ORIGINAL ARTICLE Chemistry and Biochemistry

Molecular cloning and expression profiling of procollagen a1(I) of cultured Pacific bluefin tuna

Teruyoshi Tanaka • Kenji Takahashi • Kohsuke Adachi • Haruki Ohta •

Yukihiro Yoshimura • Yasuo Agawa • Yoshifumi Sawada • Osamu Takaoka •

Amal Kumar Biswas • Kenji Takii • Nobuhiro Zaima • Tatsuya Moriyama •

Yukio Kawamura

Received: 20 November 2013 / Accepted: 13 February 2014 / Published online: 22 April 2014

� The Japanese Society of Fisheries Science 2014

Abstract Type I collagen is widely distributed in most

organs in teleosts. It plays a role not only in intercellular

adhesion, but also in molecular signaling. In this study,

Pacific bluefin tuna (PBT) procollagen a1 (I) cDNA was

cloned and characterized. The nine fragments of a pro-

collagen a1 (I) chain cDNA clone were prepared and

spliced together to create the complete coding region. The

resulting amino acid sequence was homologous with that

of other teleosts. The mRNA expression profile of PBT

procollagen a1 (I) in various tissues and the phylogenetic

analysis with other vertebrate procollagen a1 (I) chains

suggest that PBT procollagen a1 (I) could be a precursor

form of the PBT type I collagen a1 chain. In addition, its

level of expression in PBT larvae and early juveniles

gradually increased with somatic growth. This increase was

related to the standard length, wet body weight, and protein

content of each individual fish. Therefore, the expression

profile of procollagen a1 (I) may be a useful indicator for

somatic growth in fish larvae and juveniles.

Keywords Pacific bluefin tuna � Type I collagen �Procollagen � Somatic growth

Introduction

The Pacific bluefin tuna Thunnus orientalis (PBT) is an

important food resource species in aquaculture, particularly

in Japan. Many companies have engaged in PBT farming,

and its domestic production is quickly growing. However,

further development of PBT aquaculture is still needed to

maintain sustainable fisheries. In 2002, we successfully

developed the first full-cycle aquaculture of PBT at the

Oshima Fishery Experiment Station, Kinki University in

Kushimoto, Wakayama, Japan [1].

The next step for sustainable fisheries is the devel-

opment of mass seeding techniques. During the rapid

somatic growth stage in their early life, high mortality

was found in laboratory-reared PBT [1–3]. One reason

T. Tanaka and K. Takahashi contributed equally to this work.

The nucleotide sequence data reported are available in the DDBJ/

EMBL/GenBank databases under the accession number AB824735.

T. Tanaka � K. Takahashi � H. Ohta � Y. Yoshimura �N. Zaima � T. Moriyama � Y. Kawamura (&)

Department of Applied Biological Chemistry, Graduate School

of Agriculture, Kinki University, 3327-204, Nakamachi,

Nara 631-8505, Japan

e-mail: kawamury@kyoto-wu.ac.jp

T. Tanaka � O. Takaoka � A. K. Biswas � K. Takii

Uragami Experiment Station, Fisheries Laboratories,

Kinki University, Uragami, Nachikatsuura, Higashimuro,

Wakayama 649-5145, Japan

K. Adachi

Graduate School of Agriculture, Kochi University,

Monobe Otsu 200, Nankoku, Kochi 783-8502, Japan

Y. Agawa � Y. Sawada

Oshima Experiment Station, Fisheries Laboratories,

Kinki University, Oshima, Kushimoto, Higashimuro,

Wakayama 649-3633, Japan

Present Address:

Y. Kawamura

Department of Food Nutrition and Biochemistry, Kyoto

Women’s University, Kitahiyoshi-cho 35, Imakumano,

Higashiyama-ku, Kyoto 605-8501, Japan

123

Fish Sci (2014) 80:603–612

DOI 10.1007/s12562-014-0737-7

for this death is the cannibalism that occurs during the

10–30 days after hatching (DAH) of larvae and juveniles

[1, 4, 5]. The early mass mortality of PBT is also

potentially a result of different individual growth per-

formances [6]. Therefore, somatic growth is a key factor

for their survival. Another reason for this early death is

that PBT have very weak skin, which makes handling

difficult for the researchers that constantly rear this

species. Indeed, over 40 % of PBT juveniles died of skin

injuries that occurred during transportation with hand

nets that takes place during the 1st week of transfer of

these land-based farmed juveniles to open net cages [7].

Therefore, it is important to understand the characteris-

tics of their skin and type I collagen which is the major

component of PBT skin.

Collagen is a well-known major fibrillar protein in

vertebrates [8]. It is widely distributed in most organs,

such as skin, bone, and muscle. Fibril-forming collagen

is divided into the following five types: I, II, III, V, and

XI [9, 10]. The molecules are composed of three sub-

units, called a chains, which are initially secreted as

procollagen a chains from fibroblasts, chondrocytes,

osteoblasts, etc. The structure of the pro-a chain contains

a central, triple-helical domain. This domain has a

repeated Gly-X-Y motif, with a proline (Pro) residue at

the X position and hydroxyproline (Hyp) residue at the Y

position in many cases. There is also an N- and C-telo-

peptide domain and an N- and C-terminal propeptide

domain. The assembled two procollagen a1 and one

procollagen a2 chain in teleosts, one procollagen a1, one

procollagen a2, and one procollagen a3 chain in some

fishes configure the triple helix [11, 12]. The molecules

then form covalent cross-links between the telopeptide

and adjacent helical domains of the collagen molecules

and the propeptides are cleaved, resulting in a mature

collagen fibril [13].

