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Comprehensive interaction of dicalcin with annexinsin frog olfactory and respiratory ciliaTatsuya Uebi1, Naofumi Miwa1,2,* and Satoru Kawamura1,21 Department of Biology, Graduate School of Science, Osaka University, Japan2 Graduate School of Frontier Biosciences, Osaka University, JapanCalcium ions are known to modulate signal transduc-tion in various cells. This effect is usually mediatedby Ca2+-binding proteins. For example, in olfactoryreceptor cells, odorant stimuli induce Ca2+influxthrough a cyclic nucleotide gated channel [1]. Theincrease in the Ca2+concentration is detected bycalmodulin, a well-known Ca2+-binding protein. TheCa2+-bound form of calmodulin has essential roles inolfactory adaptation [2,3]. In photoreceptor cells, sev-eral Ca2+-binding proteins are known to be presentand to modulate phototransduction signals [4].We previously found a Ca2+-binding protein, dical-cin (renamed from p26olf [5]), in frog olfactory epithe-lium, and reported that dicalcin is expressed in theolfactory epithelium, lung, and spleen [6,7]. In theolfactory epithelium and lung, dicalcin localizes in thecilia. Dicalcin has partial homology to S100 proteins, afamily of EF-hand Ca2+-binding proteins, and consistsof two S100A11-like regions aligned in sequence. Theamino 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 variouscellular functions, such as cell cycle progression andcell survival [9–11]. S100 proteins show no enzymaticactivities by themselves and, instead, modulate thefunction of other proteins through direct binding toKeywordsannexin; dicalcin; olfactory cilia; respiratorycilia; S100CorrespondenceS. Kawamura, Graduate School of FrontierBiosciences, Osaka University, Yamada-oka1–3, Suita, Osaka 565-0871, JapanFax: +81 6 6879 4614Tel: +81 6 6879 4610E-mail: kawamura@fbs.osaka-u.ac.jp*Present addressDepartment of Physiology, School ofMedicine, Toho University, Tokyo, JapanDatabaseAmino acid sequences have been submittedto DDBJ under the following accessionnumbers: frog annexin A1, AB286845; frogannexin A2, AB286846; frog annexin A4,AB286848; frog annexin A5, AB286847(Received 8 May 2007, revised 20 July2007, accepted 24 July 2007)doi:10.1111/j.1742-4658.2007.06007.xDicalcin (renamed from p26olf) is a dimer form of S100 proteins foundin frog olfactory epithelium. S100 proteins form a group of EF-handCa2+-binding proteins, and are known to interact with many kinds of tar-get protein to modify their activities. To determine the role of dicalcin inthe olfactory epithelium, we identified its binding proteins. Several proteinsin frog olfactory epithelium were found to bind to dicalcin in aCa2+-dependent manner. Among them, 38 kDa and 35 kDa proteins weremost abundant. Our analysis showed that these were a mixture of annex-in A1, annexin A2 and annexin A5. Immunohistochemical analysis showedthat dicalcin and all of these three subtypes of annexin colocalize in theolfactory cilia. Dicalcin was found to be present in a quantity almost suffi-cient to bind all of these annexins. Colocalization of dicalcin and the threesubtypes of annexin was also observed in the frog respiratory cilia. Dicalcinfacilitated Ca2+-dependent liposome aggregation caused by annexin A1 orannexin A2, and this facilitation was additive when both annexin A1 andannexin A2 were present. In this facilitation effect, the effective Ca2+con-centrations were different between annexin A1 and annexin A2, and there-fore the dicalcin–annexin system in frog olfactory and respiratory cilia cancover a wide range of Ca2+concentrations. These results suggested thatthis system is associated with abnormal increases in the Ca2+concentrationin the olfactory and other motile cilia.FEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4863these proteins. p53, RAGE and annexins are known tobe binding proteins of S100 proteins. S100 proteins areknown to form dimers, and the dimer form binds tothe binding protein to exert the effect. Because dicalcinconsists of two S100-like domains aligned in sequence,the function of dicalcin is probably similar to that ofan S100 dimer.Although the Ca2+-binding property has been inves-tigated in