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Genome-Wide Survey of Genes Encoding Muscle Proteins in the Pearl Oyster,Pinctada fucataAuthor(s): Daisuke Funabara, Daiki Watanabe, Nori Satoh and Satoshi KanohSource: Zoological Science, 30(10):817-825. 2013.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.30.817URL: http://www.bioone.org/doi/full/10.2108/zsj.30.817

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2013 Zoological Society of JapanZOOLOGICAL SCIENCE 30: 817–825 (2013)

Genome-wide Survey of Genes Encoding Muscle Proteins

in the Pearl Oyster, Pinctada fucata

Daisuke Funabara1*, Daiki Watanabe1, Nori Satoh2, and Satoshi Kanoh1

1Graduate School of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie 514-8507, Japan2Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University,

Onna, Okinawa 904-0495, Japan

The mechanisms of contraction of molluscan striated and smooth muscles differ from those in ver-

tebrates. Molluscan striated muscles adopt a myosin-linked regulation, unlike vertebrates. Smooth

muscles in these species show a unique form of contraction, in which the tension is maintained

for a long time with little energy consumption, called catch. The available gene information is insuf-

ficient to elucidate the mechanism of contraction of molluscan muscles at the molecular level.

BLAST searching was thus used to annotate genes encoding proteins related to muscle contraction

in the completely determined genome of the pearl oyster Pinctada fucata using partial nucleotide

sequences obtained by 3' RACE. We identified genes that encode components of the thick-filament,

such as myosin heavy chain, myosin essential and regulatory light chains, paramyosin and

twitchin; of the thin-filament, such as actin, tropomyosin, troponin-T, troponin-I, troponin-C and

calponin; and the PKA catalytic subunit, which is a key player in the regulation of catch contraction.

The analysis indicated that isoforms of myosin heavy chain, paramyosin, and calponin are

produced by alternative splicing.

Key words: adductor muscle, catch contraction, myosin heavy chain, myosin light chain, paramyosin,

twitchin, actin, tropomyosin, troponin, calponin, PKA catalytic subunit

INTRODUCTION

The genome of the bivalve pearl oyster Pinctada fucata

has been sequenced (Takeuchi et al., 2012). The pearl

oyster P. fucata, which produces Akoya pearls, is one of the

most important organisms in the pearl farming industry. The

genome sequence of the pearl oyster should facilitate the

understanding of the mechanisms of pearl formation. In

addition, it should provide information that will improve our

understanding of the biological phenomena of bivalve mol-

lusks, including muscle contraction.

Molluscan smooth muscles, such as the bivalve adduc-

tor muscle and the mussel anterior byssus retractor muscle

(ABRM), show a unique form of muscle contraction called

catch (Uexküll, 1912). Bivalve adductor muscles are com-

posed of two parts: semi-translucent and white opaque mus-

cles. The former is thought to be responsible for the quick

closure of shells, and the latter for catch contraction that

keeps shells tightly closed for many hours. Catch muscle

can maintain this tension for long periods with little energy

consumption.

Catch muscles begin to contract in response to secreted

acetylcholine, which induces an increase in the intracellular

concentration of Ca2+. After tension has developed, intracel-

lular [Ca2+] decreases to the resting level, and then the mus-

cle enters the catch state, in which the tension is maintained

for a long time. Secretion of serotonin breaks the catch

state, followed by relaxation of the muscle (Funabara et al.,

2007).

Three muscle proteins, myosin, actin and twitchin, are

thought to be responsible for catch contraction (Yamada et

al., 2001; Funabara et al., 2007). Molluscan myosin, a con-

tractile protein essential for muscle contraction, is activated

by binding of Ca2+. It then interacts with actin to initiate the

contraction (Szent-Györgyi, 2007). This active state makes

possible the transition to the catch state after intracellular

[Ca2+] decreases to the resting level (Ishii et al., 1989). At

the breaking of the catch state, twitchin is phosphorylated by

cAMP-dependent protein kinase activated by cAMP, which

is increased by the secretion of serotonin (Siegman et al.,

1997; Siegman et al., 1998). In vitro experiments showed

that inactivated myosin, actin and twitchin form a complex

that collapses when twitchin is phosphorylated, suggesting

that the complex is responsible for maintaining tension dur-

ing catch (Funabara et al., 2007; Funabara et al., 2009).

The main components of molluscan muscles have been

physiologically, biochemically and molecular biologically

analyzed. However, although the regulation of muscle con-

traction has been studied in various species, the complete

molecular mechanism of molluscan catch contraction

remains obscure.