Several studies have investigated the sequences of col-

lagen peptides found in various fish. Saito et al. [14]

reported the primary structure of a fish type I procollagen

using fibroblasts isolated from the rainbow trout On-

corhynchus mykiss. A full-length cDNA of the type I

procollagen a1 from the skate Raja kenojei was shown to

be an elasmobranch by Hwang et al. [15]. The cDNA

cloning of type I collagen a1, 2, and 3 in the flounder

Paralichthys olivaceus was also performed by Suzuki et al.

[16]. However, while there are some reports on fish col-

lagen sequences, little is known about PBT type I collagen.

In the present study, PBT procollagen a1 (I) cDNA was

identified and expression profiling was performed in dif-

ferent tissues of a 1-year-old fish and in the whole body of

PBT larvae and early juveniles.

Materials and methods

RNA isolation and cDNA synthesis

Skin, muscle (o-toro, chu-toro, akami, and chiai: see description

of these four muscle types in the Discussion), and liver were

isolated from a 1-year-old PBT and immediately frozen in liquid

nitrogen. Total RNA was prepared with the RNeasy Lipid Tissue

Mini Kit (Qiagen; Hilden, Germany) according to the manu-

facturer’s instructions. The total RNA extracted was treated with

DNase I (Takara Bio; Otsu, Japan) and then purified with the

NucleoTrap mRNA Mini kit (Takara Bio; Otsu, Japan). The full-

length cDNA was obtained using the SMARTer PCR cDNA

Synthesis Kit (Takara Bio; Otsu, Japan).

To extract the total RNA from the whole body of the

PBT larvae and juveniles, a Sepasol-RNA Super kit

(Nacalai Tesque; Kyoto, Japan) was used according to

manufacturer’s instructions. The total RNA was subse-

quently treated with the TURBO DNase-free kit (Ambion;

Austin, TX, USA) to remove any genomic DNA contam-

ination. One microgram of each sample was reverse tran-

scribed into cDNA with the High-Capacity cDNA Reverse

Transcription kit (Applied Biosystems; Foster, CA, USA).

Cloning of PBT procollagen a1 (I) cDNA

The cloning of procollagen a1 (I) cDNA from PBT skin was

carried out according to the design shown in Fig. 1. Briefly,

polymerase chain reaction (PCR) was performed to obtain the

Fig. 1 cDNA cloning design for the procollagen a1 (I) chains from

PBT skin. The upper figure displays a diagram of the PBT

procollagen a1 (I) chain. The lower nine fragments show the cDNA

clones obtained by PCR (I–VII) and RACE (VIII and IX). The gene-

specific primers used in this study are listed in Table 1. Forward and

reverse primers are shown as right and left arrows, respectively.

Arrows with dotted lines represent the adaptor promoter. 1, N-pro-

peptide; 2, N-telopeptide; 3, triple-helical domain; 4, C-telopeptide; 5,

C-propeptide

604 Fish Sci (2014) 80:603–612

123

cDNA fragment using the PBT skin cDNA as the template

and PrimeSTAR Max Premix (29) (Takara Bio; Otsu, Japan).

The PCR program was 40 cycles at 94 �C for 0.5 min, 60 �C

for 0.5 min, and 72 �C for 1.5 min. The PCR primers were

designed based on the alignment of the cDNA of procollagen

a1 (I) chains and various vertebrate sequences. The sequences

of the PCR primers are shown in Table 1. The PCR product

was confirmed using agarose gel electrophoresis. The single

band that corresponded to the amplified fragment was excised

from the 1 % agarose gel and then purified with the QIAquick

Gel Extraction Kit (Qiagen; Hilden, Germany). The DNA

sequence was determined with a 3500/35009L Genetic

Analyzer (Applied Biosystems; Foster, CA, USA) using a

BigDye Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems; Foster, CA, USA) according to the manufac-

turer’s instructions.

Rapid amplification of cDNA ends (RACE)

Using the SMARTer PCR cDNA Synthesis Kit (Takara

Bio; Otsu, Japan), 30- and 50-RACE PCR was performed to

amplify the procollagen a1 (I) cDNA ends. The gene-

specific IX and adaptor primers were used for the 30-RACE, and the adaptor and gene-specific VIII primers for

the 50-RACE, according to the manufacturer’s instructions.

The PCR program was 40 cycles at 94 �C for 0.5 min,

60 �C for 0.5 min, and 72 �C for 1.5 min. The fragments

were separated using a 1 % agarose gel, were excised from

it, and were subsequently sequenced to verify their identity.

