Tài liệu Báo cáo khoa học: Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I pdf

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Comparative studies on the functional roles of N- andC-terminal regions of molluskan and vertebrate troponin-IHiroyuki Tanaka1, Yuhei Takeya1, Teppei Doi1, Fumiaki Yumoto2,3, Masaru Tanokura3,Iwao Ohtsuki2, Kiyoyoshi Nishita1and Takao Ojima11 Laboratory of Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Japan2 Laboratory of Physiology, The Jikei University School of Medicine, Tokyo, Japan3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, JapanTroponin is a Ca2+-dependent regulatory protein com-plex, which constitute thin filaments together withactin and tropomyosin [1]. It is composed of three dis-tinct subunits: troponin-C (TnC), which binds Ca2+,troponin-T (TnT), which binds tropomyosin, and trop-onin-I (TnI), which binds actin and inhibits actin–myo-sin interaction [2–4]. In relaxed muscle, TnI binds toactin and inhibits contraction. Upon muscle stimula-tion, Ca2+binds to TnC and induces the release of theinhibition by TnI, resulting in muscle contraction. Tounderstand the molecular mechanisms of this Ca2+switching, extensive studies of the structure, function,and Ca2+-dependent conformational changes of tropo-nin subunits have been carried out.In vertebrate muscles, TnC has a dumbbell-likeshape with the N- and C-terminal globular domainslinked by a central helix [5,6]. Each domain containstwo EF-hand Ca2+-binding motifs [7], thus TnC hasfour possible Ca2+-binding sites, sites I and II in theN-domain and sites III and IV in the C-domain [8,9].Keywordsinvertebrate; mollusk; regulatorymechanism; troponin; troponin-ICorrespondenceTakao Ojima, Laboratory of Biochemistryand Biotechnology, Graduate School ofFisheries Sciences, Hokkaido University,Hakodate, Hokkaido 041–8611, JapanTel ⁄ Fax: +81 138 408800E-mail: ojima@fish.hokudai.ac.jpNoteThe nucleotide sequences of cDNAs enco-ding Akazara scallop 52K-TnI and 19K-TnIare available in DDBJ ⁄ EMBL ⁄ GenBankdatabases under accession numbers,AB206837 and AB206838, respectively(Received 24 March 2005, revised 13 June2005, accepted 15 July 2005)doi:10.1111/j.1742-4658.2005.04866.xVertebrate troponin regulates muscle contraction through alternative bind-ing of the C-terminal region of the inhibitory subunit, troponin-I (TnI), toactin or troponin-C (TnC) in a Ca2+-dependent manner. To elucidate themolecular mechanisms of this regulation by molluskan troponin, we com-pared the functional properties of the recombinant fragments of Akazarascallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akaz-ara scallop TnI (ATnI232)292), which contains the inhibitory region (resi-dues 104–115 of rabbit TnI) and the regulatory TnC-binding site (residues116–131), bound actin-tropomyosin and inhibited actomyosin-tropomyosinMg-ATPase. However, it did not interact with TnC, even in the presenceof Ca2+. These results indicated that the mechanism involved in the alter-native binding of this region was not observed in molluskan troponin. Onthe other hand, ATnI130)252, which contains the structural TnC-binding site(residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly toboth actin and TnC. Moreover, the ternary complex consisting of this frag-ment, troponin-T, and TnC activated the ATPase in a Ca2+-dependentmanner almost as effectively as intact Akazara scallop troponin. Therefore,Akazara scallop troponin regulates the contraction through the activatingmechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observationthat corresponding rabbit TnI-fragment (RTnI1)116) shows similar activa-ting effects, these findings suggest the importance of the TnI N-terminalregion not only for maintaining the structural integrity of troponin com-plex but also for Ca2+-dependent activation.AbbreviationsTnC, troponin-C; TnI, troponin-I; TnT, troponin-T; IPTG, isopropyl-1-thio-b-D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride.FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4475Sites III and IV also show affinity for Mg2+and arethought to be always occupied by sarcoplasmic Mg2+,whereas Ca2+binding to site I and ⁄ or II is believedto trigger muscle contraction [10]. TnC interacts withboth TnI and TnT. The TnC–TnI interaction andchanges in the interaction upon Ca2+binding to TnChave been intensively studied as the central mecha-nisms of Ca2+switching. It has been revealed that TnIhas three major TnC-binding sites [11–14], namely astructural TnC-binding site (residues 1–30 in rabbitfast skeletal TnI), an inhibitory region (residues 104–115), and a regulatory TnC-binding site (residues 116–131). In the relaxed state, the inhibitory region bindsto actin and inhibits actin–myosin interaction [11,12],while in the contractile state, Ca2+-binding to site Iand ⁄ or II of TnC causes the exposure of a hydropho-bic patch on the surface of the N-domain [15], result-ing in hydrophobic interaction between the N-domainand the regulatory TnC-binding site [16]. This inter-action induces the dissociation of the inhibitory region,which is adjacent to the regulatory TnC-binding site,from actin, resulting in the release of the inhibitionand