Comparative studies on the functional roles of N- and
C-terminal regions of molluskan and vertebrate troponin-I
Hiroyuki Tanaka
1
, Yuhei Takeya
1
, Teppei Doi
1
, Fumiaki Yumoto
2,3
, Masaru Tanokura
3
,
Iwao Ohtsuki
2
, Kiyoyoshi Nishita
1
and Takao Ojima
1
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 Ca
2+
-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 Ca
2+
,
troponin-T (TnT), which binds tropomyosin, and trop-

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 Ca
2+
-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 (ATnI
232)292
), which contains the inhibitory region (resi-
dues 104–115 of rabbit TnI) and the regulatory TnC-binding site (residues
116–131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin
Mg-ATPase. However, it did not interact with TnC, even in the presence
of Ca
2+
. These results indicated that the mechanism involved in the alter-

,
whereas Ca
2+
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 Ca
2+
binding to TnC
have been intensively studied as the central mecha-
nisms of Ca
2+
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, Ca
2+
-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-

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 Ca
2+
-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. ATnI

by TnI-fragments
The inhibition of actomyosin-tropomyosin Mg-ATPase
by TnI fragments was compared. The inhibitory effects
of RTnI, RTnI
1)116
and RTnI
96)181
differed greatly
from one another, although all of these proteins
contained the inhibitory region (Fig. 2A). RTnI
1)116
inhibited 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 been
reported previously [18,28,29], weaker inhibitory effects
of RTnI
1)116
revealed the importance of residues
117–181 for maximal inhibition. In particular, residues
Functional regions of molluskan TnI H. Tanaka et al.
4476 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
140–148 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 RTnI
96)181
was the strongest (94% of
the ATPase was inhibited), suggesting that residues
1–95 may decrease the inhibitory effects of residues
96–181.

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, RTnI
1)116
and RTnI
96)181
cosedimented with molar ratios of
approximately 0.23, 0.048, and 0.35, respectively, to
actin. On the other hand, ATnI-19K, ATnI
130)252
and
ATnI
232)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).
H. Tanaka et al. Functional regions of molluskan TnI

1)128
and ATnI
232)292
did not form a complex with
Akazara scallop TnC under any of the tested conditions,
whereas ATnI-52K, ATnI-19K, and ATnI
130)252
did
under both urea concentrations in the presence of Ca
2+
(Fig. 3B). It was interesting that ATnI
232)292
did not
form a complex, as ATnI
232)292
corresponds to
RTnI
96)181
and 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
ATnI
130)252
showed complex formation even in the
absence of Ca
2+
(Fig. 3B, upper panels), suggesting that

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 Ca
2+
.
As has been reported for similar vertebrate TnI frag-
ments [14,18,29], the inhibitory effect of RTnI
96)181
in
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; RTnI
1)116
, n; RTnI
96)181
, h. (B) ATnI-52K, d; ATnI-
19K, s; ATnI
1)128
, e; ATnI
130)252
, n; ATnI
232)292
, h.
Functional regions of molluskan TnI H. Tanaka et al.
4478 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS

; lanes l and q, ATnI
232)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
,
RTnI
96)181
, ATnI-19K, ATnI
130)252
, and ATnI
232)292
did not migrate into the gels, while free ATnI-52K and ATnI
1)128
exhibited 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

232)292
was not neutralized
by adding Akazara scallop TnC, irrespective of Ca
2+
concentrations (Fig. 5B, upper panel). Moreover, the
amount of ATnI
232)292
cosedimented with actin-tropo-
myosin was unaffected by the presence and absence of
TnC and Ca
2+
(Fig. 5B, lower panel). Therefore, the
Ca
2+
-switching mechanisms involving the alternative
binding of the C-terminal region of TnI were not pre-
sent in Akazara scallop troponin.
Ca
2+
-regulatory effects of troponins containing
TnI fragments
The Ca
2+
-regulatory effects of troponins composed of
TnI-fragments, native TnT, and TnC on actomyosin-
AB
Fig. 5. Functional differences between RTnI
96)181
(A) and ATnI
232)292