In the present study, a genome-wide survey was per-

formed to clarify the catch contraction at the molecular level

by identifying genes related to muscle contraction, such as

actin-associated proteins and myosin-associated proteins.

* Corresponding author. Tel. : +81-59-231-9564;

Fax : +81-59-231-9540;

E-mail : funabara@bio.mie-u.ac.jp

doi:10.2108/zsj.30.817

D. Funabara et al.818

MATERIALS AND METHODS

Target genes

Genes were selected that encode proteins involved in muscle

contraction in the pearl oyster Pinctada fucata. Myosin heavy chain,

myosin regulatory light chain, myosin essential light chain, paramy-

osin, and twitchin were selected as myosin-associated proteins.

Actin, calponin, tropomyosin, troponin C, troponin I, and troponin T

were selected as actin-associated proteins. cAMP-dependent pro-

tein kinase catalytic subunit, which plays an important role in catch

contraction, was also selected.

cDNA cloning

3’-rapid amplification of cDNA ends (RACE) was conducted to

determine the partial nucleotide sequences of the target genes,

using total RNA purified from the adductor muscle of live specimens

of pearl oyster Pinctada fucata harvested in the Mikimoto Pearl

Farm owned by K. Mikimoto & Co., Ltd., Mie Prefecture, Japan.

DNA primers for RACE were designed based on the partial nucle-

otide sequences of the above genes obtained from the P. fucata

genome database (Takeuchi et al., 2012). cDNA templates were

synthesized using a 3’-Full RACE Core Set (Takara Bio, Otsu,

Japan). 3’ RACE products were sequenced on an ABI PRISM 3100

Genetic Analyzer (Life Technologies, Carlsbad, CA, USA). The

nucleotide sequence data reported are available in DDBJ/EMBL/

GenBank databases under accession numbers AB735586 (Pifuc-

MHC-1), AB735587 (Pifuc-MHC-2), AB735588 (Pifuc-MRLC),

AB735589 (Pifuc-PM-1), AB735590 (Pifuc-PM-2), AB735591 (Pifuc-

PM-3), AB735592 (Pifuc-TW-1), AB735593 (Pifuc-TW-2),

AB735594 (Pifuc-TW-3), AB735595 (Pifuc-TM-1), AB735596 (Pifuc-

TM-2), AB735597 (Pifuc-TN-I), AB735598 (Pifuc-TN-T), AB735599

(Pifuc-CP-1), AB735600 (Pifuc-CP-2), AB735601 (Pifuc-CP-3),

AB735602 (Pifuc-CPKA).

Gene identification from Pinctada fucata genome

Gene identification was performed as described in Miyamoto et

al. (2013, present issue), except that the nucleotide sequences

determined above were used in the BLAST search against the gene

models (predicted transcripts) from the pearl oyster genome

sequence.

Domain searching

Domain searching was performed as described in Miyamoto et

al. (2013, present issue). Briefly, motif structures of protein

sequences in the gene models obtained by BLAST searching were

analyzed with the Pfam database to confirm the given gene models

as target genes.

Alignment and molecular phylogeny

Alignment and molecular phylogeny were analyzed as

described in Miyamoto et al. (2013, present issue). Briefly, protein

sequences of the gene models annotated in the present study and

those of counterpart genes from other species were aligned with

ClustalW and phylogenetic trees were drawn using the neighbor-

joining method.

RESULTS AND DISCUSSION

Myosin heavy chain

Myosin is a contractile protein responsible for muscle

contraction, and is composed of six subunits: two heavy

chains and four light chains (Holmes, 2008). Myosin has two

head regions that contain the sites of the ATPase, and a rod

region containing the coiled-coil structure that is essential for

assembly of myosin molecules into thick filaments. Light

chains are wound around the neck region of the head of the

heavy chain.

Two distinct sequences encoding partial myosin heavy

chain (MHC) were obtained from a cDNA library of the

adductor muscle of P. fucata. We named these Pifuc-MHC-

1 and -2, respectively. The Pifuc-MHC-1 cDNA was 2730

nucleotides (nt) and the Pifuc-MHC-2 cDNA was 1365 nt;

both cDNAs contained stop codons in their sequences.