muscle contraction [17]. The structural TnC-bind-ing site interacts with the C-domain of TnC in boththe relaxed and contractile states, which plays a rolein maintaining the structural integrity of the troponincomplex [17,18]. These switching mechanisms wererecently confirmed by crystallographic studies of ver-tebrate troponins [19,20], which demonstrated that theCa2+-saturated N- and C-domains of TnC bind to theregulatory and structural TnC-binding sites, respect-ively, of TnI, and suggested that the C-terminal regionof TnI (including the inhibitory region and the regula-tory TnC-binding site) exhibits a positional changefrom actin-tropomyosin filament to the N-domain ofTnC in a Ca2+-dependent manner.However, a significant discrepancy exists betweenthe above schemes and the structural and functionalfeatures of some invertebrate troponins. MolluskanTnC binds only one mole of Ca2+per mole of proteinat site IV in the C-domain because of amino acid sub-stitutions at sites I–III [21,22]. Nevertheless, ternarytroponin complex combined with molluskan tropomyo-sin can regulate the Mg-ATPase activity of vertebrateactomyosin in a physiologically significant Ca2+-dependent manner [21]. Moreover, the troponin regu-lates the ATPase of molluskan myofibril together witha well known myosin light chain-linked regulatory sys-tem, especially under low temperature conditions [23].Therefore, the molecular mechanisms of regulation bymolluskan troponin are expected to be somewhat dif-ferent from those described above. A previous studyrevealed that the C-domain of molluskan TnC isresponsible not only for Ca2+-binding but also for theinteraction with TnI, although the presence of boththe N- and C-domains is essential for full regulatoryability [24,25].In the present study, we compared the functionalsites of molluskan and vertebrate TnI by using therecombinant fragments of Akazara scallop Chlamysnipponensis TnI and rabbit fast skeletal TnI. Theresults provide evidence that molluskan troponin func-tions through a mechanism in which the region span-ning from the structural TnC-binding site to theinhibitory region of TnI plays an important role.ResultsEscherichia coli expression of TnI-fragmentsFigure 1A shows a schematic representation of therecombinant TnI-fragments used in this study. ATnI-52K, ATnI-19K and RTnI are the recombinantAkazara scallop 52K-TnI, 19K-TnI (isoforms; seeExperimental procedures section and [27]), and rabbitfast skeletal TnI, respectively. ATnI1)128is the frag-ment corresponding to the N-terminal extending regionof 52K-TnI. ATnI130)252and RTnI1)116are the frag-ments, corresponding to the regions spanning from thestructural TnC-binding sites to the inhibitory regionsof Akazara scallop and rabbit TnI, respectively.ATnI232)292and RTnI96)181correspond to the regionsspanning from the inhibitory regions to the C-terminiof these TnI. Figure 1B shows an SDS ⁄ PAGE ofthese purified recombinant proteins. ATnI-52K andATnI1)128showed anomalously low mobility due tothe high fraction of hydrophilic residues in the N-ter-minal extending region as described previously [26].The initiator Met at the N-terminus was removed bythe bacterial cell for all these proteins except forRTnI96)181.Inhibition of Mg-ATPase of actomyosinby TnI-fragmentsThe inhibition of actomyosin-tropomyosin Mg-ATPaseby TnI fragments was compared. The inhibitory effectsof RTnI, RTnI1)116and RTnI96)181differed greatlyfrom one another, although all of these proteinscontained the inhibitory region (Fig. 2A). RTnI1)116inhibited only 33% of rabbit-actomyosin–rabbit-tropo-myosin Mg-ATPase at a 3 : 1 molar ratio with tropo-myosin, compared with 82% for RTnI. As has beenreported previously [18,28,29], weaker inhibitory effectsof RTnI1)116revealed the importance of residues117–181 for maximal inhibition. In particular, residuesFunctional regions of molluskan TnI H. Tanaka et al.4476 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS140–148 had been proven to bind to actin-tropomyosinand thus are referred to as the second actin-tropo-myosin-binding site [14]. Moreover, in our results, theinhibition by RTnI96)181was the strongest (94% ofthe ATPase was inhibited), suggesting that residues1–95 may decrease the inhibitory effects of residues96–181.On the other hand, Akazara scallop TnI isoformsand their fragments showed somewhat different pro-perties (Fig. 2B). ATnI130)252, which corresponds toRTnI1)116, inhibited about 70% of rabbit-actomyosin-scallop-tropomyosin Mg-ATPase at a 3 : 1 molar ratiowith tropomyosin. Moreover, the inhibition byATnI232)292, which corresponds to RTnI96)181, wasweaker (51%) than that by ATnI-19K (88%) orATnI130)252. Therefore, the effects of the N- or C-ter-minal region of TnI on the function of the inhibitoryregion appeared to differ between rabbit and Akazarascallop TnI. Interestingly, ATnI-52K showed weakerinhibition (65%) than ATnI-19K, suggesting thatthe N-terminal extending region of 52K-TnI coulddecrease the inhibitory effects, although ATnI1)128,which corresponds to the N-terminal extendingregion, on its own, exhibited neither activation norinhibition.To determine whether the inhibitory effect correlateswith the binding affinity to actin-tropomyosin, weexamined each TnI for its ability to cosediment withactin-tropomyosin. When TnI-fragments were mixed at2 : 1 molar ratios with tropomyosin, RTnI, RTnI1)116and RTnI96)181cosedimented with molar ratios ofapproximately 0.23, 0.048, and 0.35, respectively, toactin. On the other hand, ATnI-19K, ATnI130)252andATnI232)292cosedimented with molar ratios of 0.49,0.44, and 0.065, respectively, to actin (the extent of thecosedimentation of ATnI-52K could not be deter-mined because it precipitated even in the absence ofactin-tropomyosin in a control experiment due to thelow solubility). Therefore, the observed differencein the inhibitory effects of TnI-fragments might beABFig. 