Phylogenetic analysis

Vertebrate procollagen amino acid sequences were obtained

from the DNA Data Bank of Japan (DDBJ). The species and

their accession numbers are as follows: human Homo sapiens

(BC059281), mouse Mus musculus (AA008230), dog Canis

lupus familiaris (AF153062), African clawed frog Xenopus

laevis (AB034701), Japanese fire belly newt Cynops pyrr-

hogaster (AB015438), Japanese flounder P. olivaceus

(AB196513), rainbow trout O. mykiss (AB052835), and skate

R. kenojei (AB195842). For the phylogenetic analysis with

other animal a chains, the following species and their

accession numbers were used: human a1 (I) (P02452), human

a2 (I) (P08123), human a1 (II) (P02458), human a1 (III)

(EAX10911), human a1 (V) (EAW88131), human a2

(V) (EAX10906), human a3 (V) (EAW84061), human a1

(XI) (AAA51891), human a2 (XI) (CAI95623), zebrafish

Danio rerio a1 (I) (XP_005156036), zebrafish a2

(I) (AAH71278), zebrafish a3 (I) (NP_958886), torafugu

Takifugu rubripes a1 (I) (XP_003961060), and tilapia Ore-

ochromis niloticus a1 (I) (XP_005458232). An amino acid

alignment and phylogenetic tree was generated using the

ClustalW (2.1) program with the neighbor-joining method

(DDBJ, http://clustalw.ddbj.nig.ac.jp [17]). The robustness of

the topologies was evaluated using a bootstrap analysis with

1,000 bootstrap replications.

Fish rearing and sampling for the PBT procollagen a1

(I) gene expression analysis

PBT eggs were collected at the Oshima Experiment Sta-

tion, Fisheries Laboratories, Kinki University. The eggs

were transferred to a 40-t concrete tank (diameter 6.0 m,

depth 1.7 m) for hatching and rearing. The larvae and

juveniles were reared at the Uragami Experiment Station,

Fisheries Laboratories, Kinki University. The water tem-

perature was maintained at approximately 27.5 �C. The

feeding design for the larvae and juveniles was as follows:

rotifers Brachionus rotundiformis were used from 2 to 18

DAH, brine shrimp Artemia nauplii were used from 11 to

24 DAH, the yolk sac larvae of striped beakperch Ople-

gnathus fasciatus were used from 13 to 31 DAH, and

formula feed was used from 18 to 32 DAH. A sample of the

PBT larvae and juveniles was taken at 6, 9, 12, 15, 18, 21,

Table 1 Primer sequence

Primer sequence

I

Forward 50-ATGTTCAGCTTTGTGGATATTCGC-30

Reverse 50-CTCCATTCTTTCCAGGGGG-30

II

Forward 50-CTGGTCCTCAGGGTTTCACTGG-30

Reverse 50-TCCTCTCTTTCCTTCCTCACC-30

III

Forward 50-TGGTTTCCCTGGTGCTCGTGGTC-30

Reverse 50-CACCACGCTCACCAGGGAAACC-30

IV

Forward 50-CCAAGGGAGTGACTGGTAGCCCTGG-30

Reverse 50-CAGCGTCTCCCTTGGGTCCAGTATC-30

V

Forward 50-GACAACGGGAGCCGAGACTAGAG-30

Reverse 50-TTTGCTGGACCTCCTGGTGCTGA-30

VI

Forward 50-CCAAGGGAGACCGTGGTGAGA-30

Reverse 50-GCAAGTCTCACCAGTCTCCATGTTG-30

VII

Forward 50-TGCCGTGACCTGAAGATGTGCCACC-30

Reverse 50-TTACAAGAAGCAGACTGGGCCAA-30

VIII

Forward Adaptor primer

Reverse 50-GAGAGAAGTTTCCGCCAAGGCCAGG-30

IX

Forward 50-TGCCGTGACCTGAAGATGTGCCACC-30

Reverse Adaptor primer

Fish Sci (2014) 80:603–612 605

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25, 29, and 32 DAH. Samples for gene expression analysis

were immediately frozen in liquid nitrogen and then stored

at -80 �C until analysis. The standard length (SL) was

measured using a projector. In each sample, 5–300 larvae

and juveniles were taken for measurements of the body

weight (BW) and the average weight per individual.

Protein concentration estimation

The fish samples were extracted with radioimmunoprecipita-

tion assay buffer (Nacalai Tesque; Kyoto, Japan). The protein

concentration was determined using the Modified Lowry Pro-

tein Assay Kit (Thermo Scientific; Rockford, IL, USA).

Quantitative real-time PCR

For a quantitative analysis of gene expression, cDNA

amplification was performed using SYBR Premix Ex Taq

(Takara Bio; Otsu, Japan) and procollagen a1 (I) primers

(forward primer, 50-CATCATCCCAGACGGAGAGT-30;reverse primer, 50-CATCCTCTCCCTTTTCCACA-30),which corresponds to 230–328 base pairs (bp) of the cDNA

sequence discovered in this study. The b-actin primers

were designed according to Adachi et al. [18]. The

dynamic range of the procollagen a1 (I) mRNA expression

was evaluated by the following method. The mRNA con-

centration was quantified at an ultraviolet wavelength of

260 nm and diluted by ten orders of magnitude. The

quantitative real-time PCR was performed at the range that

showed a linear relationship between the concentration and

threshold values (CT). The quantitation of procollagen a1

(I) gene expression was performed within the dynamic

range. The expression data was normalized to b-actin

expression for each individual sample. The specificity of

the amplification products was analyzed using the disso-

ciation curve analysis at the end of each PCR reaction.