BLAST searching with the Pifuc-MHC-1 cDNA against the

gene models resulted in matches to pfu_aug1.0_74379.1_

49164, pfu_aug1.0_301043.1_35996 and pfu_aug1.0_

6251.1_23985. BLAST searching with Pifuc-MHC-2 cDNA

also matched the same three gene models. These results

show that myosin heavy chain isoforms are produced from

a single gene by alternative splicing.

The myosin heavy chain phylogenetic tree showed that

the two myosin heavy chain genes of P. fucata cluster with

the same genes from other mollusks (Fig. 1A).

It is unknown whether actin-linked regulation similar to

that seen in vertebrates occurs in molluscan striated muscle.

In Atlantic bay scallop Argopecten irradians adductor mus-

cles, myosin heavy chain isoforms are expressed in striated

and catch muscles by alternative splicing (Nyitray et al.,

1994). Interestingly, a myosin-rod like protein, called catchin

or myorod, is produced from the myosin heavy chain gene

by alternative splicing in catch muscle (Yamada et al.,

2000). The function of catchin/myorod has remained

obscure. In the present study, sequences encoding catchin/

myorod were not identified. However, it is likely that they are

also expressed in P. fucata catch muscle, as catch muscles

from various other species contain catchin/myorod variants.

Myosin light chain

Each of the myosin heads has two types of light chains:

a regulatory light chain and an essential light chain, each of

which has EF-hand motifs and is classified as a small Ca2+-

binding protein (Sellers, 1999). In vertebrate striated muscle,

not all light chains are essential for the ATPase activity of

the myosin head, whereas in scallop adductor muscle, the

regulatory light chain plays a role in myosin activation

(Szent-Györgyi, 2007). In the absence of the regulatory light

chain, scallop myosin loses the ability to bind to Ca2+. Direct

binding of Ca2+ to the essential light chain activates scallop

myosin.

cDNA cloning resulted in one clone encoding the full-

length amino acid sequence of myosin regulatory light chain

(MRLC), which we named Pifuc-MRLC. BLAST searching of

the Pifuc-MRLC cDNA against the gene models matched

one model pfu_aug1.0_9091.1_67985, which contained an

undetermined region represented with NNN…NN in the

sequence of the gene model, which includes exon 3 of the

gene. The Pinctada myosin regulatory light chain is pre-

dicted to be composed of 10 exons.

We were unable to determine the sequence of the myo-

sin essential light chain (MELC) by using 3’ RACE in the

present study; therefore, we conducted a BLAST search

using sequences from other molluscan species. One gene

model, pfu_aug1.0_68031.1_24087, was identified and

named Pifuc-MELC.

The phylogenetic tree of the myosin light chains (Fig.

1B) showed that the genes of the regulatory and essential

light chains were clearly separated into different groups, and

were clustered with the corresponding genes of other mol-

Pearl Oyster Muscle Protein Genes 819

lusks.

It is unknown whether myosin light chains are involved

in the regulation of catch contraction. Further study is

needed to elucidate the function of myosin light chains in

catch contraction.

Paramyosin

The thick filaments of molluscan muscles comprise a

core of paramyosin covered by a monolayer of myosin

(Szent-Györgyi et al., 1971; Epstein et al., 1975; Cohen,

1982). The function of paramyosin is not known, but is

Fig. 1. Continued.

D. Funabara et al.820

thought to be required to sustain high force production of up

to 15 kg/cm2 for catch muscles (Millman, 1964). Paramyosin,

an enzymatically inert protein, forms rod-like 220 kDa

molecules with a length of 130 nm and a diameter of 2 nm,

consisting of two entirely α-helical polypeptide chains

(Kendrick-Jones et al., 1969). Although paramyosin is widely

distributed in invertebrate muscles, only catch muscles con-

tain a large amount of paramyosin (Winkelman, 1976). The

parallel correlation of thick filament length to paramyosin

content and to force production raise the possibility that a

high paramyosin content is related to catch by the genera-

tion of high levels of force (Lowy et al., 1964; Levine et al.,

1976). Paramyosin is readily phosphorylated in vitro, and

molecular cloning of paramyosin from Mytilus anterior

byssus retractor muscle (ABRM), a typical catch muscle,

suggested that the phosphorylation sites reside in the C-

terminal region (Watabe et al., 1989; Watabe et al., 2000).

Drosophila melanogaster paramyosin isoforms, called mini-

paramyosin, are produced from the same gene as paramy-

osin, which is regulated by two promoters for paramyosin

and miniparamyosin (Maroto et al., 1995). In Drosophila, it

is considered that paramyosin isoforms are involved in mat-

uration of muscles.