1. (A) Schematic representation of recombinant TnI-fragments. The numbers preceding and following each box indicate the amino acidpositions of Akazara scallop 52K-TnI (Swiss-Prot #Q7M3Y3) and rabbit fast skeletal TnI (Swiss-Prot #P02643). The N-terminal extendingregion of 52K-TnI and the functional regions that have been previously identified in vertebrate TnI are indicated by bars. The inhibitoryregions are shaded. (B) SDS ⁄ PAGE of recombinant TnI-fragments used in this study. Each protein (1.5 lg) was run on a 10% (w/v) acryl-amide gel. Molecular mass markers are also shown (M).H. Tanaka et al. Functional regions of molluskan TnIFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4477attributable to the difference in their binding affinitiesfor actin-tropomyosin. In addition, ATnI1)128did notcosediment and remained in the supernatant (data notshown). This suggested that the N-terminal extendingregion of 52K-TnI was not involved in binding toactin-tropomyosin, although this region showedsequence homology to the N-terminal tropomyosinbinding site of vertebrate TnT [26].Interactions of TnI-fragments with TnCWe compared the ability of TnI-fragments to form acomplex with TnC by alkaline urea PAGE. The experi-ments were performed under either 6 or 3 m urea condi-tions in the presence of either 2 mm EDTA or 2 mmCaCl2. RTnI and both rabbit TnI-fragments formed acomplex with rabbit TnC in 2 mm CaCl2but not in2mm EDTA under both urea conditions (Fig. 3A).These results agreed with those reported by Farah et al.for chicken skeletal TnI-fragments [18], and were com-patible with the fact that all of these proteins have atleast two of three known TnC-binding sites, namely thestructural TnC-binding site, the inhibitory region, andthe regulatory TnC-binding site. On the other hand,ATnI1)128and ATnI232)292did not form a complex withAkazara scallop TnC under any of the tested conditions,whereas ATnI-52K, ATnI-19K, and ATnI130)252didunder both urea concentrations in the presence of Ca2+(Fig. 3B). It was interesting that ATnI232)292did notform a complex, as ATnI232)292corresponds toRTnI96)181and should have two TnC-binding sites, theinhibitory region and the regulatory TnC-binding site.Therefore, this suggests that TnC-binding affinities ofthese regions of the Akazara scallop TnI were muchweaker than those of rabbit TnI. Moreover, underthe 3 m urea condition, ATnI-52K, ATnI-19K, andATnI130)252showed complex formation even in theabsence of Ca2+(Fig. 3B, upper panels), suggesting thatin the absence of Ca2+, the Akazara scallop TnI bindsto TnC more strongly than rabbit due to the propertiesof the interaction between residues 130–252 and TnC.We also performed affinity chromatography to con-firm the interaction of TnI-fragments with immobilizedrabbit or Akazara scallop TnC under nondenaturingconditions (Fig. 4). ATnI232)292binding to Akazarascallop TnC was not observed, even in the absence ofboth urea and KCl and the presence of 0.5 mm CaCl2,whereas ATnI130)252, RTnI1)116, and RTnI96)181strongly bound to TnCs. These results suggested thatthe inhibitory region and the regulatory TnC-bindingsite of Akazara scallop TnI essentially cannot interactwith TnC.Ca2+-dependent alternative binding of C-terminalTnI fragments to actin-tropomyosin and TnCTo understand the biological significance of the differ-ence in TnI–TnC interactions, we compared the abilityof TnC to neutralize the inhibitory effects of the C-ter-minal fragments in the presence and absence of Ca2+.As has been reported for similar vertebrate TnI frag-ments [14,18,29], the inhibitory effect of RTnI96)181inFig. 2. Inhibition of actomyosin-tropomyosin Mg-ATPase by rabbit(A) or Akazara scallop (B) TnI-fragments. The actomyosin-tropo-myosin Mg-ATPase was measured at increasing ratios of TnIor TnI-fragments to tropomyosin as indicated on the abscissa.The measurements were performed at 15 °C. The results wereexpressed as a percentage of the ATPase activity obtained in theabsence of TnI. Each point is an average of three determinations.(A) RTnI, d; RTnI1)116, n; RTnI96)181, h. (B) ATnI-52K, d; ATnI-19K, s; ATnI1)128, e; ATnI130)252, n; ATnI232)292, h.Functional regions of molluskan TnI H. Tanaka et al.4478 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBSa 2 : 1 molar ratio with tropomyosin was effectivelyneutralized by rabbit TnC in the presence of Ca2+,but not in its absence (Fig. 5A, upper panel). In addi-tion, the cosedimentation experiment performed undera 4 : 4 : 2 : 7 molar ratio of RTnI96)181–TnC–tropo-myosin–actin showed that the amount of RTnI96)181BAFig. 