Statistical analysis

The data are presented as mean ± standard deviation (SD).

All data were analyzed with Excel-Toukei 2006 (SSRI;

Tokyo, Japan). One-way analysis of variance, followed by

post hoc Bonferroni correction, was used to examine the

differences in the various tissues and fish stages. P \ 0.05

was considered significant.

Results

cDNA cloning of the PBT procollagen a1 (I) gene

Based on the homologous sequences of the procollagen a1

(I) gene in vertebrates, we designed primers for that of the

PBT. Double-stranded cDNA was prepared from the PBT

skin and used for PCR, which was successfully performed.

When the homology of all the cloned fragments was ana-

lyzed using BLAST on the National Center for Biotech-

nology Information server (http://blast.ncbi.nlm.nih.gov/

Blast.cgi), the top hits for each fragment were the collagen

a1 chain, not the a3 chain. The top hits and E values for

fragments I, II, III, IV, V, VI, VII, VIII, and IX were Pun-

damilia nyererei, 0.0; P. nyererei, 0.0; P. nyererei, 0.0; O.

niloticus, 0.0; O. niloticus, 0.0; T. rubripes, 0.0; O. niloticus,

0.0; Haplochromis burtoni, 1e-115; and O. niloticus, 0.0,

respectively. These results suggest that the cloned fragments

were derived from the PBT procollagen a1 chain. Based on

the cDNA sequence of the cloned PBT skin procollagen a1

(I), the amino acid sequence was determined and consisted

of 1,454 amino acids (4,362 bp), as shown in Fig. 2. We

estimated the location of the N- and C-terminal regions by

comparison of the protein sequence with the procollagen a1

sequences in other animals. The N- and C-terminal domains

were composed of 166 and 274 amino acids (498 and

822 bp), respectively. The helical region of the PBT pro-

collagen a1 (I) consisted of 1,014 amino acids.

The amino acid sequence of the PBT procollagen a1 (I) is

closely similar to the human, skate, and rainbow trout

sequences. Nile tilapia O. niloticus was the closest match,

with 85 % amino acid identity in the complete primary

structure. Animals with the closest amino acid sequence

identity to the PBT procollagen a1 (I) N- and C-terminal

regions were the zebrafish D. rerio (65 % identity) and Nile

tilapia O. niloticus (93 % identity), respectively. The

C-terminal region was more highly conserved than the

N-terminal region, as described in previous reports [19, 20].

Dion and Myers [20] hypothesized that the Cys residues

in the C-terminal propeptide play a crucial role in intra- and

interchain disulfide bond formation, and thus in the inter-

action of the procollagen a chains. In the C-terminal region

of the PBT procollagen a1 (I) chain, two Ser residues (1,253

and 1,270) were replaced with Cys residues. This could

result in distortion of the PBT collagen, because of the

missing intrachain disulfide bond. In contrast, Cys residues

at positions 1,247, 1,279, 1,287, 1,358, 1,403, and 1,450 in

the PBT procollagen are conserved in vertebrates, and the

residues likely participate in intrachain disulfide bonds.

We found six amino acid residues in the N-terminal

sequence of the procollagen with the Met-Phe-Ser-Phe-

Val-Asp motif. Interestingly, these residues are conserved

in all procollagen a1 (I) chains that have been previously

reported. The PBT procollagen a1 (I) chain also has ten

Cys residues (33, 51, 54, 56, 63, 68, 74, 84, 85, and 88) in

the N-terminal propeptide region in common with the skate

and rainbow trout.

The sequences of the cell adhesion motif (Arg-Gly-

Asp), the integrin a1b2 recognition motif (Gly-Phe-Hyp-

606 Fish Sci (2014) 80:603–612

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Gly-Gln-Arg), and the Lys-related cross-linking site were

also present in the PBT procollagen, although these were

not perfectly conserved amongst the other species.

Therefore, the amino acid sequence of PBT is similar to

that of other fish, although there were some differences as

well.

Fig. 2 The cloned PBT procollagen a1 (I). The amino acid sequence

of PBT procollagen a1 (I) was compared to human, skate, and

rainbow trout using multiple alignments. Right and left arrows show

the start and end points of the triple-helical domain. The closed

circles show the position of Cys residues in the N- and C-terminal

domains. The open circles show positions where conserved Cys are

replaced by Ser in PBT. The residues surrounded by a box highlight

the cell adhesion site (Arg-Gly-Asp) and the integrin a1b2 recogni-

tion site (Gly-Phe-Hyp-Gly-Gln-Arg). The overbar shows the Lys

related to the cross-linking site

Fish Sci (2014) 80:603–612 607

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Phylogenetic analysis of the PBT procollagen a1 (I)

To clarify the relationship between the procollagen a1

(I) chain of PBT and that of other vertebrates, phylo-

genetic analysis was performed; the results are shown

in Fig. 3a. The phylogenetic tree generated for PBT and

other vertebrates clustered the procollagen a1

(I) sequences into several groups, all of which had a

bootstrap value higher than 997, indicating their

significance. The procollagen a1 (I) of PBT was similar

to that of rainbow trout, Japanese flounder, and skate.