Three distinct clones encoding paramyosin (PM) were

obtained from adductor muscle by 3’ RACE. They were

named Pifuc-PM-1, -2 and -3. The Pifuc-PM-1 cDNA

matched five gene models: pfu_aug1.0_26361.1_04515,

pfu_aug1.0_590429.1_65134, pfu_aug1.0_585598.1_21872,

pfu_aug1.0_1385.1_07922 and pfu_aug1.0_13919.1_18060.

The cDNA sequence of Pifuc-PM-2 matched three gene mod-

els: pfu_aug1.0_590429.1_65134, pfu_aug1.0_585598.1_

21872 and pfu_aug1.0_26361.1_04515. The cDNA sequence

of Pifuc-PM-3 matched four gene models: pfu_aug1.0_

1385.1_07922, pfu_aug1.0_13919.1_18060, pfu_aug1.0_

31733.1_12073 and pfu_aug1.0_245498.1_50234. Gene

models pfu_aug1.0_13851.1_07922 and pfu_aug1.0_

13919.1_18060 are predicted to be located at the same

locus on homologous chromosomes, as the same region of

the paramyosin gene sequence corresponds to them.

Comparison of the sequences of Pifuc-PM-1, -2 and -3 with

the corresponding gene models, appeared to show that

paramyosin isoforms are produced by one gene by alterna-

tive splicing.

The Pifuc-PM-1 and -2 phylogenetic tree (Fig. 1C)

showed that Pinctada paramyosin forms a clade indepen-

dent from the known paramyosins. On the other hand, Pifuc-

PM-3 was separated into the same clade as Mytilus

paramyosin, which was isolated from the catch muscle.

Pifuc-PM-3 may be expressed in catch muscle and play a

role in catch contraction.

Twitchin

Twitchin, a giant protein belonging to titin/connectin fam-

ily, was first identified as the unc-22 gene product of

Caenorhabditis elegans, which showed abnormal twitching

movement (Waterston et al., 1980; Benian et al., 1989;

Benian et al., 1993). C. elegans individuals lacking the gene

encoding twitchin had abnormal sarcomere structures.

In the bivalve catch muscle, Mytilus ABRM, it was

reported that a high molecular weight protein is involved in

catch contraction. The protein was subsequently identified

by partial cDNA cloning as twitchin (Siegman et al., 1997;

Siegman et al., 1998). Phosphorylation of twitchin releases

the catch state, leading to muscle relaxation; it also prevents

Fig. 1. Muscular protein phylogenetic trees showing amino acid sequence relationships between isoform gene families from Pinctada fucata

and other species. (A) myosin heavy chain; (B) myosin light chain; (C) paramyosin; (D) twitchin; (E) actin; (F) tropomyosin; (G) troponin-C; (H)

troponin-I; (I) troponin-T; (J) calponin; (K) catalytic subunit of PKA. The sequences used are represented as accession numbers, plus species

and protein names. Pinctada proteins are marked by large black dots. The trees were generated by the neighbor-joining method. The number

at each branch indicates the percentage of times that a node was supported in 1000 bootstrap pseudoreplications. The scale bars indicate frac-

tional sequence divergence.

Pearl Oyster Muscle Protein Genes 821

the muscle from re-entering the catch state. Dephosphory-

lation of twitchin allows the muscle to enter the catch state.

This evidence clearly indicated that twitchin is a regulator of

catch contraction.

Twitchin comprises multiple repeats of immunoglobulin

(Ig) and fibronectin type 3 (Fn3) motifs, and has a single

kinase domain similar to the catalytic domain of myosin light

chain kinase (Funabara et al., 2003). Twitchin is phosphory-

lated by PKA and three molecules of phosphate are incor-

porated into one twitchin protein (Funabara et al., 2001a);

two of the phosphorylation sites have been identified.

Twitchin forms a complex with myosin and actin in a phos-

phorylation-sensitive manner, implying that the formation of

the complex is essential for maintenance of tension in the

catch state (Funabara et al., 2007; Funabara et al., 2009).

Twitchin isoforms are expressed in tissues other than catch

muscle in Mytilus (Kusaka et al., 2008).