3. Complex formation between TnI-fragments and TnC detected by alkaline urea PAGE. TnI-fragments were combined with TnC as des-cribed under ‘Experimental procedures’. The final concentration of the proteins was 13.8 lM. Twenty-microliter aliquots of the mixture wereelectrophoresed on the gel containing either 6 or 3M urea and either 2 mM EDTA (– Ca; upper panels) or 2 mM CaCl2(+ Ca; lower panels).(A) Rabbit TnI or TnI-fragments were run on the gels in the absence (lanes a–c) or presence (lanes d–f) of equimolar amounts of rabbit TnC.Lanes a and d, RTnI; lanes b and e, RTnI1)116; lanes c and f, RTnI96)181; lane g, rabbit TnC. (B) Akazara scallop TnI or TnI-fragmentswere run in the absence (lanes h–l) or presence (lanes m–q) of equimolar amounts of Akazara scallop TnC. Lanes h and m, ATnI-52K; lanesi and n, ATnI-19K; lanes j and o, ATnI1)128; lanes k and p, ATnI130)252; lanes l and q, ATnI232)292; lane r, Akazara scallop TnC. Complex forma-tion was detected by the bands of the TnI–TnC complex (arrowheads) and weakening of the free TnC bands. Free RTnI, RTnI1)116,RTnI96)181, ATnI-19K, ATnI130)252, and ATnI232)292did not migrate into the gels, while free ATnI-52K and ATnI1)128exhibited a band near theorigin and at the middle of the gel, respectively. The bands corresponding to the free rabbit or Akazara scallop TnC were found in the middleto bottom of the gels (indicated as RTnC or ATnC, respectively).Fig. 4. TnC-affinity chromatography of TnI-fragments. The fragments of rabbit or Akaz-ara scallop TnI were applied onto the affinitycolumns prepared by immobilizing eitherrabbit (A) or Akazara scallop (B) TnC onFormyl-Cellulofine. The fragments wereeluted with a stepwise gradient of KClconcentrations indicated at the top of thefigures. Each fraction contains 1.0 mL.Eluted protein was detected by the methodof Bradford [40] and identified bySDS ⁄ PAGE (data not shown). Due to lowsolubility, RTnI1)116was applied at a KClconcentration of 0.1M.H. Tanaka et al. Functional regions of molluskan TnIFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4479cosedimented with actin-tropomyosin was greatlyreduced in the presence of Ca2+but not in its absence.The amount that remained with TnC in the super-natant was greater in the presence of Ca2+than in itsabsence (Fig. 5A, lower panel). Therefore, this sugges-ted that RTnI96)181bound actin and TnC in theabsence and presence, respectively, of Ca2+. Thesephenomena should directly reflect the mechanism ofCa2+switching involving the alternative binding of theC-terminal region of TnI to actin or TnC in a Ca2+-dependent manner [17,19]. On the other hand, theinhibitory effect of ATnI232)292was not neutralizedby adding Akazara scallop TnC, irrespective of Ca2+concentrations (Fig. 5B, upper panel). Moreover, theamount of ATnI232)292cosedimented with actin-tropo-myosin was unaffected by the presence and absence ofTnC and Ca2+(Fig. 5B, lower panel). Therefore, theCa2+-switching mechanisms involving the alternativebinding of the C-terminal region of TnI were not pre-sent in Akazara scallop troponin.Ca2+-regulatory effects of troponins containingTnI fragmentsThe Ca2+-regulatory effects of troponins composed ofTnI-fragments, native TnT, and TnC on actomyosin-ABFig. 5. Functional differences between RTnI96)181(A) and ATnI232)292(B). Upper panels, effects of TnC on inhibition by the C-terminal TnI-fragments. TnI-fragments were present at a 2 : 1 molar ratio of TnI-fragments ⁄ tropomyosin. The Mg-ATPase activity was measured atincreasing ratios of TnCs to the fragments in the presence (d) or absence (s )ofCa2+. The measurements were performed at 15 °C. Theresults were expressed as a percentage of the ATPase activity obtained in the absence of both TnI and TnC. Lower panels, change in C-ter-minal TnI-fragment affinity for actin-tropomyosin tested by cosedimentation experiments. The fragments were added to actin-tropomyosin ata molar ratio of 4 : 2 : 7 (fragment ⁄ tropomyosin ⁄ actin) with or without an equimolar amount of TnC in the presence or absence of Ca2+.Thepellets (P) and supernatants (S) were redissolved in equivalent volumes of 5M urea solution and then run on SDS ⁄ PAGE. Lanes a and d, inthe absence of both TnC and Ca2+; lanes b and e, in the presence of TnC and the absence of Ca2+; lanes c and f, in the presence of bothTnC and Ca2+. Ac, actin; Tm, tropomyosin; RTnC, rabbit TnC; ATnC, Akazara scallop TnC. The relative staining intensities of the C-terminalTnI-fragments on lanes a–c were expressed as a percentage of that on lane a and were shown on the right.Functional regions of molluskan TnI H. Tanaka et al.4480 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBStropomyosin Mg-ATPase were compared. The assayswere performed at different temperatures, 15 °C, whichis the normal ambient temperature for Akazara scal-lops and is suitable for functionalizing the molluskantroponin [23], and 25 °C, at which many assays ofCa2+regulation by vertebrate troponin have been con-ducted [14,18,28–30]. At 15 °C, all the ternary com-plexes consisting of rabbit TnI or TnI fragments,rabbit