detail in dicalcin [12], little is known aboutits physiologic function. To investigate this, in thepresent study we first tried to determine the bindingproteins of dicalcin. We found that several of the pro-teins in frog olfactory epithelium bind to dicalcin in aCa2+-dependent manner. Among them, 38 kDa and35 kDa proteins were the major proteins. We identifiedthem as annexin A1, annexin A2 and annexin A5. Wefurther examined their localizations and the effect ofdicalcin on the activities of these annexins by measur-ing liposome aggregation.ResultsPurification of binding proteins of dicalcinBinding proteins of dicalcin were searched for amongthe soluble and membrane-associated proteins of frogolfactory cilia. Because dicalcin is an S100-likeEF-hand Ca2+-binding protein, we expected that thebinding proteins would bind to dicalcin in a Ca2+-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-Sepharosecolumn at 1 mm Ca2+. Most of the proteins werefound in the pass-through fraction (Fig. 1, elutionpeak A and lane A), but some of the proteins wereretained, and eluted by reducing the Ca2+concentra-tion (Fig. 1, elution peak B and lane B). Several pro-teins were found in lane B, but the major proteinswere 38 kDa and 35 kDa proteins. The latter couldbe 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 whendicalcin was not attached to the Sepharose beads(Fig. 1C). Although the amount of each of the elutedproteins varied among preparations, 38 kDa and35 kDa proteins were always the major constituents.We therefore focused on these proteins in the follow-ing study. Essentially similar binding proteins weredetected when we used the soluble protein fraction,but the amounts of the proteins were greater in theChaps-solubilized fraction. For this reason, we usedthis fraction in the following studies.Amino acid sequence analysis of 38 kDa and35 kDa proteinsDuring the course of this study, we realized that35 kDa proteins contained proteolytic fragments of38 kDa proteins: in the presence of protease inhibi-tors, the amount of 38 kDa proteins was larger thanthat in the absence of the inhibitors. However, wecould not inhibit the proteolysis completely: even inthe presence of a cocktail of inhibitors, our immuno-logic study detected signals of 38 kDa proteins at the35 kDa position (see Fig. 3A below). In addition, thedegree of inhibition was variable, depending on eachpreparation. 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 theiramino acid sequences were determined. The resultsuggested that the 38 kDa and the 35 kDa proteinsare 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 annexinfamily protein. Instead, the sequence of a fragmentshowed some similarity to the sequence of annex-in A1, annexin A2, annexin A4 or annexin A5 ofFig. 1. Purification of binding proteins of dicalcin by affinity columnchromatography. The Chaps-solubilized protein fraction of the ciliaof frog olfactory epithelium was loaded to a dicalcin-Sepharose col-umn at 1 mM Ca2+. Most of the proteins passed through the col-umn (A in the elution profile) at a high (1 mM)Ca2+concentration,but some proteins remained in the column and came out only afteraddition of 5 mM EGTA (B in the profile). Inset: SDS ⁄ PAGE patternsof the Chaps-solubilized cilia protein fraction (cilia), the pass-throughfraction (A) and the eluate in the presence of 5 mM EGTA (B). As acontrol, an eluate was obtained similarly as in (B), but with the useof Sepharose beads without dicalcin conjugated (C). Proteins werestained 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 FEBSother animal species, which suggested that the38 kDa and the 35 kDa proteins were a mixture ofthese annexins. We therefore tried to isolate cDNAsof annexin A1, annexin A2, annexin A4 and annex-in A5 to identify which annexins were in the fractionof the 38 kDa and the 35 kDa proteins.Cloning of annexin cDNAsOn the basis of the partial amino acid sequences of theproteolytic fragments as determined above, we synthe-sized oligonucleotide degenerate primers and usedthem 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 wereamplified, and the frog olfactory cDNA librarywas screened with these fragments. The