in general. Annexin A1, annexin A2, annexin A5 and dicalcin are present in the olfactory cilia at a ratio of 1 : 0.42 ± 0.09 : 0.54 ± 0.15 : 1.9 ± 0.6, and dicalcin may be present in greater amounts (see Results). A molecular modeling study showed that the structure of dicalcin is similar to that of an S100 dimer [8]. Because a dimer form of S100 protein binds two annexin mole- cules [21], one dicalcin molecule would bind to two molecules of annexins. If it is the case, the amount of dicalcin is stoichiometrically sufficient to form com- plexes with annexin A1, annexin A2 and annexin A5. Facilitation by dicalcin of membrane aggregation induced by annexins The half-maximal dicalcin effects were observed at <5 lm Ca 2+ with annexin A2 and at about 30 lm with annexin A1 (Fig. 6). These Ca 2+ concentrations are the effective ranges of annexin A2 and annexin A1 of other animal species [32]. The dissociation constant of Ca 2+ binding to dicalcin has been reported to be 10–20 lm [12]. A simple expectation, therefore, was that the Ca 2+ concentration effective for liposome aggregation in the presence of annexin A2 and dicalcin would be determined by dicalcin, which shows lower affinity for Ca 2+ than does annexin A2. Similarly, one could expect that the effective Ca 2+ concentration in the presence of annexin A1 and dicalcin would be determined by annexin A1. However, the results were different from what we expected. The effective Ca 2+ concentrations did not change significantly in the presence or absence of dicalcin. The results indicated that the Ca 2+ dependency of liposome aggregation in the presence of dicalcin is determined by annexins, not by dicalcin. The result therefore suggested that there is cooperative regulation of Ca 2+ binding to dicalcin by annexins. The increase in the degree of Ca 2+ binding in the presence of binding proteins is known for S100A4 [33] and has been suggested for S100A11 [34]. We measured liposome aggregation in a mixture of dicalcin, annexin A1, annexin A2 and annexin A5 (Fig. 6D). The observed liposome aggregation profile could be explained by the sum of each of the constitu- ents in the mixture. In this study, we mixed all of these proteins at once. If, as we assumed, dicalcin binds to two molecules of annexin, a dicalcin molecule would be able to bind two annexin molecules of different sub- types, such as annexin A1 plus annexin A2, and ann- exin A1 plus annexin A5. However, the aggregation profile obtained in the mixture could be explained by the sum of the results obtained independently using single species of annexin. This result suggests that even when all of the annexins are present in the mixture, annexins of a homomeric pair, not a heteromeric one, tend to bind to dicalcin to form a complex. Possible physiologic functions of dicalcin and annexins in the cilia It has been estimated that the intracellular Ca 2+ con- centration in the olfactory cilia is about 40 nm at the resting level, and increases to higher levels after odorant stimulation [35]. In respiratory cilia, the intra- cellular Ca 2+ concentration increases up to a sub- micromolar level at the maximum [36]. The range of Ca 2+ concentration where the dicalcin–annexin com- plex has an effect seems to be higher than these ‘physi- ologic’ Ca 2+ concentrations. Therefore, we believe that the dicalcin–annexin complex exerts its effect when the Ca 2+ concentration is abnormally increased. The cell membranes of motile cilia are subject to mechanical stress and are often disrupted [37]. In addition to this, the olfactory cilia are exposed to environmental chemi- cals, microorganisms and viruses, etc., so that the cil- ium membrane is likely to be damaged. In these cases, the cytoplasmic Ca 2+ concentration at the disrupted site could possibly be quite high. Because (a) the effec- tive Ca 2+ concentrations are different between annex- in A1 and annexin A2 (Fig. 6), (b) dicalcin is present in a quantity sufficient to bind all of the annexins (see Results), and (c) all these molecules colocalize in the same cilia (Figs 4 and 5), it is possible that the dical- cin–annexin system could cover a wide range of Ca 2+ T. Uebi et al. Role of dicalcin in frog olfactory cilia FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4871 concentrations inside the cell to reseal the disrupted membranes. It has been reported that annexin A1 [38] and annexin A1 and annexin A2 [39] have important roles in membrane repair. Annexin A5 did not show liposome aggregation activity, in agreement with the findings of a previous study [14], even in the presence of dicalcin (Fig. 6D). Because antibody against annexin A5 has been reported to