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, although
they exhibited quite different Ca
2+
-dependence curves
(Fig. 6A). The complex containing RTnI
1)116
(repre-
sented as RTn
1)116
) showed no inhibition, even under
low Ca
2+
concentrations, although it strongly activa-
ted the ATPase at Ca
2+
concentrations higher than
pCa 4.5. RTn
96)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 ATnI
232)292
, Akazara scallop TnT and TnC
(ATn
232)292
) inhibited the ATPase irrespective of Ca

whereas RTn
96)181
more effectively regulated the
ATPase than at 15 °C (Fig. 6C). These results
obtained at 25 °C 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. Ca
2+
-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; RTn
1)116
, n;
RTn

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 conditions is required, the
results of the alkaline urea gel electrophoresis indicate
that this interaction is strengthened by Ca
2+
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
Ca
2+
-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 Ca
2+
,
and Ca
2+
-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 Ca

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
Ca
2+
-dependent activation. In addition, at 15°C, 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
Ca
2+
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
Ca
2+
binding to site I or II of TnC. However, we can-
not rule out the possibility that the substitution of
Mg
2+
at site III or IV of vertebrate TnC with Ca

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
Functional regions of molluskan TnI H. Tanaka et al.
4482 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
discomfort 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), ATnI
1)128
(recombinant frag-
ment consisting of residues 1–128 of 52K-TnI), ATnI-19K
(recombinant 19K-TnI; residues 130–292), ATnI
130)252
(fragment; residues 130–252), and ATnI
232)292
(fragment;

GAAG-3¢) and RTnI181R (5¢-CCCCGGAGCC
GGATCC
CCAGCCCC-3¢). These primers were designed based on
the sequence retrieved from the GenBank database under
accession number L04347, and NcoIorBamHI 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 RTnI
1)116
(fragment; residues 1–116 of rabbit fast skeletal TnI) and
RTnI
96)181
(fragment; residues 96–181), using the primer
sets RTnI1F and RTnI116R (5¢-GAGCATGGCG
GGAT
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.

umn chromatography under the conditions used for CM-
Toyopeal chromatography. RTnI, RTnI
1)116
, and ATnI-
19K were also purified by hydroxyapatite (Wako Pure
Chemicals, Osaka, Japan) column chromatography per-
formed using 6 m urea, 10 mm KH
2
PO
4
(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) acryl-
H. Tanaka et al. Functional regions of molluskan TnI
FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4483
amide and 0.48% (w/v) bis-acrylamide slab gel containing
either 6 m or 3 m urea and either 2 mm CaCl
2
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 CaCl

1mm EGTA. The proteins in the effluents were detected
by the method of Bradford [40], and identified by
SDS ⁄ PAGE. RTnI
1)116
, which was insoluble in 10 mm
Tris ⁄ HCl (pH 7.6) and 0.5 mm CaCl
2
, 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 MgCl
2
, and 0.2 mm EGTA (in the absence
of Ca
2+
) or 0.2 mm EGTA plus 0.3 mm CaCl
2
(in the pres-
ence of Ca
2+

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 Mg
2+
-ATPase activity
The inhibition of actomyosin-tropomyosin Mg
2+
-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 15 °C in a medium
containing 50 mm KCl, 2 mm MgCl
2
,20mm Tris maleate
(pH 6.8), 1 mm ATP, and 0.2 mm EGTA (in the absence of
Ca
2+
) or 0.2 mm EGTA plus 0.3 mm CaCl
2
(in the pres-

for the Ca
2+
–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].
Functional regions of molluskan TnI H. Tanaka et al.
4484 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
Acknowledgements
This study was supported by Special Coordination
Funds from the Ministry of Education, Culture,
Sports, Science and Technology, of the Japanese
Government.
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