full-lengthsequences of frog annexin cDNAs were obtained, andthe amino acid sequences were deduced (supplemen-tary Fig. S1). The amino acid sequences detected inthe proteolytic fragments were found in the deducedamino 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 wasnot present, or the content of annexin A4 was small inthe fraction of the 38 kDa and 35 kDa proteins.Among our recombinant annexins (see below), theapparent molecular mass of annexin A4 was slightlylower than 35 kDa on our SDS ⁄ PAGE gel. Becausethe density of the corresponding position on theSDS ⁄ PAGE gel of the binding proteins of dicalcin wasfaint, this result also suggested that the content ofannexin A4 in the 35 kDa proteins was small even ifit was present. For these reasons, we did not studyannexin A4 further.Identification of annexin A1, annexin A2 andannexin A5 as the binding proteins of dicalcinOur results were so far consistent with the notion thatthe 38 kDa and the 35 kDa proteins are annexin A1,annexin A2, and annexin A5. However, we were nottotally sure of this at this stage. Therefore, we firsttried to confirm that annexin A1, annexin A2 and an-nexin A5 show Ca2+-dependent binding to dicalcin, asthe 38 kDa and the 35 kDa proteins do. For this, weobtained recombinant annexin A1, annexin A2 andannexin A5 expressed in Escherichia coli. The apparentmolecular masses of recombinant annexin A1 and ann-exin A2 were both 38 kDa, and that of annexin A5was 35 kDa (Fig. 2), and all of them bound to the di-calcin-Sepharose beads in a Ca2+-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 againstannexin A1, annexin A2 or annexin A5 in mouse andrabbit using recombinant annexins (supplementaryFig. S2). Antiserum against annexin A1 recognized boththe 38 kDa and the 35 kDa proteins (Fig. 3A, low Ca2+eluate), and antiserum against annexin A2 also detectedthe 38 kDa and the 35 kDa proteins. Antiserum againstannexin A5 detected only the 35 kDa proteins.From the above results, it became evident that the38 kDa proteins contained both full-length annexin A1and annexin A2, and the 35 kDa proteins containedfull-length annexin A5 together with proteolyticfragments 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 the38 kDa and 35 kDa proteins (Fig. 3B). In Fig. 3B,there are weak signals of annexin A1 at aroundpH 5.1. They are probably the signals of annexin A1that was not focused in our two-dimensional electro-phoresis.Fig. 2. Ca2+-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 mM Ca2+. The beads were washed 10 times bycentrifugation with K-gluc buffer supplemented with 1 mM Ca2+,and the 1st and the 10th extracts were subjected to SDS ⁄ PAGE(high-Ca2+wash 1 and high-Ca2+wash 10). The beads were finallywashed with K-gluc buffer supplemented with 5 mM EGTA, andthe extract was subjected to SDS ⁄ PAGE (low-Ca2+wash).T. Uebi et al. Role of dicalcin in frog olfactory ciliaFEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4865Colocalization of annexins and dicalcin in frogolfactory and respiratory epitheliumDicalcin has been reported to localize in the cilia offrog olfactory and respiratory epithelium [7]. Tounderstand the possible association of annexin A1,annexin A2 and annexin A5 with the function of dical-cin, we examined the colocalization of each annexinwith dicalcin, using specific antisera (supplementaryFig. S2). In addition, we also examined whether differ-ent subtypes of the annexins colocalize in the samecilia. Figures 4 and 5 show the immunohistochemicalstudies of dicalcin and annexin A1, annexin A2 andannexin A5. In Fig. 4, the olfactory cilia, which wereidentified immunohistochemically with olfactory cilia-specific Golfantibody (Fig. 4M), were found to bereactive to antiserum against dicalcin (Fig. 4A,D,G).The cilia were also positively stained with antiserumagainst annexin A1 (Fig. 4B), annexin A2 (Fig. 4E),and annexin A5 (Fig. 4H). The merged image clearlyshowed colocalization of dicalcin with each of the an-nexins (Fig. 4C,F,I). In this study, the conditions forobtaining immunofluorescence were kept constant ineach of the observations with rabbit antiserum (TexasRed) or mouse antiserum (fluorescein isothiocyanate),and therefore the color in the