Keywords invertebrate; mollusk; regulatory mechanism; troponin; troponin-I Correspondence Takao Ojima, Laboratory of Biochemistry and Biotechnology, Graduate School of Fisheries Sciences
Trang 1C-terminal regions of molluskan and vertebrate troponin-I Hiroyuki Tanaka1, Yuhei Takeya1, Teppei Doi1, Fumiaki Yumoto2,3, Masaru Tanokura3,
Iwao Ohtsuki2, Kiyoyoshi Nishita1and Takao Ojima1
1 Laboratory of Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Japan
2 Laboratory of Physiology, The Jikei University School of Medicine, Tokyo, Japan
3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan
Troponin is a Ca2+-dependent regulatory protein
com-plex, which constitute thin filaments together with
actin 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 to
actin and inhibits contraction Upon muscle
stimula-tion, Ca2+binds to TnC and induces the release of the
inhibition by TnI, resulting in muscle contraction To
understand 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-like shape with the N- and C-terminal globular domains linked by a central helix [5,6] Each domain contains two EF-hand Ca2+-binding motifs [7], thus TnC has four possible Ca2+-binding sites, sites I and II in the N-domain and sites III and IV in the C-domain [8,9]
Keywords
invertebrate; mollusk; regulatory
mechanism; troponin; troponin-I
Correspondence
Takao Ojima, Laboratory of Biochemistry
and Biotechnology, Graduate School of
Fisheries Sciences, Hokkaido University,
Hakodate, Hokkaido 041–8611, Japan
Tel ⁄ Fax: +81 138 408800
E-mail: ojima@fish.hokudai.ac.jp
Note
The nucleotide sequences of cDNAs
enco-ding Akazara scallop 52K-TnI and 19K-TnI
are available in DDBJ ⁄ EMBL ⁄ GenBank
databases under accession numbers,
AB206837 and AB206838, respectively
(Received 24 March 2005, revised 13 June
2005, accepted 15 July 2005)
doi:10.1111/j.1742-4658.2005.04866.x
Vertebrate troponin regulates muscle contraction through alternative bind-ing of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca2+-dependent manner To elucidate the molecular mechanisms of this regulation by molluskan troponin, we com-pared the functional properties of the recombinant fragments of Akazara scallop 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 (resi(resi-dues 116–131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase However, it did not interact with TnC, even in the presence
of Ca2+ These results indicated that the mechanism involved in the alter-native binding of this region was not observed in molluskan troponin On the other hand, ATnI130)252, which contains the structural TnC-binding site (residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC Moreover, the ternary complex consisting of this frag-ment, troponin-T, and TnC activated the ATPase in a Ca2+-dependent manner almost as effectively as intact Akazara scallop troponin Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI Together with the observation that corresponding rabbit TnI-fragment (RTnI1)116) shows similar activa-ting effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin com-plex but also for Ca2+-dependent activation
Abbreviations
TnC, troponin-C; TnI, troponin-I; TnT, troponin-T; IPTG, isopropyl-1-thio-b- D -galactopyranoside; PMSF, phenylmethylsulfonyl fluoride.