TnT and TnC, regulated the ATPase, althoughthey exhibited quite different Ca2+-dependence curves(Fig. 6A). The complex containing RTnI1)116(repre-sented as RTn1)116) showed no inhibition, even underlow Ca2+concentrations, although it strongly activa-ted the ATPase at Ca2+concentrations higher thanpCa 4.5. RTn96)181did not activate the ATPasebeyond the level observed in the absence of troponin,even at pCa 4.0. On the other hand, the complex con-sisting of ATnI232)292, Akazara scallop TnT and TnC(ATn232)292) inhibited the ATPase irrespective of Ca2+concentration, and could not regulate it at all(Fig. 6B). This property could be explained by the factthat the inhibitory region and the regulatoryTnC-binding site of Akazara scallop TnI bind to actin-tropomyosin, but not to TnC, irrespective of Ca2+concentration, as described above. Moreover,ATn130)252regulated the ATPase almost as effectivelyas intact troponins (ATn-52K or ATn-19K), suggestingthat the region spanning from the regulatory TnC-binding site to the C-terminus of Akazara scallop TnIis not important for this regulation, and that Akazarascallop troponin acts through mechanisms in which theregion spanning from the structural TnC-binding siteto the inhibitory region plays an important role. Itshould also be mentioned that ATn-52K more stronglyactivated the ATPase than ATn-19K under high Ca2+concentrations. Thus, the N-terminal extending regionof ATnI-52K may be involved in the activation of theATPase in the presence of Ca2+. When we performedsimilar assays at 25 °C, the regulation by RTn1)116,which was observed at 15 °C, became unremarkable,whereas RTn96)181more effectively regulated theATPase than at 15 °C (Fig. 6C). These resultsobtained at 25 °C were essentially the same as thosereported by Farah et al. [18] for the chicken skeletaltroponins containing similar TnI fragments. On theother hand, the regulatory ability of Akazara scalloptroponins dramatically decreased (Fig. 6D), suggestingthat Akazara scallop troponin does not function at thetemperature appropriate for vertebrate troponins.DiscussionThe vertebrate TnI is known to interact with TnC inan antiparallel manner such that the regulatory andFig. 6. Ca2+-regulation of actomyosin-tropo-myosin Mg-ATPase by rabbit (A and C) andAkazara scallop (B and D) reconstituted tropo-nins. The effects of the troponin containingTnI or TnI fragments on the actomyosin-tropomyosin Mg-ATPase were measured asa function of pCa ()10g[Ca2+]). The assayswere performed at 15 °C (A and B) or 25 °C(C and D). A and C: RTn, d; RTn1)116, n;RTn96)181, h. B and D: ATn-52K, d; ATn-19K, s; ATn130)252, n;ATn232)292, h. Theactivities in the absence of troponin are indi-cated by dashed lines.H. Tanaka et al. Functional regions of molluskan TnIFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4481structural TnC-binding sites of TnI interact with theN- and C-domains, respectively, of TnC [18,19]. Theinhibitory region is known to interact with boththe N- and C-domains, but preferentially with theC-domain [18,20,31]. In the present study, we revealeda striking difference in the TnI–TnC interactions ofvertebrate and mollusk. We showed that ATnI232)292,which is the Akazara scallop TnI-fragment containingthe inhibitory region and the regulatory TnC-bindingsite, does not bind to Akazara scallop TnC, whereasATnI130)252, which contains the structural TnC-bind-ing site and the inhibitory region, strongly binds toTnC. The antiparallel structural features of vertebrateTnI–TnC complex and previous observations that theN-domain of Akazara scallop TnC did not bind toTnI while the C-domain bound strongly [24], suggest asingle interaction between the structural TnC-bindingsite of TnI and the C-domain of TnC in Akazara scal-lop TnI–TnC complex. Although the further verifica-tion under nondenaturing conditions is required, theresults of the alkaline urea gel electrophoresis indicatethat this interaction is strengthened by Ca2+and isstronger than the corresponding interaction in rabbitTnI–TnC in the absence of divalent cation. Therefore,this interaction potentially participates in both theCa2+-dependent activation of the contraction and themaintenance of structural integrity of the troponincomplex in the relaxed state.Troponin-tropomyosin based regulation exhibits twocomponents [32]: inhibition and removal of inhibitionin the absence and presence, respectively, of Ca2+,and Ca2+-dependent activation. The regulatory mech-anism involving the alternative binding of the C-ter-minal region of TnI to actin or TnC should beresponsible for the former. However, it cannot accountfor the latter, namely the phenomenon that, in thepresence of Ca2+, troponin activates actomyosin-tropomyosin Mg-ATPase beyond the level observablein the absence of troponin. This activation is promin-ent, especially for molluskan troponin, which confersCa2+sensitivity on the ATPase predominantlythrough its activation in the presence of Ca2+, ratherthan by inhibition due to its absence. In contrast, thevertebrate troponin regulates the ATPase mainly byinhibition in the absence of Ca2+(Fig. 6 and [21,32]).The difference in Ca2+sensitization between verte-brates and