Three distinct but partial sequences of twitchin (TW)

were obtained by 3’ RACE in the present study. The three

clones were designated as Pifuc-TW-1, -2 and -3. BLAST

searching of the Pifuc-TW-1 cDNA against the gene models

resulted in one matching model, pfu_aug1.0_7452.1_60249,

which did not include the full-length Pifuc-TW-1 sequence. A

further survey of the genome sequence database was then

conducted using the region that did not match the gene

models. As a result, segmented sequences of Pifuc-TW-1

were found in scaffold: 155607, scaffold: 227476, scaffold:

403848, scaffold: 427943 and scaffold: 218340. The whole

sequence of Pifuc-TW-2 was found in gene model

pfu_aug1.0_7452.1_60248. BLAST searching with the Pifuc-

TW-3 cDNA matched two gene models, pfu_aug1.0_

42945.1_05233 and pfu_aug1.0_40924.1_05151. These

two gene models are thought to be located in the same

locus on homologous chromosomes, as the same region of

Pifuc-TW-3 matches both of them. A further survey using the

region of the Pifuc-TW-3 cDNA that did not match the gene

models indicated that scaffold: 278718 and scaffold: 154940

contained the sequence of Pifuc-TW-3. The open reading

frames predicted from the nucleotide sequences of Pifuc-

TW-1, -2 and -3 had no stop codons in their sequences;

however, the deduced protein sequences have a motif struc-

ture comprising Ig and Fn3 motifs, which are observed in

Mytilus twitchin. This evidence raises the possibility that

Pifuc-TW-1, -2, and -3 are partial sequences derived from a

single twitchin gene.

The twitchin phylogenetic tree made with Pifuc-TW-1,

which is the longest sequence, and members of the titin/

connectin family (Fig. 1D) showed that Pinctada twitchin

can be placed in a clade with Mytilus twitchin. It is difficult

to obtain the full-length sequence of twitchin due to its large

size; therefore, the whole genome sequence of P. fucata will

aid in the determination of the sequence and function of

molluscan twitchin.

Actin

Actin, the main component of thin-filament in muscle,

participates in muscle contraction by interacting with myosin

to bring about sliding between thin- and thick-filaments. One

actin gene of P. fucata has been registered in the DNA data-

base; therefore, no actin cDNA cloning was done in the

present study. The known actin sequence was used for

BLAST searching against the gene models.

Thirteen actin genes were identified in the gene models:

pfu_aug1.0_1723.1_37096, pfu_aug1.0_3120.1_51891, pfu_

aug1.0_3120.1_51892, pfu_aug1.0_3120.1_51894, pfu_aug

1.0_3120.1_51895, pfu_aug1.0_3120.1_51896, pfu_aug1.0_

352.1_36511, pfu_aug1.0_352.1_36513, pfu_aug1.0_

44590.1_12559, pfu_aug1.0_46037.1_05345, pfu_aug1.0_

6682.1_02287, pfu_aug1.0_86513.1_42174 and pfu_aug1.0_

9193.1_17448. The actin phylogenetic tree (Fig. 1E) showed

that Pinctada actins are clustered with known molluscan

actins and are clearly separated from the vertebrate actins.

Tropomyosin

Tropomyosin, one of the main components of thin fila-

ments, binds to actin filaments along the longitudinal axis in

rod-shape and coiled-coil structures. In striated muscle, tro-

pomyosin is involved in regulation of muscle contraction,

together with the troponin complex. A variety of tropomyosin

isoforms are expressed in various tissues from four genes

in vertebrates using alternative promoters and alternative

splicing (Lees-Miller and Helfman, 1991; Pittenger et al.,

1994). It is not known, however, whether tropomyosin is

involved in the regulation of catch contraction.

Two distinct tropomyosin (TM) isoforms were obtained

from adductor muscle by 3’ RACE. The isoforms were des-

ignated as Pifuc-TM-1 and -2. The sequences of the two

isoforms were identical, except for a partial sequence in the

middle region of the molecules, indicating that the two iso-

forms are produced by alternative splicing from a single

gene. BLAST searching with the two cDNA sequences

matched one gene model, pfu_aug1.0_6509.1_67448,

which contained the full-length sequence of Pifuc-TM-1. The

gene model did not contain the partial sequence of Pifuc-

TM-2, indicating that the region is an exon that was not pre-

dicted in the gene model.

The tropomyosin phylogenetic tree (Fig. 1F) showed

that Pifuc-TM-1 and -2 are clustered with known molluscan

tropomyosins.

The expression patterns of Pifuc-TM-1 and -2 may be

different, depending on muscle type (catch or non-catch) in

the adductor, reflecting muscle properties.