Trang 2Sites III and IV also show affinity for Mg2+ and are
thought to be always occupied by sarcoplasmic Mg2+,
whereas Ca2+ binding to site I and⁄ or II is believed
to trigger muscle contraction [10] TnC interacts with
both TnI and TnT The TnC–TnI interaction and
changes in the interaction upon Ca2+ binding to TnC
have been intensively studied as the central
mecha-nisms of Ca2+switching It has been revealed that TnI
has three major TnC-binding sites [11–14], namely a
structural TnC-binding site (residues 1–30 in rabbit
fast skeletal TnI), an inhibitory region (residues 104–
115), and a regulatory TnC-binding site (residues 116–
131) In the relaxed state, the inhibitory region binds
to actin and inhibits actin–myosin interaction [11,12],
while in the contractile state, Ca2+-binding to site I
and⁄ 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-domain
and 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 inhibition
and muscle contraction [17] The structural
TnC-bind-ing site interacts with the C-domain of TnC in both
the relaxed and contractile states, which plays a role
in maintaining the structural integrity of the troponin
complex [17,18] These switching mechanisms were
recently confirmed by crystallographic studies of
ver-tebrate troponins [19,20], which demonstrated that the
Ca2+-saturated N- and C-domains of TnC bind to the
regulatory and structural TnC-binding sites,
respect-ively, of TnI, and suggested that the C-terminal region
of TnI (including the inhibitory region and the
regula-tory TnC-binding site) exhibits a positional change
from actin-tropomyosin filament to the N-domain of
TnC in a Ca2+-dependent manner
However, a significant discrepancy exists between
the above schemes and the structural and functional
features of some invertebrate troponins Molluskan
TnC binds only one mole of Ca2+per mole of protein
at site IV in the C-domain because of amino acid
sub-stitutions at sites I–III [21,22] Nevertheless, ternary
troponin complex combined with molluskan
tropomyo-sin can regulate the Mg-ATPase activity of vertebrate
actomyosin in a physiologically significant Ca2+
-dependent manner [21] Moreover, the troponin
regu-lates the ATPase of molluskan myofibril together with
a well known myosin light chain-linked regulatory
sys-tem, especially under low temperature conditions [23]
Therefore, the molecular mechanisms of regulation by
molluskan troponin are expected to be somewhat
dif-ferent from those described above A previous study
revealed that the C-domain of molluskan TnC is
responsible not only for Ca2+-binding but also for the interaction with TnI, although the presence of both the N- and C-domains is essential for full regulatory ability [24,25]
In the present study, we compared the functional sites of molluskan and vertebrate TnI by using the recombinant fragments of Akazara scallop Chlamys nipponensis TnI and rabbit fast skeletal TnI The results provide evidence that molluskan troponin func-tions through a mechanism in which the region span-ning from the structural TnC-binding site to the inhibitory region of TnI plays an important role
Results
Escherichia coli expression of TnI-fragments Figure 1A shows a schematic representation of the recombinant TnI-fragments used in this study ATnI-52K, ATnI-19K and RTnI are the recombinant Akazara scallop 52K-TnI, 19K-TnI (isoforms; see Experimental procedures section and [27]), and rabbit fast skeletal TnI, respectively ATnI1)128 is the frag-ment corresponding to the N-terminal extending region
of 52K-TnI ATnI130)252 and RTnI1)116 are the frag-ments, corresponding to the regions spanning from the structural TnC-binding sites to the inhibitory regions
of Akazara scallop and rabbit TnI, respectively ATnI232)292 and RTnI96)181correspond to the regions spanning from the inhibitory regions to the C-termini
of these TnI Figure 1B shows an SDS⁄ PAGE of these purified recombinant proteins ATnI-52K and ATnI1)128 showed anomalously low mobility due to the 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 by the bacterial cell for all these proteins except for RTnI96)181
Inhibition of Mg-ATPase of actomyosin
by TnI-fragments The inhibition of actomyosin-tropomyosin Mg-ATPase
by TnI fragments was compared The inhibitory effects
of RTnI, RTnI1)116 and RTnI96)181 differed greatly from one another, although all of these proteins contained the inhibitory region (Fig 2A) RTnI1)116 inhibited only 33% of rabbit-actomyosin–rabbit-myosin Mg-ATPase at a 3 : 1 molar ratio with tropo-myosin, compared with 82% for RTnI As has been reported previously [18,28,29], weaker inhibitory effects
of RTnI1)116 revealed the importance of residues 117–181 for maximal inhibition In particular, residues
Trang 3140–148 had been proven to bind to actin-tropomyosin
and thus are referred to as the second
actin-tropo-myosin-binding site [14] Moreover, in our results, the
inhibition by RTnI96)181 was the strongest (94% of
the ATPase was inhibited), suggesting that residues
1–95 may decrease the inhibitory effects of residues