inhibit the survival of oxidation-damaged cells [40], the dicalcin–annexin A5 complex may possibly contri- bute to a recovery process after chemical damage. Dicalcin in other species So far, we have found dicalcin in Rana catesbeiana [6] and Xenopus laevis [5]. In addition, the sequence of dicalcin mRNA of X. tropicalis has been registered in a database (NM_001016706). Thus, dicalcin has been found only in the three species of frogs. The Mexican salamander, Ambystoma mexicanum, has an S100A11- like protein with an insertion of four amino acid resi- dues in its C-terminal half EF hand (supplementary Fig. S3), and this insertion is characteristically observed in dicalcin. Nevertheless, this S100A11-like protein is a monomer form of an S100 protein and is not like dical- cin. Therefore, dicalcin might be derived from a unique S100 protein of ancestral amphibia, and could be a frog-specific protein. Members of the Caudata, includ- ing the Mexican salamander, have a tendency to stay either in an aquatic or a terrestrial environment. In contrast, most frogs are more biphasic, and actively move between land and water. Because the olfactory motile cilia in these frogs could be exposed to vigorous mechanical stress very often, they might have needed to have a very effective membrane repair system. Dicalcin, a homodimer form of S100 proteins, could be the form of S100 protein that exerts this effect most efficiently. Experimental procedures Solutions The standard buffer solution contained 115 mm potassium gluconate, 2.5 mm KCl, and 10 mm Hepes (pH 7.5) (K-gluc buffer). Low-salt K-gluc buffer (LS-K-gluc buffer) con- tained 50 mm potassium gluconate and 20 mm Hepes (pH 7.5). Either 1 mm CaCl 2 or 5 mm EGTA was added to the LS-K-gluc buffer. Ringer’s solution contained 115 mm NaCl, 3 mm KCl, 2 mm MgCl 2 ,2mm CaCl 2 ,10mm glu- cose, and 5 mm Tris ⁄ HCl (pH 7.5). Tris-buffered saline (NaCl ⁄ Tris) contained 0.9% NaCl and 100 mm Tris ⁄ HCl (pH 7.5). NaCl ⁄ P i contained 137 mm NaCl, 2.7 mm KCl, 8.1 mm Na 2 HPO 4 , and 1.5 mm NaH 2 PO 4 (pH 7.4). Preparation of Chaps-solubilized proteins of the olfactory cilia Animal care was carried out in accordance with the institu- tional guidelines of Osaka University. Partially purified cilia from frog olfactory epithelium were obtained as described previously [13]. Briefly, olfactory cilia were detached from the epithelia by abruptly raising the Ca 2+ concentration to 10 mm. The deciliated epithelia were removed by brief centrifugation (1500 g, 5 min; TOMY MRX-150, TMA-11 rotor, TOMY SEIKO, Tokyo, Japan), and the supernatant containing the cilia was further centrifuged at 12 000 g for 15 min (TOMY MRX-150, TMA-11 rotor). The supernatant was removed and used as the soluble protein fraction of frog olfactory epithelium. The resulting pellet containing the isolated cilia was washed twice with K-gluc buffer, resuspended in LS-K-gluc buffer containing 4% Chaps, and kept at 4 °C overnight to solubi- lize the membrane-associated proteins of the isolated cilia. The Chaps-solubilized proteins were then obtained in the supernatant after centrifugation at 440 000 g for 5 min (Hitachi CS100, RP100AT4 rotor, Hitachi Koki, Tokyo, Japan). The supernatant was diluted with LS-K-gluc buffer containing 1 mm Ca 2+ so that the concentration of Chaps was reduced to 0.05%. The diluted fraction was centrifuged at 12 000 g for 30 min (TOMY MRX-150, TMA-11 rotor) to remove any precipitates before subjecting it to affinity column chromatography as described below. A cocktail of protease inhibitors (leupeptin, 5 lgÆmL )1 ; phenyl- methanesulfonyl fluoride, 5 lgÆmL )1 ; aprotinin, 5 lgÆmL )1 ; bestatin, 40 lgÆmL )1 ) was present at the indicated final con- centrations during the preparation of the above fraction. Affinity purification of binding proteins of dicalcin A dicalcin-Sepharose column was prepared as previously described [13]. Chaps-solubilized proteins of the isolated cilia were loaded on the dicalcin-Sepharose column pre- equilibrated with LS-K-gluc buffer containing 1 m m CaCl 2 and 0.05% Chaps. After elution of unbound proteins, pro- teins that were bound to the column at 1 mm Ca 2+ were eluted by reducing the Ca 2+ concentration with LS-K-gluc buffer containing 5 mm EGTA and 0.05% Chaps. In some studies, K-gluc buffer was used instead of LS-K-gluc buffer to isolate the binding proteins, but no significant differences were observed in the detected proteins. Determination of partial amino acid sequences of binding proteins of dicalcin Purified binding proteins of dicalcin were digested with lysyl endopeptidase (Wako, Osaka, Japan) at an enzyme ⁄ substrate ratio of 1 : 100 in 1 mL of a Tris