mollusks should also be closely related tothe difference in the inhibitory effects of vertebrateand molluskan tropomyosins [33], which inhibit rab-bit actomyosin Mg-ATPase activity to 0.043 and0.021 lmolÆmin)1Æmg myosin)1, respectively, at 15°C(Fig. 6A,B). In the present study, we compared thefunctional roles of the N- and C-terminal regions ofmolluskan and vertebrate TnI and revealed for thefirst time that (a) the alternative binding of the TnIC-terminal region is not observed in molluskan tropo-nin, as the C-terminal region of molluskan TnI doesnot interact with TnC; and (b) molluskan troponinregulates the ATPase by a mechanism in which theTnI N-terminal region (from the structural TnC-bind-ing site to the inhibitory region) participates in theCa2+-dependent activation. In addition, at 15°C, sim-ilar activation is observed for the troponin containingthe corresponding vertebrate TnI-fragment, suggestingthe presence of a common activating mechanismbetween vertebrates and mollusks. In molluskantroponin, the activation is probably induced by streng-thening of the interaction between the structural TnC-binding site and the C-domain of TnC accompanyingCa2+binding to site IV of TnC. In vertebrate tropo-nin, the activation may be a result of the interactionbetween the inhibitory region and TnC accompanyingCa2+binding to site I or II of TnC. However, we can-not rule out the possibility that the substitution ofMg2+at site III or IV of vertebrate TnC with Ca2+causes the activation in vitro. Several observationshave indicated that the N-terminal region of vertebrateTnI is involved in the activating process [14,28,30]. Inparticular, Malnic et al. [30] suggested that the activa-ting effects of the N-terminal region of TnT are exer-ted in the presence of Ca2+by the TnI N-terminalregion (from the structural TnC-binding site to theTnT-binding site) and TnC.In summary, we propose a novel view of the generalarchitecture of TnI. In vertebrate muscles, the C-ter-minal region plays a role in the inhibition ⁄ removal ofinhibition by alternative binding, while the N-terminalregion is responsible for the Ca2+-dependent activa-tion. This view replaces the general and conventionalview that the N-terminal region of TnI only plays arole in maintaining the structural integrity of the tro-ponin complex. In molluskan muscles, the C-terminalregion does not function and troponin regulatescontraction only through the activation exerted by theN-terminal region of TnI.Experimental proceduresMuscle proteinsTropomyosin, TnT, and TnC from Akazara scallop striatedadductor muscle or rabbit fast skeletal muscle were pre-pared by the method of Ojima and Nishita [21,34]. Rabbitfast skeletal myosin and F-actin were prepared by themethod of Perry [35] and Spudich and Watt [36], respect-ively. All measures were taken to minimize pain andFunctional regions of molluskan TnI H. Tanaka et al.4482 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBSdiscomfort of animals. The procedures were conducted inaccordance with the institutional guidelines by HokkaidoUniversity.Construction of plasmids expressing TnI fragmentsBased on the partial nucleotide sequence (GenBank acces-sion number AB009368), we cloned the cDNA includingthe entire coding region for Akazara scallop TnI by5¢-RACE [37] from the striated adductor muscle. As aresult, two cDNA clones encoding isoforms, namely52K-TnI and 19K-TnI [27], were obtained. The deducedamino acid sequence of 19K-TnI was identical to that ofC-terminal 163 residues of 52K-TnI. The 52K-TnI-cDNAwas subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad,CA, USA), and used as a template for PCR to amplify theDNAs encoding various regions of 52K-TnI. For theamplification of the DNAs encoding ATnI-52K (recombin-ant 52K-TnI; residues 1–292), ATnI1)128(recombinant frag-ment consisting of residues 1–128 of 52K-TnI), ATnI-19K(recombinant 19K-TnI; residues 130–292), ATnI130)252(fragment; residues 130–252), and ATnI232)292(fragment;residues 232–292), combinations of the forward and reverseprimers, ATnI1F (5¢-CATATCACCATGGGTTCCCTTG-3¢)and ATnI292R (5¢-CTTGATTTGGATCCTTTAAGGTATAGC-3¢), ATnI1F and ATnI128R (5¢-GTTCCGGATCCTATCTTCTGGCTTCC-3¢), ATnI130F (5¢-GCCAGAACCATGGCGGAGGAAC-3¢) and ATnI292R, ATnI130Fand ATnI252R (5¢-CAAGTTTGGGATCCTATTTGTTAACTTTTC-3¢), and ATnI232F (5¢-CGAGATTAATGCCATGGCACTTAAGG-3¢) and ATnI292R, respectively,were used. These forward and reverse primers introducedNcoI and BamHI restriction sites (underlined), respectively,into the PCR products. These primers also introduced theinitiation or termination codons (bold), except inATnI292R, which would anneal to the 3¢-noncoding region.It should be noted that in ATnI1F and ATnI232F, the Ser1and Thr232 codons in the template were replaced by Gly1and Ala232, respectively, in addition to introducing theNcoI site. The PCR products were digested with NcoI andBamHI and then ligated into the NcoI-BamHI site of theexpression vector, pET-16b (Novagen, Madison, WI,USA).We also cloned the cDNA encoding rabbit fast skeletalTnI from the back muscle of rabbit by RT-PCR using theprimer set, RTnI1F (5¢-CAAACCTCACCATGGGAGATGAAG-3¢) and RTnI181R (5¢-CCCCGGAGCCGGATCCCCAGCCCC-3¢). These