96–181
On the other hand, Akazara scallop TnI isoforms
and their fragments showed somewhat different
pro-perties (Fig 2B) ATnI130)252, which corresponds to
RTnI1)116, inhibited about 70% of
rabbit-actomyosin-scallop-tropomyosin Mg-ATPase at a 3 : 1 molar ratio
with tropomyosin Moreover, the inhibition by
ATnI232)292, which corresponds to RTnI96)181, was
weaker (51%) than that by ATnI-19K (88%) or
ATnI130)252 Therefore, the effects of the N- or
C-ter-minal region of TnI on the function of the inhibitory
region appeared to differ between rabbit and Akazara
scallop TnI Interestingly, ATnI-52K showed weaker
inhibition (65%) than ATnI-19K, suggesting that
the N-terminal extending region of 52K-TnI could decrease the inhibitory effects, although ATnI1)128, which corresponds to the N-terminal extending region, on its own, exhibited neither activation nor inhibition
To determine whether the inhibitory effect correlates with the binding affinity to actin-tropomyosin, we examined each TnI for its ability to cosediment with actin-tropomyosin When TnI-fragments were mixed at
2 : 1 molar ratios with tropomyosin, RTnI, RTnI1)116 and RTnI96)181 cosedimented with molar ratios of approximately 0.23, 0.048, and 0.35, respectively, to actin On the other hand, ATnI-19K, ATnI130)252and ATnI232)292 cosedimented with molar ratios of 0.49, 0.44, and 0.065, respectively, to actin (the extent of the cosedimentation of ATnI-52K could not be deter-mined because it precipitated even in the absence of actin-tropomyosin in a control experiment due to the low solubility) Therefore, the observed difference
in the inhibitory effects of TnI-fragments might be
A
B
Fig 1 (A) Schematic representation of recombinant TnI-fragments The numbers preceding and following each box indicate the amino acid positions of Akazara scallop 52K-TnI (Swiss-Prot #Q7M3Y3) and rabbit fast skeletal TnI (Swiss-Prot #P02643) The N-terminal extending region of 52K-TnI and the functional regions that have been previously identified in vertebrate TnI are indicated by bars The inhibitory regions 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).
Trang 4attributable to the difference in their binding affinities
for actin-tropomyosin In addition, ATnI1)128did not
cosediment and remained in the supernatant (data not
shown) This suggested that the N-terminal extending
region of 52K-TnI was not involved in binding to
actin-tropomyosin, although this region showed
sequence homology to the N-terminal tropomyosin
binding site of vertebrate TnT [26]
Interactions of TnI-fragments with TnC
We compared the ability of TnI-fragments to form a complex 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 mm CaCl2 RTnI and both rabbit TnI-fragments formed a complex with rabbit TnC in 2 mm CaCl2 but not in
2 mm 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 at least two of three known TnC-binding sites, namely the structural TnC-binding site, the inhibitory region, and the regulatory TnC-binding site On the other hand, ATnI1)128and ATnI232)292did not form a complex with Akazara scallop TnC under any of the tested conditions, whereas ATnI-52K, ATnI-19K, and ATnI130)252 did under both urea concentrations in the presence of Ca2+ (Fig 3B) It was interesting that ATnI232)292 did not form a complex, as ATnI232)292 corresponds to RTnI96)181and should have two TnC-binding sites, the inhibitory region and the regulatory TnC-binding site Therefore, this suggests that TnC-binding affinities of these regions of the Akazara scallop TnI were much weaker than those of rabbit TnI Moreover, under the 3 m urea condition, ATnI-52K, ATnI-19K, and ATnI130)252 showed complex formation even in the absence of Ca2+(Fig 3B, upper panels), suggesting that
in the absence of Ca2+, the Akazara scallop TnI binds
to TnC more strongly than rabbit due to the properties
of the interaction between residues 130–252 and TnC
We also performed affinity chromatography to con-firm the interaction of TnI-fragments with immobilized rabbit or Akazara scallop TnC under nondenaturing conditions (Fig 4) ATnI232)292 binding to Akazara scallop TnC was not observed, even in the absence of both urea and KCl and the presence of 0.5 mm CaCl2, whereas ATnI130)252, RTnI1)116, and RTnI96)181 strongly bound to TnCs These results suggested that the inhibitory region and the regulatory TnC-binding site of Akazara scallop TnI essentially cannot interact with TnC
Ca2+-dependent alternative binding of C-terminal TnI fragments to actin-tropomyosin and TnC
To understand the biological significance of the differ-ence in TnI–TnC interactions, we compared the ability
of 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)181in
Fig 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 TnI
or TnI-fragments to tropomyosin as indicated on the abscissa.
The measurements were performed at 15 C The results were
expressed as a percentage of the ATPase activity obtained in the
absence of TnI Each point is an average of three determinations.