Troponin

In vertebrate skeletal muscles, binding of Ca2+ to tro-

ponin, which is located on tropomyosin in thin filaments, is

the trigger for tension development and the start of muscle

contraction. Troponin comprises three subunits, troponin-C,

-I and -T. Ca2+ binding to troponin-C induces a structural

change, the signal of which is transmitted to troponin-I, the

inhibitory subunit, through troponin-T, the tropomyosin-bind-

ing subunit, and then the thin filament is allowed to interact

with thick filament (Farah and Reinach, 1995).

In mollusks, troponin from striated muscle has been

studied in the Akazara scallop Chlamys nipponensis

akazara, and its characteristics are quite different from those

in vertebrates. Rabbit skeletal troponin-C has four Ca2+-

binding sites (Collins et al., 1977), whereas only one binding

site is found in scallop troponin-C (Nishita et al., 1994; Ojima

et al., 1994). Scallop troponin-I also has a unique structure,

with an additional sequence of about 130 amino acids at the

N-terminus, resulting in the molecular weight being twice as

D. Funabara et al.822

high as vertebrate troponin-I (Tanaka et al., 1998). The N-

and C-terminal regions of scallop troponin-T are about 20

amino acids shorter and about 80 amino acids longer,

respectively, than those of rabbit (Inoue et al., 1996; Inoue

et al., 1998). The functional implications of the variants of

mollusk troponin subunits are unknown. Catch muscle of the

Giant Ezo scallop Patinopecten yessoensis contains tro-

ponin (Nishita et al., 1997). It is unknown whether troponin

is involved in catch contraction.

One clone of 905 nt encoding troponin-C (TN-C) was

obtained by cDNA cloning. BLAST searching with the cDNA

against the gene models resulted in no matches. Troponin

C sequences from the other species were BLAST searched

against the gene models, resulting in a match for one gene

model, pfu_aug1.0_1306.1_22530. The gene model was

annotated as Pifuc-TN-C in the present study.

The Pifuc-TN-C phylogenetic tree showed that the

troponin-C subunit was clustered with those from other mol-

lusks (Fig. 1G).

One clone of 2389 nt encoding troponin-I (TN-I) was

obtained by cDNA cloning and named Pifuc-TN-I. BLAST

searching with this cDNA against the gene models matched

one gene model, pfu_aug1.0_13173.1_39463. Part of the

sequence of Pifuc-TN-I did not match any gene models;

therefore, BLAST searching was conducted against the

genome. The rest of sequence of Pifuc-TN-I is distributed in

scaffold: 183989, scaffold: 241700, scaffold: 229747 and

scaffold: 608865. Predictions based on the cDNA sequence

and the results of BLAST searching indicate that the Pifuc-

TN-I gene is located in scaffold: 13173, scaffold: 229747,

scaffold: 183989, scaffold: 241700 and scaffold: 608865 and

these scaffolds align in this order.

The troponin-I phylogenetic tree showed that the

troponin-I subunit clusters with those from other mollusks

(Fig. 1H).

One clone of 1178 nt encoding troponin-T (TN-T) was

obtained by cDNA cloning and named Pifuc-TN-T. BLAST

searching with the Pifuc-TN-T cDNA against the gene mod-

els matched two gene models pfu_aug1.0_38623.1_26842

and pfu_aug1.0_59807.1_70898. Part of the sequence of

Pifuc-TN-T did not match any gene models; therefore,

BLAST searching was conducted against the genome. The

rest of sequence of troponin-T is distributed in scaffold:

266319 and scaffold: 9514. Predictions based on the cDNA

sequence and the results of BLAST searching indicate that

the troponin-T gene is located in scaffolds 38623, 59807,

266319 and 1514 and these scaffolds align in this order.

The Pifuc-TN-T phylogenetic tree showed that the

troponin-T subunit is clustered with those from other mol-

lusks (Fig. 1I).

Calponin

Calponin, a basic protein first found in vertebrate

smooth muscle, binds to F-actin (Takahashi et al., 1986).

Calponin has a calponin homology (CH) domain and multi-

ple repeats (Takahashi and Nadal-Ginard, 1991). In vitro

experiments have raised the possibility that it has inhibitory

effects on the binding of myosin to actin, regulating the con-

traction of smooth muscle (Winder and Walsh, 1990).