primers were designed based onthe sequence retrieved from the GenBank database underaccession number L04347, and NcoIorBamHI sites (under-lined) and the initiation codon (bolded) were introducedinto the sequences. The cDNA subcloned into pCR2.1-TOPO was first subjected to mutagenesis for deactivatingthe native NcoI site in the coding region by using Mutan-Super Express Km kit (Takara-bio, Ohts, Japan). Themutated DNA was cut out with NcoI and BamHI andligated into pET-16b for the construction of the plasmidexpressing RTnI (recombinant rabbit fast skeletal TnI; resi-dues 1–181). The expression plasmid was also used as atemplate for PCR to amplify the DNA encoding RTnI1)116(fragment; residues 1–116 of rabbit fast skeletal TnI) andRTnI96)181(fragment; residues 96–181), using the primersets RTnI1F and RTnI116R (5¢-GAGCATGGCGGGATCCTACATGCGCAC-3¢) and RTnI96F (5¢-GCTGGAGGCCATGGACCAGAAGC-3¢) and RTnI181R, respectively(BamHI ⁄ NcoI sites and termination ⁄ initiation codons areindicated by underlines and bold type face, respectively). InRTnI96F the Asn96 of the template was replaced byAsp96, and an NcoI site was introduced. The PCR prod-ucts were used for the construction of expression plasmidsby the method described above.Expression and purification of recombinant TnIfragmentsThe expression plasmids were introduced into E. coliBL21(DE3) cells (Novagen) and cultivated at 37 °C for 9 hin LB medium, and then TnI fragments were expressed byinduction with 1 mm IPTG. The cells were harvested bycentrifugation (10 000 g, 10 min), and resuspended in STETbuffer (8% (w/v) sucrose, 50 mm Tris ⁄ HCl (pH 8.0),50 mm EDTA, and 5% (v/v) Triton X-100), and then lysedby three freeze-thaw cycles. After centrifugation (10 000 g,10 min), ATnI1)128, ATnI232)292, and RTnI96)181werefound in the supernatant, and purified by CM-Toyopearl650 m (Tosoh, Tokyo, Japan) column chromatography inthe presence of 6 m urea [34]. ATnI-52K, ATnI-19K,ATnI130)252, RTnI, and RTnI1)116, which were found inthe precipitate, were dissolved in 7 m guanidine hydrochlo-ride, 10 m m Tris ⁄ HCl (pH 7.6), 1 mm EDTA, and 5 mm 2-mercaptoethanol, and then subjected to CM-Toyopeal col-umn chromatography as described above. ATnI-52K wasfurther purified by DEAE-Toyopearl 650 m (Tosoh) col-umn chromatography under the conditions used for CM-Toyopeal chromatography. RTnI, RTnI1)116, and ATnI-19K were also purified by hydroxyapatite (Wako PureChemicals, Osaka, Japan) column chromatography per-formed using 6 m urea, 10 mm KH2PO4(pH 7.0), 5 mm 2-mercaptoethanol, and a linear gradient of 0–500 mm KCl.The N-terminal sequences of these recombinant proteinswere analyzed on an ABI 492HT protein sequencer(Applied Biosystems, Foster City, CA, USA).Polyacrylamide gel electrophoresisSDS ⁄ PAGE was carried out using the method of Porzioand Pearson [38] on a 10% (w/v) acrylamide and 0.1% bis-acrylamide slab gel. Alkaline urea PAGE was performed bythe method of Head and Perry [39] on a 6% (w/v) acryl-H. Tanaka et al. Functional regions of molluskan TnIFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4483amide and 0.48% (w/v) bis-acrylamide slab gel containingeither 6 m or 3 m urea and either 2 mm CaCl2or 2 mmEDTA. The samples were prepared as follows: TnI-frag-ment and TnC were mixed to a 1 : 1 molar ratio in themedium containing 0.125 m KCl, 10 mm Tris ⁄ HCl(pH 7.6), and either 5 mm CaCl2or 5 mm EDTA, and thendiluted with 1.5 volumes of either 10 or 5 m urea, 41.5 mmTris, 133 mm glycine (pH 8.6), 0.02% (w/v) bromophenolblue, and 8% (v/v) 2-mercaptoetanol. The samples wereallowed to stand for 2 h on ice before application to thegels. The electrophoresis was carried out at room tempera-ture by using 25 mm Tris and 80 mm glycine (pH 8.6) as arunning buffer.The gels were stained with 0.2% (w/v) Coomassie brilli-ant blue R250. Fluorescent staining using SYPRO Red(Cambrex, East Rutherford, NJ, USA) was also performedfor densitometric analysis on a fluorescent imager, FLA-3000G (Fuji Photo Film, Tokyo, Japan).Affinity chromatographyRabbit or Akazara scallop TnC was immobilized onFormyl-Cellulofine (Chisso, Tokyo, Japan) according tothe procedure suggested by the manufacturer. The TnC-Cellulofine was packed into a column (0.8 · 4.0 cm) andequilibrated with 10 mm Tris ⁄ HCl (pH 7.6) and 0.5 mmCaCl2. About 50 nmol of TnI-fragment was dialyzedagainst the same solution and then applied onto the col-umn. The fragment was eluted with a stepwise gradient ofKCl at a flow rate of 0.16 mLÆmin)1. The fragment thatwas not eluted under these conditions was removed with6 m urea, 0.5 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and1mm EGTA. The proteins in the effluents were detectedby the method of Bradford [40], and identified bySDS ⁄ PAGE. RTnI1)116, which was insoluble in 10 mmTris ⁄ HCl (pH 7.6) and 0.5 mm CaCl2, was applied at aKCl concentration of 0.1 m.Actin-tropomyosin centrifugation studiesThe binding of the TnI-fragment to actin-tropomyosin wasanalyzed by a cosedimentation assay. The assay conditionswere as follows: 0.15 mgÆmL)1(3.6 lm) rabbit F-actin,0.075 mgÆmL)1(1.1 lm) rabbit or Akazara scallop tropo-myosin, 2.2 lm recombinant TnI-fragment with or