As shown in Fig. 3b, further phylogenetic analysis

clarified the relationship between the PBT procollagen

a (I) chains and other vertebrate fibrillar procollagen achains. The procollagen a1 (I) of PBT was closest to the

tilapia a1 (I). These results indicate that the cloned

fragment was derived from the PBT procollagen a1

chain.

Fig. 3 Procollagen a1

(I) phylogenetic tree,

constructed using the neighbor-

joining method and based on the

amino acid sequences of

vertebrates. The bootstrap value

of 1,000 repeated calculations is

shown at the branches. The

scale bar indicates 10 %

sequence dissimilarity

608 Fish Sci (2014) 80:603–612

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PBT procollagen a1 (I) gene expression in various

tissues

Type I collagen is widely distributed in most teleost tis-

sues. To further investigate the characteristics of the PBT

procollagen a1 (I), the mRNA expression level in different

tissues was determined. We quantified the relative

expression levels of the procollagen a1 (I) using b-actin, a

housekeeping gene, as a control. As shown in Fig. 4, pro-

collagen a1 (I) gene expression was detected in PBT skin,

muscle, and liver. The highest expression of procollagen

a1 (I) was in the skin, suggesting that type I collagen may

play an important role there.

Changes in SL, BW, and protein content during PBT

larval and early juvenile development

To examine the utility of procollagen a1 (I) gene expres-

sion as a somatic growth marker, larval and early juvenile

stages of PBT were collected. We measured the SL and

BW and the results are shown in Fig. 5. The SL and BW

increased from 4.5 ± 0.5 mm and 0.0009 ± 0.00004 g at

6 DAH to 49.1 ± 5.7 mm and 1.4 ± 0.05 g at 32 DAH,

respectively. That is, the SL and BW increased 11- and

1,530-fold during the 27 days.

Ehrlich reported that protein content increased at a faster

rate than neutral lipid in the early developmental stages of

plaice Pleuronectes platessa [21] and Pacific herring Clu-

pea harengus [22]. It is likely that larvae and early stage

juveniles prioritize somatic growth, and the growth

depends on protein accumulation. Therefore, we deter-

mined the protein content as a function of morphological

development. The protein level for each individual fish

(9104 lg/fish) was slightly increased until 18 DAH

(Fig. 6a). After this time point, the increase accelerated

until 32 DAH. The protein composition (9104 lg/fish) at

6–32 DAH of the PBT larvae and juveniles corresponded

to incremental increases in the SL and wet BW. These

results suggest that the growth of the PBT larvae and early

Fig. 4 Procollagen a1 (I) gene expression profile. The total RNA

from various tissues of a 1-year-old PBT were used to synthesize

cDNA for PCR amplification using PBT procollagen a1 (I)-specific

primers and b-actin as the control. Each assay was done in triplicate

for each tissue. Each data value is presented as the mean ± SD. The

letters on the bar graphs denote significant differences (P \ 0.05)

Fig. 5 Changes in the standard length (SL, closed circle) and body

weight (BW, open circle) of laboratory-reared PBT larvae and

juveniles and their morphological changes. Three fish at each

sampling point were randomly selected and analyzed. Each data

value is presented as the mean ± SD. The letters above the points

denote significant differences (P \ 0.05)

Fig. 6 Changes in the protein level per a fish and b wet weight

(WW). Each data value is presented as the mean ± standard

deviation. Three fish at each sampling point were randomly selected

and analyzed. The letters above the points denote significant

differences (P \ 0.05)

Fish Sci (2014) 80:603–612 609

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juveniles depends on protein accumulation, in accordance

with the results seen in other fish.

The changes in protein content per wet weight (lg/mg

WW) in the fish showed different patterns from individual

to individual (Fig. 6b). The protein level (lg/mg WW) was

slightly increased until 25 DAH and remained constant

from 25 DAH onward. These protein profiles illustrated a

similar pattern to that seen in a report by Tanaka et al. [3].

Changes in procollagen a1 (I) gene expression

during development of PBT larvae and early juveniles

We further examined the possibility for procollagen a1

(I) gene expression to act as an index of somatic growth in

PBT larvae and early juveniles. The dissociation curve of the

PCR products was a single peak, suggesting that the PCR

specifically amplified the target sequence. Figure 7 shows

the changes in procollagen a1 (I) gene expression during

PBT growth. The expression level gradually increased from

15 to 18 DAH and then rapidly increased during 18–25 DAH.

The level remained constant during 25–29 DAH and then

rapidly increased during 25–32 DAH again. When the cor-

relations between procollagen a1 (I) gene expression level

and protein content (9104 lg/fish), SL, and BW were

examined, the correlations were statistically significant: the

R2 were 0.90, 0.89, and 0.89, respectively (Fig. 8). These

results suggest that procollagen a1 (I) gene expression may

be a useful index for somatic growth.