In mollusks, calponin has been found in Mytilus ABRM

and has one CH domain and five calponin repeats (Funabara

et al., 2001b). ABRM calponin showed inhibitory effects on

the Mg2+-ATPase activity of ABRM myofibrils, implying that

calponin may have a role in regulation of catch contraction

of molluscan smooth muscle (Funabara et al., 2001b).

Three distinct clones encoding partial sequences of cal-

ponin (CP) were obtained by 3’ RACE and were designated

Pifuc-CP-1, -2 and -3. BLAST searching of the Pifuc-CP

cDNAs against the gene models identified models pfu_aug

1.0_33531.1_12152 for Pifuc-CP-1, pfu_aug1.0_8205.1_

53126 for Pifuc-CP-2 and pfu_aug1.0_8205.1_53127 for

Pifuc-CP-3. Some of the Pifuc-CP-1 sequence did not

match any gene models; therefore, further BLAST searching

was performed against the genome. Pifuc-CP-1 sequence

was distributed on scaffold: 58487 in addition to scaffold:

33531 and scaffold: 8205, which contain the above gene

models. Judging from the comparison of the sequences of

Pifuc-CP-1, -2, and -3, these three scaffolds align on the

same chromosome and constitute the calponin gene. Thus,

the calponin isoforms of P. fucata are probably expressed

by alternative splicing from one gene.

The calponin phylogenetic tree (Fig. 1J) showed that all

calponin isoforms were clustered with those of other mol-

lusks and parasites. Pifuc-CP-2, -3 and Mytilus calponin,

which is found in catch muscle, were separated into the

same clade, implying that the two isoforms might be selec-

tively expressed in the catch muscle.

cAMP-dependent protein kinase catalytic subunit

cAMP-dependent protein kinase (PKA) is one of the

best characterized protein kinases in mammals, but there is

little information on PKA in molluscan muscles (Taylor et al.,

1990). Only the biochemical features of PKA from catch

muscle and other tissues of the bivalve Mytilus have been

analyzed (Bejar and Villamarin, 2006; Bardales et al., 2008;

Bardales et al., 2011). PKA is not a muscular protein, but

has a key role in the regulation of catch contraction in

bivalve smooth muscle, where PKA, activated by the

increased cAMP induced by the secretion of serotonin,

phosphorylates twitchin to break the catch state (Funabara

et al., 2001a; Funabara et al., 2005). PKA from catch mus-

cle has not been molecularly characterized.

One clone of 1835 nt encoding the catalytic subunit of

PKA (CPKA) was obtained by cDNA cloning and named

Pifuc-CPKA. A BLAST search of the cDNA against the gene

models matched two gene models: pfu_aug1.0_19091.1_

18605 and pfu_aug1.0_1567.1_58573. Predicted from the

high homology of the sequences, these gene models are

thought to reside on the same locus on homologous chro-

mosomes. Part of the cDNA sequence showed no matches

to the gene models; therefore it was used in a BLAST

against the genome. Scaffold: 44192 and scaffold: 686920

contained the sequence of Pifuc-CPKA.

The phylogenetic tree (Fig. 1K) for the catalytic subunit

of PKA showed that Pifuc-CPKA clustered with those of ani-

mal phyla.

Conclusions

We identified a number of genes encoding muscle pro-

teins that play major roles in muscle contraction in the P.

fucata genome (Table 1). The results indicated that isoforms

of myosin heavy chain, paramyosin, tropomyosin and cal-

Pearl Oyster Muscle Protein Genes 823

ponin are expressed by alternative splicing, raising the pos-

sibility that the isoforms are used selectively in catch and

non-catch muscles. In the present study, the whole adduc-

tor, composed of catch and non-catch muscles, was used

for cDNA cloning. Further study is needed to identify which

spliced transcripts are expressed in the catch muscle and

elucidate their roles in catch contraction.

As described above, little information on molluscan mus-

cle proteins responsible for catch contraction was available.

The present study shows that it has now become easier to

obtain gene information for muscle proteins using genome

databases. The findings of the present study provide tools

for determining the function of muscle proteins in mollusks

and for studying of the molecular mechanism of contraction

of molluscan muscles.