withoutequimolar amount of TnC, 50 mm KCl, 20 mm Tris maleate(pH 6.8), 2 mm MgCl2, and 0.2 mm EGTA (in the absenceof Ca2+) or 0.2 mm EGTA plus 0.3 mm CaCl2(in the pres-ence of Ca2+). The proteins were mixed in the presence of0.3 m KCl and then diluted to the above conditions. Thesamples (0.5 mL) were incubated at 15 °C for 30 min andthen centrifuged at 100 000 g for 30 min on an OptimaTL-100 ultracentrifuge (Beckman Coulter, Fullerton, CA,USA). The pellets and supernatants were redissolved inequivalent volumes (0.1 mL) of 5 m urea, 5 mm Tris ⁄ HCl(pH 8.9), 0.5% (w ⁄ v) SDS, and 5% (v ⁄ v) 2-mercaptoetha-nol, and then analyzed by SDS ⁄ PAGE. The amount of theTnI-fragment bound to actin-tropomyosin was estimated bydensitometry, using known amounts of protein run on thesame gel, as a standard. The amount of nonspecificprecipitation of the TnI-fragment was also monitored bysimultaneous centrifugation of the sample containing noactin-tropomyosin under the same conditions.Reconstitution of troponinsRecombinant TnI-fragment and native TnC and TnT weremixed at a 1 : 1 : 1 molar ratio and dialyzed against 6 murea, 0.5 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and 5 mm2-mercaptoethanol. The urea and KCl concentrations werereduced stepwise by the following changes of dialysis buf-fer: (a) buffer B (3 m urea, 0.5 m KCl, 10 mm Tris maleate(pH 6.8), 2 mm MgCl2, 0.2 mm EGTA, 0.3 mm CaCl2,0.01% NaN3(w/v), and 5 mm 2-mercaptoethanol); (b) buf-fer B containing 1 m urea and 0.5 m KCl; (c) buffer B con-taining 0.5 m KCl; and (d) buffer B containing 0.25 m KCl.After dialysis, the complexes were centrifuged and the sup-ernatants were used immediately.Measurements of Mg2+-ATPase activityThe inhibition of actomyosin-tropomyosin Mg2+-ATPaseby the TnI-fragment and the release of the inhibition byTnC were measured in the presence of 0.05 mgÆmL)1(1.2 lm) rabbit F-actin, 0.1 mgÆmL)1(0.19 lm) rabbit myo-sin, 0.025 mgÆmL)1(0.38 lm) rabbit or Akazara scalloptropomyosin, and various concentrations of TnI-fragmentand TnC. The assays were performed at 15 °C in a mediumcontaining 50 mm KCl, 2 mm MgCl2,20mm Tris maleate(pH 6.8), 1 mm ATP, and 0.2 mm EGTA (in the absence ofCa2+) or 0.2 mm EGTA plus 0.3 mm CaCl2(in the pres-ence of Ca2+). The Ca2+regulatory effect of the recon-stituted troponin was measured in the presence of0.03 mgÆmL)1(0.71 lm) rabbit F-actin, 0.06 mgÆmL)1(0.11 lm) rabbit myosin, 0.015 mgÆmL)1(0.23 lm) rabbitor Akazara scallop tropomyosin, and 0.23 lm reconstitutedtroponin. The assays were performed at 15 or 25 °Cinamedium containing 50 mm KCl, 2 mm MgCl2,20mmTris maleate (pH 6.8), 1 mm ATP, 0.1 mm CaCl2and0–3.84 mm EGTA. The concentrations of EGTA requiredto attain the desired final free Ca2+concentrations (pCa7.5–4.0) were calculated by using the stability constant of8.45 · 105m)1for the Ca2+–EGTA complex [41].The reaction was initiated by adding 0.5 mL of 10 mmATP to 4.5 mL of the solution containing all the compo-nents except for ATP. After 2, 4, 6, and 8 min incubation,1 mL aliquots were withdrawn from the reaction mixtureand added to 4 mL of acidic malachite green solution todetermine the liberated inorganic phosphate concentrationsby the method of Chan et al. [42].Functional regions of molluskan TnI H. Tanaka et al.4484 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS[...]... 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Amino acid sequence of troponin-I from Akazara scallop striated adductor muscle J Biochem (Tokyo) 124, 304–310 27 Ojima T & Nishita K (1991) A binary complex of troponin-I and troponin-T from Akazara scallop striated adductor muscle J Biochem (Tokyo) 110, 847–850 28 Van Eyk JE, Thomas LT, Tripet B, Wiesner RJ, Pearlstone JR, Farah CS, Reinach FC & Hodges RS (1997) Distinct regions of troponin I regulate... et al Functional regions of molluskan TnI Acknowledgements This study was supported by Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and Technology, of the Japanese Government References 1 Ebashi S & Kodama A (1965) A new protein factor promoting aggregation of tropomyosin J Biochem (Tokyo) 58, 107–108 2 Ohtsuki I, Maruyama K & Ebashi S (1986) Regulatory and cytoskeletal... Ebashi S (1986) Regulatory and cytoskeletal proteins of vertebrate skeletal muscle Adv Protein Chem 38, 1–67 3 Zot AS & Potter JD (1987) Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction Annu Rev Biophys Biophys Chem 16, 535–559 4 Farah CS & Reinach FC (1995) The troponin complex and regulation of muscle contraction FASEB J 9, 755– 767 5 Sundaralingam M, Bergstrom... Pearson AM (1977) Improved resolution of myofibrillar proteins with sodium dodecyl sulphate– polyacrylamide gel electrophoresis Biochim Biophys Acta 490, 27–34 39 Head JF & Perry SV (1974) The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I) Biochem J 137, 145–154 40 Bradford MM (1976) A rapid and sensitive method for the quantitation of . Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I Hiroyuki Tanaka1,. 6A,B). In the present study, we compared the functional roles of the N- and C-terminal regions of molluskan and vertebrate TnI and revealed for the first
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