Discussion

In this study, we cloned PBT procollagen a1 (I) cDNA.

From this, we were able to determine the amino acid

sequence of PBT procollagen a1 (I), which shares close

similarity to the human, skate, and rainbow trout sequences

(Fig. 2). The helical region of the PBT procollagen a1

(I) consists of 1,014 amino acid residues, which is the same

as other fibrillar collagen a chains from higher vertebrates.

The phylogenetic tree revealed that procollagen a1 (I) from

PBT is similar to rainbow trout, Japanese flounder, and

skate (Fig. 3a). When the phylogenetic analyses among

other vertebrate procollagen a chains were compared, the

PBT procollagen a1 (I) was the closest to tilapia procol-

lagen a1 (I) and not to the other class of a chains (Fig. 3b).

These results indicated that the cloned fragment was

derived from the PBT procollagen a1 chain.

Fig. 7 The relative gene expression pattern of procollagen a1 (I)/b-

actin in the PBT whole body as a function of somatic growth. Three

fish at each sampling point were randomly selected and analyzed.

Each data value is presented as the mean ± SD. Letters above the

points denote significant differences (P \ 0.05)

Fig. 8 The correlation of the relative gene expression pattern of

procollagen a1 (I)/b-actin and the a standard length (SL), b body

weight (BW), and c protein content (9104 lg/fish)

610 Fish Sci (2014) 80:603–612

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The thermal denaturation temperature of collagen is

related to the proline and hydroxyproline content [23].

Therefore, collagen thermal stability depends on the

amount of the Gly-Pro-Hyp motifs within a given protein

sequence. Because the Gly-Pro-Hyp motif occurs post-

translationally, the Gly-Pro-Pro content in the cDNA

sequence is a good estimation of the Gly-Pro-Hyp content

in the mature protein [24]. The number of Gly-Pro-Pro

motifs in the triple helical region of the PBT procollagen

a1 (I) was 30, while those of zebrafish, rainbow trout, and

torafugu are 31, 23, and 19, respectively. Therefore, PBT

procollagen a1 (I) has the second highest number of Gly-

Pro-Pro motifs, after zebrafish, among the previously

reported fish. PBT maintains a high body temperature, even

in ambient water temperatures, through the internalization

of aerobic locomotor muscles, higher aerobic capacities,

and elaboration of vasculature into countercurrent heat

exchangers [25]. Therefore, the high body temperature

might correlate with the higher number of Gly-Pro-Pro

motifs in PBT procollagen a1 (I), which is also seen in

zebrafish, a tropical, thermophilic fish [26]. Because Hyp

cannot be found in the cDNA sequence, we determined the

amino acid composition of the extracted PBT skin collagen

using an amino acid auto-analyzer. The Pro and Hyp

content in the PBT skin type I collagen was 12.9 and

7.9 %, respectively (data not shown); the ratio in PBT is

higher than in rainbow trout and skate, suggesting that the

high content of Gly-Pro-Pro in PBT procollagen a1

(I) contributes to its higher body temperature.

On the other hand, Gly-Gly sequences (Gly-Gly and

Gly-Gly-Gly) tend to be found in phylogenetically lower

classes of animals and frequently appear in frogs and

invertebrates, such as urchins and abalones [27]. Because

Gly is the smallest amino acid, the Gly-Gly sequence likely

contributes to the partial skew in the triple helix and the

decline in thermal stability. The number of Gly-Gly

sequences in the triple-helical regions of the PBT procol-

lagen a1 (I) was 14, while in zebrafish, rainbow trout, and

torafugu the number is 4, 22, and 11, respectively. Indeed,

the number of Gly-Gly sequences in PBT is fewer than the

number in rainbow trout, which is a freshwater fish that

lives in low-temperature water. In addition, two Ser resi-

dues (1,253 and 1,270) in the C-terminal region of the PBT

procollagen a1 (I) chain were replaced by Cys residues that

play a crucial role in the interaction of the procollagen achains [20]. This could indicate that PBT collagen might

easily accrue distortion in its protein structure. PBT have

very weak skin, which makes handling difficult for

researchers when rearing this species. One explanation for

such a weak skin in PBT is the primary and higher order

structure of the skin’s collagen.

This study also shows the mRNA expression in the PBT

skin, muscle, and liver. The highest expression of

procollagen a1 (I) was in the skin. These data are supported

by our recent study showing that roughly 80 % of the PBT

skin is type I collagen [28]. We also examined the pro-

collagen a1 (I) gene expression in the following four types

of muscle: o-toro is the white ventral muscle with the

highest fat content; akami is the red proximal muscle with

the lowest fat content; the color and lipid content of chu-

toro is between that of o-toro and akami; and chiai is the

dark red muscle with a low lipid content similar to akami

[29, 30]. The expression of collagen in the four types of

muscle may be correlated with the fat content. Adipocytes

induce hypertrophy through the incorporation of triglyc-

erides into the cell. The hypertrophic cell induces further

hypertrophy and cell division through activation of per-

oxisome proliferator-activated receptor c, which is a

ligand-activated transcription factor that belongs to the

nuclear hormone receptor superfamily [31]. We believe

procollagen a1 (I) gene expression levels reflect both fat

cell number and size because collagen fibers constitute the

extracellular matrix [8].