merged picture wasdependent on the relative intensities of red and greenfluorescence, namely, the titers of antisera against di-calcin and annexins. Preabsorption of the specific anti-bodies by recombinant proteins significantly reducedthe signals (Miwa et al. [13] for anti-dicalcin serumand 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 inthe same cell. Figure 5 shows the immunohistochemi-cal study of colocalization of annexin A1, annexin A2,and annexin A5. For any combination of these threesubtypes of annexin, colocalization was demonstrated(Fig. 5). Therefore, it was evident that all three sub-types of annexin are present in the same olfactorycilium. From the results in Figs 4 and 5, it becameevident that dicalcin, annexin A1, annexin A2 andannexin A5 all colocalize in the same olfactory cilium.In the respiratory epithelium, similar colocalizationwas observed (supplementary Fig. S3), although thesignal of Golf, a marker protein of olfactory cells, wasnot seen.Estimation of the relative molecular abundanceof dicalcin and annexins in frog olfactory ciliaThe above immunohistochemical study showed that allsubtypes of the annexins studied here colocalize withABFig. 3. Identification of annexin A1, annexin A2 and annexin A5 by western blot analysis. (A) Determination that the 38 kDa proteins are amixture 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-Ca2+eluate), were electrophoresed on an SDS ⁄ PAGE gel, and the proteins were stained with silver (silver stain). The proteins were probed withspecific 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 duringpreparation. (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 molecularmass 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 FEBSdicalcin in frog olfactory cilia. To understand the sig-nificance of this colocalization, we tried to estimate therelative molecular abundance of dicalcin and annexins.In this quantification, we used both the soluble andthe membrane fraction after detachment of the cilia(see Experimental procedures). They were solubilizedwith the SDS ⁄ PAGE sample buffer, and were directlyelectrophoresed with known amounts of recombinantdicalcin and annexins. The contents of annexins anddicalcin 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 solubleprotein, and annexins were mostly present in theChaps-solubilized fraction. Dicalcin might have beenlost during isolation of the olfactory epithelium, andtherefore the content of dicalcin could be higher thanthe value determined above. Because the number andthe 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 annexinsAs has been reported previously, annexins are knownto induce membrane aggregation in a Ca2+-dependentmanner [14], and it is also known that this activity ofannexins is enhanced by binding of S100 proteins [15].We therefore examined the effect of dicalcin onthe membrane aggregation activity of annexins. TheABCDEFGHIJKLMNOFig. 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 againstone 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) Arepresentative section treated with antibody to Golf. All positive signals against dicalcin, annexins and Golfwere observed in the cilia layer(arrowheads). (N) A control. Antiserum against annexin A2 was preabsorbed with recombinant annexin A2. (O) Frog olfactory epitheliumstained 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 ciliaFEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4867activity was measured as the increase in the absorbancedue to aggregation of phosphatidylserine liposomes(see inset in Fig. 6E, for example). The dose effect ofeach 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 ina dose-dependent manner (filled rectangles and filledcircles, respectively). Dicalcin increased their activities,and the effect was higher on annexin A2 (open circles)than on annexin A1 (open rectangles). Annexin A5 didnot show liposome aggregation activity (open and filledtriangles). Although the effect of dicalcin was obviousat annexin concentrations above 40 nm, the increase inthe absorbance was often too rapid for reliable data tobe obtained. For this reason, we used annexins at lowconcentrations. The concentrations