(A) RTnI, d; RTnI1)116, n; RTnI96)181, h (B) 52K, d;
ATnI-19K, s; ATnI 1 )128, e; ATnI130 )252, n; ATnI232 )292, h.
Trang 5a 2 : 1 molar ratio with tropomyosin was effectively
neutralized by rabbit TnC in the presence of Ca2+,
but not in its absence (Fig 5A, upper panel) In
addi-tion, the cosedimentation experiment performed under
a 4 : 4 : 2 : 7 molar ratio of RTnI96)181 –TnC–tropo-myosin–actin showed that the amount of RTnI96)181
B A
Fig 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 l M Twenty-microliter aliquots of the mixture were electrophoresed on the gel containing either 6 or 3 M urea and either 2 m M EDTA (– Ca; upper panels) or 2 m M 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, RTnI 1 )116; lanes c and f, RTnI96 )181; lane g, rabbit TnC (B) Akazara scallop TnI or TnI-fragments
were 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; lanes
i 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, RTnI 1 )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 the origin and at the middle of the gel, respectively The bands corresponding to the free rabbit or Akazara scallop TnC were found in the middle
to 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 affinity
columns prepared by immobilizing either
rabbit (A) or Akazara scallop (B) TnC on
Formyl-Cellulofine The fragments were
eluted with a stepwise gradient of KCl
concentrations indicated at the top of the
figures Each fraction contains 1.0 mL.
Eluted protein was detected by the method
of Bradford [40] and identified by
SDS ⁄ PAGE (data not shown) Due to low
solubility, RTnI 1 )116was applied at a KCl
concentration of 0.1 M
Trang 6cosedimented with actin-tropomyosin was greatly
reduced 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 its
absence (Fig 5A, lower panel) Therefore, this
sugges-ted that RTnI96)181 bound actin and TnC in the
absence and presence, respectively, of Ca2+ These
phenomena should directly reflect the mechanism of
Ca2+switching involving the alternative binding of the
C-terminal region of TnI to actin or TnC in a Ca2+
-dependent manner [17,19] On the other hand, the
inhibitory effect of ATnI232)292 was not neutralized
by adding Akazara scallop TnC, irrespective of Ca2+
concentrations (Fig 5B, upper panel) Moreover, the amount of ATnI232)292cosedimented with actin-tropo-myosin was unaffected by the presence and absence of TnC and Ca2+ (Fig 5B, lower panel) Therefore, the
Ca2+-switching mechanisms involving the alternative binding of the C-terminal region of TnI were not pre-sent in Akazara scallop troponin
Ca2+-regulatory effects of troponins containing TnI fragments
The Ca2+-regulatory effects of troponins composed of TnI-fragments, native TnT, and TnC on
Fig 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 at increasing ratios of TnCs to the fragments in the presence (d) or absence (s) of Ca 2+ The measurements were performed at 15 C The results 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 at
a molar ratio of 4 : 2 : 7 (fragment ⁄ tropomyosin ⁄ actin) with or without an equimolar amount of TnC in the presence or absence of Ca 2+ The pellets (P) and supernatants (S) were redissolved in equivalent volumes of 5 M urea solution and then run on SDS ⁄ PAGE Lanes a and d, in the absence of both TnC and Ca 2+ ; lanes b and e, in the presence of TnC and the absence of Ca 2+ ; lanes c and f, in the presence of both TnC and Ca2+ Ac, actin; Tm, tropomyosin; RTnC, rabbit TnC; ATnC, Akazara scallop TnC The relative staining intensities of the C-terminal TnI-fragments on lanes a–c were expressed as a percentage of that on lane a and were shown on the right.