buffer solution (100 mm Tris, pH 9.2) overnight at 37 °C. The Role of dicalcin in frog olfactory cilia T. Uebi et al. 4872 FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... annexin A1, annexin A2 and annexin A5 with those of mammalian orthologs Fig S2 Specificity of antisera against dicalcin and annexins Fig S3 Colocalization of dicalcin with annexin A1, annexin A2 and annexin A5 in frog respiratory epithelium Fig S4 Alignment of amino acid sequences of Rana catesbeiana dicalcin with those of S100 proteins This material is available as part of the online article from http://www.blackwell-synergy.com... described previously [6] Recombinant annexins were affinity-purified with a dicalcin- Sepharose column in a similar way as used for the isolation of native annexins Binding of recombinant annexins to dicalcin Transformed E coli cells expressing each of the annexins were suspended and sonicated in K-gluc buffer The lysate was centrifuged at 27 000 g for 15 min (Hitachi CR21, R20A2 rotor), and 1 mm CaCl2 was then... Identification of a novel interaction between the Ca2+-binding protein S, 100A, 11 and the Ca2+- and phospholipid-binding protein annexin A6 Am J Physiol Cell Physiol 292, C1417–C1430 Yamashita N, Ilg EC, Schafer BW, Heizmann CW & ¨ Kosaka T (1999) Distribution of a specific calcium-binding protein of the S100 protein family, S100A6 (calcyclin), in subpopulations of neurons and glial cells of the adult... (annexin A2), 7.5 nm (annexin A1), and 40 nm (dicalcin) , so that the ratio of the concentration of annexins was similar to that in the olfactory cilia (see Results section), but dicalcin was added in excess Acknowledgements We thank Dr H Matsumoto at the University of Oklahoma and Dr H Kurono at Kurume University for MS analysis of the binding proteins at the initial stage of this study This research... again under the same conditions to remove aggregated proteins, and a portion of the supernatant was mixed with dicalcinSepharose beads in K-gluc buffer containing 1 mm CaCl2 Role of dicalcin in frog olfactory cilia for 30 min at 4 °C After the mixture had been centrifuged (7000 g, 1 min; TOMY MRX-150, TMA-11 rotor), the supernatant was discarded The dicalcin- Sepharose beads were then washed 10 times with. .. role of the dysferlin interacting proteins annexin A1 and A2 in muscular dystrophies Hum Mutat 26, 283 Han S, Zhang KH, Lu PH & Xu XM (2004) Effects of annexins II and V on survival of neurons and astrocytes in vitro Acta Pharmacol Sin 25, 602–610 Huang K-S, McGray P, Mattaliano RJ, Burne CE, Chow P, Sinclair LK & Pepinsky RB (1987) Purification and characterization of proteolytic fragments of lipocortin... containing 1 mm CaCl2 Proteins bound to dicalcin- Sepharose beads at a high Ca2+ concentration were then eluted with K-gluc buffer containing 5 mm EGTA When truncated annexins were used, annexin A1 and annexin A2 were digested with elastase and chymotrypsin, respectively These enzymes are known to cleave the N-terminal regions of annexin A1 and annexin A2 [18,41], respectively The cleaved sites in these... Antisera against annexin A1, annexin A2 and annexin A5 were raised in both rabbit and mouse, and antiserum against dicalcin was raised in rabbit Golf antibody raised in rabbit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) For double staining, sections were reacted first with antiserum or antibody raised in rabbit overnight at 4 °C, and then were further reacted with antiserum raised in mouse... calcium- and phospholipid-binding proteins Biochim Biophys Acta 1197, 63–93 Dukhanina EA, Dukhanin AS, Lomonosov MY, Lukanidin EM & Georgiev GP (1997) Spectral studies on the calcium-binding properties of Mts1 protein and its interaction with target protein FEBS Lett 410, 403–406 Allen BG, Durussel I, Walsh MP & Cox JA (1996) Characterization of the Ca2+-binding properties of calgizzarin (S100C) isolated... collected, and the amino acid sequences of the peptides in these fractions were analyzed with a protein sequencer (G1000A; Hewlett-Packard, Palo Alto, CA, USA) Isolation of annexin cDNA clones Screening in the frog olfactory epithelium cDNA library to isolate annexin cDNAs was carried out in a similar way as described previously [6] On the basis of either the partial amino acid sequences of annexins determined . 4865 Colocalization of annexins and dicalcin in frog olfactory and respiratory epithelium Dicalcin has been reported to localize in the cilia of frog olfactory and respiratory. aggregation. Results Purification of binding proteins of dicalcin Binding proteins of dicalcin were searched for among the soluble and membrane-associated proteins of frog olfactory cilia.