of annexins werekept at 12.5 nm (annexin A1), 5 nm (annexin A2) and7.5 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 wasadded in excess.The effect of dicalcin on liposome aggregationinduced by annexins was measured at various Ca2+concentrations, and the initial rate of increase wasplotted as a function of Ca2+concentrations. Asshown in Fig. 6B, no significant aggregation wasobserved in the absence of annexins (filled triangles) ordicalcin (open triangles). In the absence of liposomes,no significant increase in absorbance was detected (notshown). In the presence of annexins alone, slightaggregation was observed, but the effect was not solarge (filled circles in Fig. 6B–E) at the annexin con-centrations used (see above). When dicalcin waspresent (open circles), the liposome aggregation activi-ties of annexin A1 or annexin A2 were facilitatedABCDEFGHIJKLFig. 5. Colocalization of annexin A1, annexin A2 and annexin A5 in frog olfactory epithelium. (A–I) Immunofluorescence double-staining ofone 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 treatedwith 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 FEBSgreatly when the Ca2+concentration was increased(Fig. 6B,C). Essentially, the effect of dicalcin was notseen with annexin A5 (Fig. 6D).The effective Ca2+concentrations depended on thesubtype of annexin: annexin A2 was more sensitive toCa2+than annexin A1. The half-maximal dicalcineffect was observed at < 5 lm Ca2+with annexin A2,but at about 30 lm with annexin A1. Although the ini-tial rate of aggregation increased to a similar level forboth annexin A1 and annexin A2 at high Ca2+con-centrations (Fig. 6B,C), this was partly because of thedifference 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 samelevel as that of annexin A1, the effect of dicalcin wasat least two times larger for annexin A2 than forannexin A1 (Fig. 6A).To simulate the effect of dicalcin in a cell, dicalcinwas added to the mixture of annexin A1, annexin A2and 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 calculatedsum of each of the activities of annexin A1, annex-in A2 and annexin A5 (Fig. 6E, thick dotted lines).Binding of truncated forms of annexinsto dicalcinIn the present study, we found that dicalcin binds toannexin A1, annexin A2 and annexin A5, and that itfacilitates the membrane aggregation activities of ann-exin A1 and annexin A2. In mammal S100 proteinsand annexins, an S100–annexin complex is formed ina subtype-specific manner: S100A10 binds to annex-in A2 [16], and S100A11 binds to annexin A1 [17]. Inthe case of mammal annexin A1 and annexin A2, thespecificity has been reported to arise in part at theirN-terminal 1–13 amino acids [18,19]. Because dicalcinbinds 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 couldbe different from those known previously. To test thispossibility, we examined the binding to dicalcin ofFig. 6. Effect of dicalcin on liposome aggregation induced by an-nexins. Time courses of annexin-induced liposome aggregationwere measured as the increase in the absorbance at 350 nm [seeinset 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 nM dicalcin at 100 lM Ca2+. The initial rate ofthe absorbance increase was plotted against the annexin concen-tration. In (B)–(E), liposome aggregation was measured at variousCa2+concentrations in the presence (open circles) and absence(filled circles) of dicalcin (DC). The initial rate of the absorbanceincrease was plotted against the Ca2+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 ± standarderror 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 asthin dotted lines in (C) and (D). The result obtained in the presenceof 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 ciliaFEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4869N-terminal-truncated forms of frog annexin A1 andannexin A2. The result showed that, indeed, dicalcinbinds to these truncated forms (Fig. 7A), which indi-cated that the N-terminal region is not essential forthe interaction of frog annexin A1 and annexin A2with dicalcin. Consistently, we observed that the35 kDa forms of annexin A1 and annexin A2 foundin the fraction of the binding proteins of dicalcin(Fig. 