Trang 7tropomyosin Mg-ATPase were compared The assays
were performed at different temperatures, 15C, which
is the normal ambient temperature for Akazara
scal-lops and is suitable for functionalizing the molluskan
troponin [23], and 25C, at which many assays of
Ca2+regulation by vertebrate troponin have been
con-ducted [14,18,28–30] At 15C, all the ternary
com-plexes consisting of rabbit TnI or TnI fragments,
rabbit TnT and TnC, regulated the ATPase, although
they exhibited quite different Ca2+-dependence curves
(Fig 6A) The complex containing RTnI1)116
(repre-sented as RTn1)116) showed no inhibition, even under
low Ca2+ concentrations, although it strongly
activa-ted the ATPase at Ca2+ concentrations higher than
pCa 4.5 RTn96)181 did not activate the ATPase
beyond 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 fact
that the inhibitory region and the regulatory
TnC-binding site of Akazara scallop TnI bind to
actin-tropomyosin, but not to TnC, irrespective of Ca2+
concentration, as described above Moreover,
ATn130)252 regulated the ATPase almost as effectively
as intact troponins (ATn-52K or ATn-19K), suggesting
that the region spanning from the regulatory TnC-binding site to the C-terminus of Akazara scallop TnI
is not important for this regulation, and that Akazara scallop troponin acts through mechanisms in which the region spanning from the structural TnC-binding site
to the inhibitory region plays an important role It should also be mentioned that ATn-52K more strongly activated the ATPase than ATn-19K under high Ca2+ concentrations Thus, the N-terminal extending region
of ATnI-52K may be involved in the activation of the ATPase in the presence of Ca2+ When we performed similar assays at 25C, the regulation by RTn1)116, which was observed at 15C, became unremarkable, whereas RTn96)181 more effectively regulated the ATPase than at 15C (Fig 6C) These results obtained at 25C were essentially the same as those reported by Farah et al [18] for the chicken skeletal troponins containing similar TnI fragments On the other hand, the regulatory ability of Akazara scallop troponins dramatically decreased (Fig 6D), suggesting that Akazara scallop troponin does not function at the temperature appropriate for vertebrate troponins
Discussion
The vertebrate TnI is known to interact with TnC in
an antiparallel manner such that the regulatory and
Fig 6 Ca2+-regulation of
actomyosin-tropo-myosin Mg-ATPase by rabbit (A and C) and
Akazara scallop (B and D) reconstituted
tropo-nins The effects of the troponin containing
TnI or TnI fragments on the
actomyosin-tropomyosin Mg-ATPase were measured as
a function of pCa ( )10g[Ca 2+
]) The assays were performed at 15 C (A and B) or 25 C
(C and D) A and C: RTn, d; RTn1)116, n;
RTn 96 )181, h B and D: 52K, d;
ATn-19K, s; ATn130)252, n; ATn232)292, h The
activities in the absence of troponin are
indi-cated by dashed lines.
Trang 8structural TnC-binding sites of TnI interact with the
N- and C-domains, respectively, of TnC [18,19] The
inhibitory region is known to interact with both
the N- and C-domains, but preferentially with the
C-domain [18,20,31] In the present study, we revealed
a striking difference in the TnI–TnC interactions of
vertebrate and mollusk We showed that ATnI232)292,
which is the Akazara scallop TnI-fragment containing
the inhibitory region and the regulatory TnC-binding
site, does not bind to Akazara scallop TnC, whereas
ATnI130)252, which contains the structural
TnC-bind-ing site and the inhibitory region, strongly binds to
TnC The antiparallel structural features of vertebrate
TnI–TnC complex and previous observations that the
N-domain of Akazara scallop TnC did not bind to
TnI while the C-domain bound strongly [24], suggest a
single interaction between the structural TnC-binding
site of TnI and the C-domain of TnC in Akazara
scal-lop TnI–TnC complex Although the further
verifica-tion under nondenaturing condiverifica-tions is required, the
results of the alkaline urea gel electrophoresis indicate
that this interaction is strengthened by Ca2+ and is
stronger than the corresponding interaction in rabbit
TnI–TnC in the absence of divalent cation Therefore,
this interaction potentially participates in both the