The SL and BW in PBT larvae and early juveniles

rapidly increased in this study during PBT development.

Similar somatic growth patterns have been reported in

previous studies for laboratory-reared PBT [3, 32]. The

RNA/DNA ratio is an indicator of somatic growth in fish

[33–35]. The rapid increase in the RNA/DNA ratio repor-

ted in the metamorphic period [3] was in agreement with

the gene expression results of procollagen a1 (I) in this

study.

There was a high correlation between procollagen a1

(I) gene expression and protein expression (9104 lg/fish),

SL, and BW. However, the total protein level was not

correlated with the relative expression of procollagen a1

(I): the R2 was 0.61 because the level of procollagen a1

(I) protein expression is low among the various PBT pro-

teins (lg/mg WW) [36]. This suggests that it may be a

useful index of somatic growth as a replacement for the

RNA/DNA ratio, although further nutritional and growth

examinations are still needed.

To summarize, we cloned the PBT procollagen a1

(I) cDNA and determined the resulting amino acid

sequence. The sequence was homologous with the pro-

collagen sequences of other teleosts. The expression of

procollagen a1 (I) is closely related to somatic growth in

PBT larvae and juveniles, suggesting that it may be a

useful indicator for somatic growth in fish larvae and

juveniles.

Acknowledgments The authors would like to thank the hatchery

staff at the Fish Nursery Centers, Kinki University, for their tre-

mendous technical support and advice. This work was supported by a

grant-in-aid for the Global COE program ‘‘International education

and research center for aquaculture science of bluefin tuna and other

fish’’ from the Ministry of Education, Culture, Sports, Science and

Fish Sci (2014) 80:603–612 611

123

Technology of Japan. The gene expression analysis was supported by

the JST/JICA SATREPS project on ‘‘Spawning ecology, nutritional

requirement, and early life history of yellowfin tuna’’.