1) were the N-terminal-truncated annexins(Fig. 7B).DiscussionIn the present study, we showed that the major bind-ing proteins of dicalcin in frog olfactory epithelium areannexin A1, annexin A2 and annexin A5 (Figs 1–3and supplementary Fig. S1). The binding does notrequire the N-terminal region of annexins (Fig. 7). Di-calcin and all these annexins colocalize in the olfactoryand respiratory cilia (Figs 4 and 5 and supplementaryFig. S3). Dicalcin was found to increase the rate ofliposome aggregation caused by annexins (Fig. 6).Specificity of the binding between dicalcin andannexinsIn the present study, we identified the 38 kDa and the35 kDa proteins as annexin A1, annexin A2 and ann-exin A5. Annexins are known to bind to a dimerform of S100 proteins. In mammals, the bindingbetween annexins and S100 dimer proteins has beenshown to be subtype-specific. S100A11 binds to annex-in A1 [17] (but see [20] also), and S100A10 binds toannexin A2 [16]. Because dicalcin in frogs shows thehighest amino acid sequence homology to S100A11(45–58%), the binding of dicalcin to annexin A1 is notsurprising. However, binding to all of annexin A1,annexin A2 and annexin A5 is a rather unique charac-teristic of dicalcin, although similar comprehensivebinding has been suggested for some of the S100 pro-teins [11]. The comprehensive binding of dicalcin tovarious 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-terminalregion and the C-terminal protein core. Although theN-terminal region has been suggested to be responsiblefor the binding to S100 proteins [21], the N-terminaltruncated forms of annexin A1 and annexin A2 bindto dicalcin (Fig. 7). The binding of these forms sug-gests that these annexins bind to dicalcin not with theN-terminal regions but with the sites that have not yetbeen identified in their core domains.In S100A10 and S100A11, the amino acid residuescontacting the corresponding annexins are known [22–24]. In dicalcin, several of them are conserved (supple-mentary Fig. S4). The amino acids thought to give thesubtype-specificity of S100 binding to annexin are alsoknown [25]. However, these residues in dicalcin are dif-ferent from those in S100A10 or S100A11 (supplemen-tary Fig. S4), which suggests that the specificity ofbinding of dicalcin to annexins is not so strict.From the above considerations, we speculate thatthe binding between annexins and dicalcin occurs viathe interaction between the conserved amino acids indicalcin and the still unknown site in the core domainof 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 iffrog annexin A5 bound to dicalcin. Recombinant frogannexin A4, which also lacks the corresponding N-ter-minal region, also showed Ca2+-dependent binding todicalcin (data not shown). Similarly, as in the presentstudy, it was reported recently that the N-terminus ofABFig. 7. Ca2+-dependent binding to dicalcin of N-terminal region-trun-cated annexin A1 and annexin A2. (A) Recombinant annexin A1 andannexin 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 ahigh Ca2+concentration, but they were eluted by reducing the Ca2+concentration (low-Ca2+wash). (B) Amino acid sequence analysisshowed 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 theN-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 FEBSannexin 6 is not required for the interaction of annexin6 with S100A11 [26].Colocalization of dicalcin and annexinsin the ciliaWe previously reported that dicalcin is present in theolfactory and the respiratory cilia [7]. Expression ofS100 proteins has been reported in the olfactory epi-thelium in teleosts and rodents [27,28], and in the ciliaof human bronchial epithelial cells [29]. Annexins havebeen detected in the tissues containing ciliated cells:the respiratory epithelium [30,31] and bronchial epithe-lial cells [29]. So far, however, localization of annexinsin 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 ofolfactory cells. In the present study, we showed thatdicalcin, annexin A1, annexin A2 and annexin A5 co-localize in the olfactory cilia. Because ciliated cellsseem to express both S100 proteins and annexins, ourresult could apply to cells that contain motile cilia ingeneral.Annexin A1, annexin