Ca2+-dependent activation of the contraction and the
maintenance of structural integrity of the troponin
complex in the relaxed state
Troponin-tropomyosin based regulation exhibits two
components [32]: inhibition and removal of inhibition
in 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 be
responsible for the former However, it cannot account
for the latter, namely the phenomenon that, in the
presence of Ca2+, troponin activates
actomyosin-tropomyosin Mg-ATPase beyond the level observable
in the absence of troponin This activation is
promin-ent, especially for molluskan troponin, which confers
Ca2+ sensitivity on the ATPase predominantly
through its activation in the presence of Ca2+, rather
than by inhibition due to its absence In contrast, the
vertebrate troponin regulates the ATPase mainly by
inhibition 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 to
the difference in the inhibitory effects of vertebrate
and molluskan tropomyosins [33], which inhibit
rab-bit actomyosin Mg-ATPase activity to 0.043 and
0.021 lmolÆmin)1Æmg myosin)1, respectively, at 15C
(Fig 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 time that (a) the alternative binding of the TnI C-terminal region is not observed in molluskan tropo-nin, as the C-terminal region of molluskan TnI does not interact with TnC; and (b) molluskan troponin regulates the ATPase by a mechanism in which the TnI N-terminal region (from the structural TnC-bind-ing site to the inhibitory region) participates in the
Ca2+-dependent activation In addition, at 15C, sim-ilar activation is observed for the troponin containing the corresponding vertebrate TnI-fragment, suggesting the presence of a common activating mechanism between vertebrates and mollusks In molluskan troponin, the activation is probably induced by streng-thening of the interaction between the structural TnC-binding site and the C-domain of TnC accompanying
Ca2+ binding to site IV of TnC In vertebrate tropo-nin, the activation may be a result of the interaction between the inhibitory region and TnC accompanying
Ca2+binding to site I or II of TnC However, we can-not rule out the possibility that the substitution of
Mg2+ at site III or IV of vertebrate TnC with Ca2+ causes the activation in vitro Several observations have indicated that the N-terminal region of vertebrate TnI is involved in the activating process [14,28,30] In particular, 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-terminal region (from the structural TnC-binding site to the TnT-binding site) and TnC
In summary, we propose a novel view of the general architecture of TnI In vertebrate muscles, the C-ter-minal region plays a role in the inhibition⁄ removal of inhibition by alternative binding, while the N-terminal region is responsible for the Ca2+-dependent activa-tion This view replaces the general and conventional view that the N-terminal region of TnI only plays a role in maintaining the structural integrity of the tro-ponin complex In molluskan muscles, the C-terminal region does not function and troponin regulates contraction only through the activation exerted by the N-terminal region of TnI
Experimental procedures
Muscle proteins Tropomyosin, TnT, and TnC from Akazara scallop striated adductor muscle or rabbit fast skeletal muscle were pre-pared by the method of Ojima and Nishita [21,34] Rabbit fast skeletal myosin and F-actin were prepared by the method of Perry [35] and Spudich and Watt [36], respect-ively All measures were taken to minimize pain and
Trang 9discomfort of animals The procedures were conducted in
accordance with the institutional guidelines by Hokkaido
University
Construction of plasmids expressing TnI fragments
Based on the partial nucleotide sequence (GenBank
acces-sion number AB009368), we cloned the cDNA including
the entire coding region for Akazara scallop TnI by
5¢-RACE [37] from the striated adductor muscle As a
result, two cDNA clones encoding isoforms, namely
52K-TnI and 19K-TnI [27], were obtained The deduced
amino acid sequence of 19K-TnI was identical to that of
C-terminal 163 residues of 52K-TnI The 52K-TnI-cDNA
was subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad,
CA, USA), and used as a template for PCR to amplify the