References

1. Sawada Y, Okada T, Miyashita S, Murata O, Kumai H (2005)

Completion of the Pacific bluefin tuna Thunnus orientalis

(Temminck et Schlegel) life cycle. Aquac Res 36:413–421

2. Kaji T, Tanaka M, Takahashi Y, Oka M, Ishibashi N (1996)

Preliminary observations on development of Pacific bluefin Tuna

Thunnus thynnus (Scombridae) larvae reared in the laboratory,

with special reference to the digestive system. Mar Freshw Res

47:261–269

3. Tanaka Y, Gwak W-S, Tanaka M, Sawada Y, Okada T, Mi-

yashita S, Kumai H (2007) Ontogenetic changes in RNA, DNA

and protein contents of laboratory-reared Pacific bluefin tuna

Thunnus orientalis. Fish Sci 73:378–384

4. Miyashita S (2006) Surfacing and bottoming death in seeding

production. Nippon Suisan Gakkaishi 72:947–948

5. Ishibashi Y, Miki T, Sawada Y, Kurata M (2014) Effects of

feeding conditions and size differences on aggressive behaviour

and cannibalism in the Pacific bluefin tuna Thunnus orientalis

(Temminck and Schlegel) larvae. Aquac Res 45:45–53

6. Tanaka Y, Satoh K, Iwahashi M, Yamada H (2006) Growth

dependent recruitment of Pacific bluefin tuna Thunnus orientalis

in the northwestern Pacific Ocean. Mar Ecol Prog Ser

319:225–235

7. Ishibashi Y, Honryo T, Saida K, Hagiwara A, Miyashita S,

Sawada Y, Okada T, Kurata M (2009) Artificial lighting prevents

high night-time mortality of juvenile Pacific bluefin tuna, Thun-

nus orientalis, caused by poor scotopic vision. Aquaculture

293:157–163

8. Shoulders MD, Raines RT (2009) Collagen structure and stabil-

ity. Annu Rev Biochem 78:929–958

9. Gelse K, Poschl E, Aigner T (2003) Collagens—structure,

function, and biosynthesis. Adv Drug Deliv Rev 55:1531–1546

10. Ramshaw JA, Shah NK, Brodsky B (1998) Gly-X-Y tripeptide

frequencies in collagen: a context for host-guest triple-helical

peptides. J Struct Biol 122:86–91

11. Gordon MK, Hahn RA (2010) Collagens. Cell Tissue Res

339:247–257

12. Ghosh AK (2002) Factors involved in the regulation of type I

collagen gene expression: implication in fibrosis. Exp Biol Med

(Maywood) 227:301–314

13. Vogel WF (2001) Collagen-receptor signaling in health and

disease. Eur J Dermatol 11:506–514

14. Saito M, Takenouchi Y, Kunisaki N, Kimura S (2001) Complete

primary structure of rainbow trout type I collagen consisting of

a1(I)a2(I)a3(I) heterotrimers. Eur J Biochem 268:2817–2827

15. Hwang JH, Yokoyama Y, Mizuta S, Yoshinaka R (2006) cDNA

cloning and characterization of type I procollagen alpha1 chain in

the skate Raja kenojei. Comp Biochem Physiol B: Biochem Mol

Biol 144:1–10

16. Suzuki T, Srivastava AS, Hashimoto H, Kurokawa T (2006)

Structural comparison of promoter and coding sequence of type I

collagen alpha 1 chain gene duplicates between zebrafish and

flounder/fugu lineages. Comp Biochem Physiol Part D Genomics

Proteomics 1:20–27

17. Saitou S, Nei M (1987) The neighbor-joining method: a new

method for reconstructing phylogenetic trees. Mol Biol Evol

4:406–425

18. Adachi K, Kato K, Yamamoto M, Ishimaru K, Kobayashi T,

Murata O, Kumai H (2008) Pulsed expression of growth hormone

mRNA in the pituitary of juvenile Pacific bluefin tuna under

aquaculture conditions. Aquaculture 281:158–161

19. Vuorio E, Crombrugghe B (1990) The family of collagen genes.

Annu Rev Biochem 59:837–872

20. Dion AS, Myers JC (1987) COOH-terminal propeptides of the

major human procollagens: structural, functional and genetic

comparisons. J Mol Biol 193:127–143

21. Ehrlich KF (1974) Chemical changes during growth and starva-

tion of larval Pleuronectes platessa. Mar Biol 24:39–48

22. Ehrlich KF (1974) Chemical changes during growth and starva-

tion of herring larvae. In: Blaxter JHS (ed) The early life history

of fish. Springer-Verlag, Berlin, pp 301–323

23. Wong DWS (1989) Mechanism and theory in food chemistry.

Van Nostrand Reinhold, New York

24. Gaill F, Mann K, Wiedemann H, Engel J, Timpl R (1995)

Structural comparison of cuticle and interstitial collagens from

annelids living in shallow seawater and at deep-sea hydrothermal

vents. J Mol Biol 246:284–294

25. Kitagawa T, Kimura S, Nakata H, Yamada H (2006) Thermal

adaptation of Pacific bluefin tuna Thunnus orientalis to temperate

waters. Fish Sci 72:149–156

26. Gerhard GS, Kauffman EJ, Wang X, Stewart R, Moore JL, Ka-

sales CJ, Demidenko E, Cheng KC (2002) Life spans and

senescent phenotypes in two strains of Zebrafish (Danio rerio).

Exp Gerontol 37:1055–1068

27. Kimura S (2002) Structure and origin of fish type I collagen.

Nippon Suisan Gakkaishi 68:637–645

28. Tanaka T, Takahashi K, Iwamoto N, Agawa Y, Sawada Y, Yo-

shimura Y, Zaima N, Moriyama T, Kawamura Y (2012) Hepa-

toprotective action of dietary bluefin tuna skin proteins on CCl4-

intoxicated mice. Fish Sci 78:911–921

29. Roy BC, Miyake Y, Ando M, Kawasaki K-I, Tsukamasa Y

(2010) Proximate and fatty acid compositions in different flesh

cuts of cultured, cultured fasted, and wild pacific bluefin tuna

(Thunnus orientalis). J Aquat Food Prod Technol 19:284–297

30. Agawa Y, Honryo T, Ishii A, Kobayashi T, Oku H, Sawada Y

(2012) Molecular identification and tissue distribution of perox-

isome proliferators activated receptor gamma transcript in cul-

tured Thunnus orientalis. Aquac Res 43:1145–1158

31. Yamauchi T, Kamon J, Waki H, Murakami K, Motojima K,

Komeda K, Ide T, Kubota N, Terauchi Y, Tobe K, Miki H,

Tsuchida A, Akanuma Y, Nagai R, Kimura S, Kadowaki T

(2001) The mechanisms by which both heterozygous peroxisome

proliferator-activated receptor gamma (PPARgamma) deficiency

and PPARgamma agonist improve insulin resistance. J Biol

Chem 276:41245–41254

32. Miyashita S, Sawada Y, Okada T, Murata O, Kumai H (2001)

Morphological development and growth of laboratory-reared

larval and juvenile Thunnus thynnus (Pisces: Scombridae). Fish

Bull (Wash.D.C.) 99:601–616

33. Bulow FJ (1970) RNA–DNA ratios as indicators of recent growth

rates of a fish. J Fish Res Board Can 27:2343–2349

34. Chıcharo MA, Chıcharo LL (2008) RNA:DNA ratio and other

nucleic acid derived indices in marine ecology. Int J Mol Sci

9:1453–1471

35. Takii K, Seoka M, Takaoka O, Furuta S, Nakamura M, Kumai H

(1994) Chemical composition, RNA and DNA contents, and

alkaline phosphatase activity with growth of striped jack larvae

through juveniles. Fish Sci 60:73–76

36. Yoshinaka R, Sato K, Anbe H, Sato M, Shimizu Y (1988) Dis-

tribution of collagen in body muscle of fishes with different

swimming modes. Comp Biochem Physiol 89B:147–151

612 Fish Sci (2014) 80:603–612

123