Table 1. Muscle protein genes identified in the Pinctada fucata genome.

gene annotationgene

number inassembly

Best hit gene model IDBLAST best hit to NCBI database

accession (species)tandem

arrangementrelatedfigure

references

myosin heavy chain(Pifuc-MHC)

1

pfu_aug1.0_301043.1_35996 EU687253 (Sepia esculenta)

no Fig. 1A

–

pfu_aug1.0_6251.1_23985 AJ249991 (Mytius galloprovincialis) Yamada et al. (2000)

pfu_aug1.0_74379.1_49164 AF134172 (Pecten maximus) Janes et al. (2000)

myosin regulatory light chain(Pifuc-MRLC)

1 pfu_aug1.0_9091.1_67985AB444943 (Placopecten magellanicus)

no

Fig. 1B

Perreault-Micale et al. (1996)

myosin essential light chain(Pifuc-MELC)

1 pfu_aug1.0_6803.1_24087 AJ563458 (Crassostrea gigas) no –

paramyosin(Pifuc-PM)

1

pfu_aug1.0_1385.1_07922

AB016070 (Mytilus galloprovincialis)

no Fig. 1C

Watabe et al. (2000)pfu_aug1.0_13919.1_18060

pfu_aug1.0_31733.1_12073

pfu_aug1.0_590429.1_65134

pfu_aug1.0_245498.1_50234 AJ547618 (Crassostrea gigas) –

pfu_aug1.0_26361.1_04515 – –

pfu_aug1.0_585598.1_21872 AB571843 (Haliotis discus discus) –

twitchin(Pifuc-TW)

2

pfu_aug1.0_28106.1_62511

AB062881 (Mytius galloprovincialis)no Fig. 1D

Funabara et al. (2003)

pfu_aug1.0_40924.1_05151

pfu_aug1.0_42945.1_05233

pfu_aug1.0_462.1_15070

pfu_aug1.0_462.1_15071

pfu_aug1.0_54844.1_56182

pfu_aug1.0_7452.1_60248

pfu_aug1.0_7452.1_60249

pfu_aug1.0_3478.1_37612 Q23551 (Caenorhabditis elegans) –

actin(Pifuc-AC)

13

pfu_aug1.0_1723.1_37096GU645236 (Penaeus japonicus)

– Fig. 1E

Zhi et al. (2011)pfu_aug1.0_9193.1_17448

pfu_aug1.0_3120.1_51891ACD99707 (Meretrix meretrix) –

pfu_aug1.0_3120.1_51892

pfu_aug1.0_3120.1_51894AM236595 (Haliotis tuberculata) –

pfu_aug1.0_352.1_36511

pfu_aug1.0_3120.1_51895 EU726273 (Pinctada fucata) Wang et al. (2008)

pfu_aug1.0_3120.1_51896 M26501 (Pisaster ochraceus) Kowbel et al. (1989)

pfu_aug1.0_352.1_36513

EU234531 (Crassostrea ariakensis) –pfu_aug1.0_44590.1_12559

pfu_aug1.0_86513.1_42174

pfu_aug1.0_46037.1_05345 AB071191 (Crassostrea gigas) Miyamoto et al. (2002)

pfu_aug1.0_6682.1_02287 AL160004 (Homo sapiens) Gregory et al. (2006)

tropomyosin(Pifuc-TM)

1 pfu_aug1.0_6509.1_67448 AB444943 (Crassostrea gigas) no Fig. 1F –

troponin-T(Pifuc-TN-T)

1pfu_aug1.0_38623.1_26842 D88423 (Chlamys nipponensis)

no Fig. 1GInoue et al. (1996)

pfu_aug1.0_59807.1_70898 AB004637 (Mizuhopecten yessoensis) –

troponin-I(Pifuc-TN-I)

1 pfu_aug1.0_13173.1_39463AB009368 (Chlamys nipponensis akazara)

no Fig. 1H Ojima et al. (1997)

troponin-C(Pifuc-TN-C)

1 pfu_aug1.0_1306.1_22530 AB049962 (Todarodes pacificus) no Fig. 1I Ojima et al. (2001)

calponin(Pifuc-CP)

1

pfu_aug1.0_33531.1_12152

AB052656 (Mytilus galloprovincialis) no Fig. 1J Funabara et al. (2001)pfu_aug1.0_8205.1_53126

pfu_aug1.0_8205.1_53127

catalytic subunit of PKA(Pifuc-CPKA)

1 pfu_aug1.0_19091.1_18605 X63421 (Aplysia californica) no Fig. 1K Beushausen et al. (1988)

D. Funabara et al.824

ACKNOWLEDGMENTS

The authors thank Dr. Kiyohito Nagai (K. Mikimoto & Co., Ltd.)

for providing pearl oysters as an experimental material.

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(Received October 2, 2012 / Accepted March 7, 2013)