DNAs encoding various regions of 52K-TnI For the
amplification 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 reverse
primers, ATnI1F (5¢-CATATCACCATGGGTTCCCTTG-3¢)
and ATnI292R (5¢-CTTGATTTGGATCCTTTAAGGTA
TAGC-3¢), ATnI1F and ATnI128R (5¢-GTTCCGGATC
CTATCTTCTGGCTTCC-3¢), ATnI130F (5¢-GCCAGAA
CCATGGCGGAGGAAC-3¢) and ATnI292R, ATnI130F
and ATnI252R (5¢-CAAGTTTGGGATCCTATTTGTTAA
CTTTTC-3¢), and ATnI232F (5¢-CGAGATTAATGCC
ATGGCACTTAAGG-3¢) and ATnI292R, respectively,
were used These forward and reverse primers introduced
NcoI and BamHI restriction sites (underlined), respectively,
into the PCR products These primers also introduced the
initiation or termination codons (bold), except in
ATnI292R, which would anneal to the 3¢-noncoding region
It should be noted that in ATnI1F and ATnI232F, the Ser1
and Thr232 codons in the template were replaced by Gly1
and Ala232, respectively, in addition to introducing the
NcoI site The PCR products were digested with NcoI and
BamHI and then ligated into the NcoI-BamHI site of the
expression vector, pET-16b (Novagen, Madison, WI,
USA)
We also cloned the cDNA encoding rabbit fast skeletal
TnI from the back muscle of rabbit by RT-PCR using the
primer set, RTnI1F (5¢-CAAACCTCACCATGGGAGAT
GAAG-3¢) and RTnI181R (5¢-CCCCGGAGCCGGATCC
CCAGCCCC-3¢) These primers were designed based on
the sequence retrieved from the GenBank database under
accession number L04347, and NcoI or BamHI sites
(under-lined) and the initiation codon (bolded) were introduced
into the sequences The cDNA subcloned into
pCR2.1-TOPO was first subjected to mutagenesis for deactivating
the native NcoI site in the coding region by using
Mutan-Super Express Km kit (Takara-bio, Ohts, Japan) The
mutated DNA was cut out with NcoI and BamHI and ligated into pET-16b for the construction of the plasmid expressing RTnI (recombinant rabbit fast skeletal TnI; resi-dues 1–181) The expression plasmid was also used as a template for PCR to amplify the DNA encoding RTnI1)116 (fragment; residues 1–116 of rabbit fast skeletal TnI) and RTnI96)181 (fragment; residues 96–181), using the primer sets RTnI1F and RTnI116R (5¢-GAGCATGGCGGGAT CCTACATGCGCAC-3¢) and RTnI96F (5¢-GCTGGAGG CCATGGACCAGAAGC-3¢) and RTnI181R, respectively (BamHI⁄ NcoI sites and termination ⁄ initiation codons are indicated by underlines and bold type face, respectively) In RTnI96F the Asn96 of the template was replaced by Asp96, and an NcoI site was introduced The PCR prod-ucts were used for the construction of expression plasmids
by the method described above
Expression and purification of recombinant TnI fragments
The expression plasmids were introduced into E coli BL21(DE3) cells (Novagen) and cultivated at 37C for 9 h
in LB medium, and then TnI fragments were expressed by induction with 1 mm IPTG The cells were harvested by centrifugation (10 000 g, 10 min), and resuspended in STET buffer (8% (w/v) sucrose, 50 mm Tris⁄ HCl (pH 8.0),
50 mm EDTA, and 5% (v/v) Triton X-100), and then lysed
by three freeze-thaw cycles After centrifugation (10 000 g,
10 min), ATnI1)128, ATnI232)292, and RTnI96)181 were found in the supernatant, and purified by CM-Toyopearl
650 m (Tosoh, Tokyo, Japan) column chromatography in the presence of 6 m urea [34] ATnI-52K, ATnI-19K, ATnI130)252, RTnI, and RTnI1)116, which were found in the precipitate, were dissolved in 7 m guanidine hydrochlo-ride, 10 mm 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 was further 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 Pure Chemicals, 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 proteins were analyzed on an ABI 492HT protein sequencer (Applied Biosystems, Foster City, CA, USA)
Polyacrylamide gel electrophoresis SDS⁄ PAGE was carried out using the method of Porzio and Pearson [38] on a 10% (w/v) acrylamide and 0.1% bis-acrylamide slab gel Alkaline urea PAGE was performed by the method of Head and Perry [39] on a 6% (w/v)
Trang 10acryl-amide and 0.48% (w/v) bis-acrylacryl-amide slab gel containing
either 6 m or 3 m urea and either 2 mm CaCl2 or 2 mm
EDTA The samples were prepared as follows:
TnI-frag-ment and TnC were mixed to a 1 : 1 molar ratio in the
medium containing 0.125 m KCl, 10 mm Tris⁄ HCl
(pH 7.6), and either 5 mm CaCl2or 5 mm EDTA, and then