A2, annexin A5 and dicalcinare present in the olfactory cilia at a ratio of1 : 0.42 ± 0.09 : 0.54 ± 0.15 : 1.9 ± 0.6, and dicalcinmay be present in greater amounts (see Results). Amolecular modeling study showed that the structure ofdicalcin is similar to that of an S100 dimer [8]. Becausea dimer form of S100 protein binds two annexin mole-cules [21], one dicalcin molecule would bind to twomolecules of annexins. If it is the case, the amount ofdicalcin is stoichiometrically sufficient to form com-plexes with annexin A1, annexin A2 and annexin A5.Facilitation by dicalcin of membrane aggregationinduced by annexinsThe half-maximal dicalcin effects were observed at<5 lm Ca2+with annexin A2 and at about 30 lmwith annexin A1 (Fig. 6). These Ca2+concentrationsare the effective ranges of annexin A2 and annexin A1of other animal species [32]. The dissociation constantof Ca2+binding to dicalcin has been reported to be10–20 lm [12]. A simple expectation, therefore, wasthat the Ca2+concentration effective for liposomeaggregation in the presence of annexin A2 and dicalcinwould be determined by dicalcin, which shows loweraffinity for Ca2+than does annexin A2. Similarly, onecould expect that the effective Ca2+concentration inthe presence of annexin A1 and dicalcin would bedetermined by annexin A1. However, the results weredifferent from what we expected. The effective Ca2+concentrations did not change significantly in thepresence or absence of dicalcin. The results indicatedthat the Ca2+dependency of liposome aggregation inthe presence of dicalcin is determined by annexins, notby dicalcin. The result therefore suggested that there iscooperative regulation of Ca2+binding to dicalcin byannexins. The increase in the degree of Ca2+bindingin the presence of binding proteins is known forS100A4 [33] and has been suggested for S100A11 [34].We measured liposome aggregation in a mixture ofdicalcin, annexin A1, annexin A2 and annexin A5(Fig. 6D). The observed liposome aggregation profilecould be explained by the sum of each of the constitu-ents in the mixture. In this study, we mixed all of theseproteins at once. If, as we assumed, dicalcin binds totwo molecules of annexin, a dicalcin molecule wouldbe 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 aggregationprofile obtained in the mixture could be explained bythe sum of the results obtained independently usingsingle species of annexin. This result suggests that evenwhen 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 dicalcinand annexins in the ciliaIt has been estimated that the intracellular Ca2+con-centration in the olfactory cilia is about 40 nm at theresting level, and increases to higher levels afterodorant stimulation [35]. In respiratory cilia, the intra-cellular Ca2+concentration increases up to a sub-micromolar level at the maximum [36]. The range ofCa2+concentration where the dicalcin–annexin com-plex has an effect seems to be higher than these ‘physi-ologic’ Ca2+concentrations. Therefore, we believe thatthe dicalcin–annexin complex exerts its effect when theCa2+concentration is abnormally increased. The cellmembranes of motile cilia are subject to mechanicalstress 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 Ca2+concentration at the disruptedsite could possibly be quite high. Because (a) the effec-tive Ca2+concentrations are different between annex-in A1 and annexin A2 (Fig. 6), (b) dicalcin is presentin a quantity sufficient to bind all of the annexins (seeResults), and (c) all these molecules colocalize in thesame cilia (Figs 4 and 5), it is possible that the dical-cin–annexin system could cover a wide range of Ca2+T. Uebi et al. Role of dicalcin in frog olfactory ciliaFEBS Journal 274 (2007) 4863–4876 ª 2007 The Authors Journal compilation ª 2007 FEBS 4871concentrations inside the cell to reseal the disruptedmembranes. It has been reported that annexin A1 [38]and annexin A1 and annexin A2 [39] have importantroles in membrane repair.Annexin A5 did not show liposome aggregationactivity, in agreement with the findings of a previousstudy [14], even in the presence of dicalcin (Fig. 6D).Because antibody against annexin A5 has been reportedto 