diluted with 1.5 volumes of either 10 or 5 m urea, 41.5 mm
Tris, 133 mm glycine (pH 8.6), 0.02% (w/v) bromophenol
blue, and 8% (v/v) 2-mercaptoetanol The samples were
allowed to stand for 2 h on ice before application to the
gels The electrophoresis was carried out at room
tempera-ture by using 25 mm Tris and 80 mm glycine (pH 8.6) as a
running 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 performed
for densitometric analysis on a fluorescent imager,
FLA-3000G (Fuji Photo Film, Tokyo, Japan)
Affinity chromatography
Rabbit or Akazara scallop TnC was immobilized on
Formyl-Cellulofine (Chisso, Tokyo, Japan) according to
the procedure suggested by the manufacturer The
TnC-Cellulofine was packed into a column (0.8· 4.0 cm) and
equilibrated with 10 mm Tris⁄ HCl (pH 7.6) and 0.5 mm
CaCl2 About 50 nmol of TnI-fragment was dialyzed
against the same solution and then applied onto the
col-umn The fragment was eluted with a stepwise gradient of
KCl at a flow rate of 0.16 mLÆmin)1 The fragment that
was not eluted under these conditions was removed with
6 m urea, 0.5 m KCl, 10 mm Tris⁄ HCl (pH 7.6), and
1 mm EGTA The proteins in the effluents were detected
by the method of Bradford [40], and identified by
SDS⁄ PAGE RTnI1)116, which was insoluble in 10 mm
Tris⁄ HCl (pH 7.6) and 0.5 mm CaCl2, was applied at a
KCl concentration of 0.1 m
Actin-tropomyosin centrifugation studies
The binding of the TnI-fragment to actin-tropomyosin was
analyzed by a cosedimentation assay The assay conditions
were 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 without
equimolar amount of TnC, 50 mm KCl, 20 mm Tris maleate
(pH 6.8), 2 mm MgCl2, and 0.2 mm EGTA (in the absence
of Ca2+) or 0.2 mm EGTA plus 0.3 mm CaCl2(in the
pres-ence of Ca2+) The proteins were mixed in the presence of
0.3 m KCl and then diluted to the above conditions The
samples (0.5 mL) were incubated at 15C for 30 min and
then centrifuged at 100 000 g for 30 min on an Optima
TL-100 ultracentrifuge (Beckman Coulter, Fullerton, CA,
USA) The pellets and supernatants were redissolved in
equivalent 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 the TnI-fragment bound to actin-tropomyosin was estimated by densitometry, using known amounts of protein run on the same gel, as a standard The amount of nonspecific precipitation of the TnI-fragment was also monitored by simultaneous centrifugation of the sample containing no actin-tropomyosin under the same conditions
Reconstitution of troponins Recombinant TnI-fragment and native TnC and TnT were mixed at a 1 : 1 : 1 molar ratio and dialyzed against 6 m urea, 0.5 m KCl, 10 mm Tris⁄ HCl (pH 7.6), and 5 mm 2-mercaptoethanol The urea and KCl concentrations were reduced 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) bufbuf-fer B con-taining 0.5 m KCl; and (d) buffer B concon-taining 0.25 m KCl After dialysis, the complexes were centrifuged and the sup-ernatants were used immediately
Measurements of Mg2+-ATPase activity The inhibition of actomyosin-tropomyosin Mg2+-ATPase
by the TnI-fragment and the release of the inhibition by TnC 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 scallop tropomyosin, and various concentrations of TnI-fragment and TnC The assays were performed at 15C in a medium containing 50 mm KCl, 2 mm MgCl2, 20 mm Tris maleate (pH 6.8), 1 mm ATP, and 0.2 mm EGTA (in the absence of
Ca2+) 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 of 0.03 mgÆmL)1 (0.71 lm) rabbit F-actin, 0.06 mgÆmL)1 (0.11 lm) rabbit myosin, 0.015 mgÆmL)1 (0.23 lm) rabbit
or Akazara scallop tropomyosin, and 0.23 lm reconstituted troponin The assays were performed at 15 or 25C in a medium containing 50 mm KCl, 2 mm MgCl2, 20 mm Tris maleate (pH 6.8), 1 mm ATP, 0.1 mm CaCl2 and 0–3.84 mm EGTA The concentrations of EGTA required
to attain the desired final free Ca2+ concentrations (pCa 7.5–4.0) were calculated by using the stability constant of 8.45· 105m)1for the Ca2+–EGTA complex [41]
The reaction was initiated by adding 0.5 mL of 10 mm ATP 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 mixture and added to 4 mL of acidic malachite green solution to determine the liberated inorganic phosphate concentrations
by the method of Chan et al [42]