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 speciesSo far, we have found dicalcin in Rana catesbeiana [6]and Xenopus laevis [5]. In addition, the sequence ofdicalcin mRNA of X. tropicalis has been registered ina database (NM_001016706). Thus, dicalcin has beenfound only in the three species of frogs. The Mexicansalamander, Ambystoma mexicanum, has an S100A11-like protein with an insertion of four amino acid resi-dues in its C-terminal half EF hand (supplementaryFig. S3), and this insertion is characteristically observedin dicalcin. Nevertheless, this S100A11-like protein is amonomer form of an S100 protein and is not like dical-cin. Therefore, dicalcin might be derived from a uniqueS100 protein of ancestral amphibia, and could be afrog-specific protein. Members of the Caudata, includ-ing the Mexican salamander, have a tendency to stayeither in an aquatic or a terrestrial environment. Incontrast, most frogs are more biphasic, and activelymove between land and water. Because the olfactorymotile cilia in these frogs could be exposed to vigorousmechanical stress very often, they might have needed tohave a very effective membrane repair system. Dicalcin,a homodimer form of S100 proteins, could be the formof S100 protein that exerts this effect most efficiently.Experimental proceduresSolutionsThe standard buffer solution contained 115 mm potassiumgluconate, 2.5 mm KCl, and 10 mm Hepes (pH 7.5) (K-glucbuffer). 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 CaCl2or 5 mm EGTA was added tothe LS-K-gluc buffer. Ringer’s solution contained 115 mmNaCl, 3 mm KCl, 2 mm MgCl2,2mm CaCl2,10mm 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 ⁄ Picontained 137 mm NaCl, 2.7 mm KCl,8.1 mm Na2HPO4, and 1.5 mm NaH2PO4(pH 7.4).Preparation of Chaps-solubilized proteins of theolfactory ciliaAnimal care was carried out in accordance with the institu-tional guidelines of Osaka University.Partially purified cilia from frog olfactory epitheliumwere obtained as described previously [13]. Briefly, olfactorycilia were detached from the epithelia by abruptly raisingthe Ca2+concentration to 10 mm. The deciliated epitheliawere 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 furthercentrifuged at 12 000 g for 15 min (TOMY MRX-150,TMA-11 rotor). The supernatant was removed and used asthe soluble protein fraction of frog olfactory epithelium.The resulting pellet containing the isolated cilia was washedtwice with K-gluc buffer, resuspended in LS-K-gluc buffercontaining 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 thesupernatant 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 buffercontaining 1 mm Ca2+so that the concentration of Chapswas reduced to 0.05%. The diluted fraction was centrifugedat 12 000 g for 30 min (TOMY MRX-150, TMA-11 rotor)to remove any precipitates before subjecting it to affinitycolumn chromatography as described below. A cocktailof 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 ofdicalcinA dicalcin-Sepharose column was prepared as previouslydescribed [13]. Chaps-solubilized proteins of the isolatedcilia were loaded on the dicalcin-Sepharose column pre-equilibrated with LS-K-gluc buffer containing 1 m m CaCl2and 0.05% Chaps. After elution of unbound proteins, pro-teins that were bound to the column at 1 mm Ca2+wereeluted by reducing the Ca2+concentration with LS-K-glucbuffer containing 5 mm EGTA and 0.05% Chaps. In somestudies, K-gluc buffer was used instead of LS-K-gluc bufferto isolate the binding proteins, but no significant differenceswere observed in the detected proteins.Determination of partial amino acid sequencesof binding proteins of dicalcinPurified binding proteins of dicalcin were digested withlysyl endopeptidase (Wako, Osaka, Japan) at anenzyme ⁄ substrate ratio of 1 : 100 in 1 mL of a Tris buffersolution (100 mm Tris, pH 9.2) overnight at 37 °C. TheRole 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 . 4865Colocalization 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.ResultsPurification of binding proteins of dicalcin Binding proteins of dicalcin were searched for amongthe soluble and membrane-associated proteins of frog olfactory cilia.
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