Marine Biotechnology

, Volume 13, Issue 2, pp 151–162

Identification and Analysis of Muscle-Related Protein Isoforms Expressed in the White Muscle of the Mandarin Fish (Siniperca chuatsi)

Authors

  • Guoqiang Zhang
    • Key Laboratory of Genome Information and Sciences, Beijing Institute of GenomicsChinese Academy of Sciences
    • Graduated University of the Chinese Academy of Sciences
  • Wuying Chu
    • Department of Bioengineering & Environmental ScienceChangsha University
    • Key Laboratory of Genome Information and Sciences, Beijing Institute of GenomicsChinese Academy of Sciences
  • Tao Meng
    • Department of Bioengineering & Environmental ScienceChangsha University
  • Linlin Pan
    • Key Laboratory of Genome Information and Sciences, Beijing Institute of GenomicsChinese Academy of Sciences
  • Renxue Zhou
    • Department of Bioengineering & Environmental ScienceChangsha University
  • Zhen Liu
    • Department of Bioengineering & Environmental ScienceChangsha University
    • Department of Bioengineering & Environmental ScienceChangsha University
Original Article

DOI: 10.1007/s10126-010-9275-1

Cite this article as:
Zhang, G., Chu, W., Hu, S. et al. Mar Biotechnol (2011) 13: 151. doi:10.1007/s10126-010-9275-1

Abstract

To identify muscle-related protein isoforms expressed in the white muscle of the mandarin fish Siniperca chuatsi, we analyzed 5,063 high-quality expressed sequence tags (ESTs) from white muscle cDNA library and predicted the integrity of the clusters annotated to these genes and the physiochemical properties of the putative polypeptides with full length. Up to about 33% of total ESTs were annotated to muscle-related proteins: myosin, actin, tropomyosin/troponin complex, parvalbumin, and Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCa). Thirty-two isoforms were identified and more than one isoform existed in each of these proteins. Among these isoforms, 14 putative polypeptides were with full length. In addition, about 2% of total ESTs were significantly homologous to “glue” molecules such as alpha-actinins, myosin-binding proteins, myomesin, tropomodulin, cofilin, profilin, twinfilins, coronin-1, and nebulin, which were required for the integrity and maintenance of the muscle sarcomere. The results demonstrated that multiple isoforms of major muscle-related proteins were expressed in S. chuatsi white muscle. The analysis on these isoforms and other proteins sequences will greatly aid our systematic understanding of the high flexibility of mandarin fish white muscle at molecular level and expand the utility of fish systems as models for the muscle genetic control and function.

Keywords

Mandarin fishESTsWhite muscleMuscle proteinIsoforms

Introduction

Fish skeletal muscles consist of two spatially separated fibers. White, fast-twitch muscles make up the bulk of the body of fish, whereas red, while slow-twitch fibers are found in a narrow midlateral band immediately under the skin (McGuigan et al. 2004). Many proteins are specifically expressed in muscle tissues, and these mainly include structure and contractile proteins (e.g., myosin, actins, and troponin) as well as soluble muscle proteins and muscle-specific regulating factors (Rodgers et al. 1987; Xu et al. 2000; Grimaldi et al. 2004).Various swimming gaits of fish depend on the different physiological properties and recruitment of the various myotomal muscle fiber types. White, fast-twitch muscles are recruited for faster swimming speeds (Maddock et al. 1994; Coughlin et al. 2005), whereas red, slow-twitch muscle for low undulatory swimming speeds (Rome et al. 1984). Variances in the physiological and biochemical properties of different muscle fiber types, responsible for the diverse contraction and relaxation of muscles, are attributable to different isoforms of muscle proteins in the muscle tissues (Berchtold et al. 2000).

The mandarin fish, popular in northeastern Asian countries, is carnivorous and naturally lives in many eastern Asian areas due to its broad temperature tolerance range. When the temperature rises above 5°C, mandarin fish begins to prey moving targets swiftly. The swiftness of the mandarin fish under broad temperature range supposedly results from different isoforms of muscle proteins, especially those in white muscles that make up the bulk of the mandarin fish's body (Rome et al. 1984; Maddock et al. 1994; Berchtold et al. 2000; Coughlin et al. 2005). Even though much work on muscle-related proteins sequences or genes is available (Guo et al. 1994; Imai et al. 1997; Hirayama et al. 1998; Xu et al. 2000; Weaver et al. 2001; Bryson-Richardson et al. 2005; Fukushima et al. 2009; Wang et al. 2008b), more detail sequence information on muscle-related proteins appears to be important for the better understanding of mandarin fish's broad adaptive capacity to diverse conditions.

EST sequencing is one of the most efficient means for gene discovery and molecular marker development, and it has been widely applied in fish biology to discover novel, uniquely expressed genes, and characterize expression profiles of distinctly expressed genes under different stimuli (Reusch et al. 2008; Wynne et al. 2008), in various organs (Li et al. 2007) and in genetic mapping and comparative genome analysis by identifying polymorphic DNA markers, such as microsatellites (Kucuktas et al. 2009) and single nucleotide polymorphisms (Sarropoulou et al. 2008; von Schalburg et al. 2008; Wang et al. 2008a; Kucuktas et al. 2009). Compared with the conventional approaches of identifying protein isoforms, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunological blotting, EST sequencing can quickly and inexpensively provide the expression profiles and sequences of different muscle-related protein isoforms on large scale (Li et al. 2007). The primary goal of this study is to identify the major isoforms of muscle-related protein expressed in the mandarin fish white muscle and compare their physiological and biochemical properties with others. Therefore, we analyzed 5,063 high-quality ESTs from the mandarin fish S. chuatsi white muscle cDNA library and predicted the integrity and the preliminary properties of the derived peptides from ESTs annotated to muscle-related genes. This first comprehensive identification of the main isoforms of muscle-related proteins should greatly deepen our understanding of the highly functional plasticity of fish muscle at molecule level, in response to varying functional demands and environmental changes.

Materials and Methods

cDNA Library Construction and EST Sequencing

Total RNA was extracted from the S. chuatsi muscle samples using Unizol reagent (Biostar, Shanghai, China). Poly(A)+mRNA was isolated from total RNA using Oligotex mRNA mini kit (Qiagen). Then poly(T)-primed cDNA was synthesized and directionally inserted into the pBluescript_II_SK(+) vector. DNA was transformed by electroporation into the Escherichia coli strain DH10B. The unnormalized library contained an average insert size of 1.7 kb. Using DYEnamic ET Terminator (GE Healthcare) with a T3 primer (5′-AATTAACCCTCACTAAAG-3′), 5,456 single-pass sequencing reactions from 5′ end of each cDNA clones were carried out. The sequencing was performed on a 3730XL DNA analyzer (Applied Biosystems, Foster City, CA).

Data Processing, Cluster Assembly, and Bioinformatics Analysis

We processed the chromatogram files sequencing from 5′ end as raw data for base-calling and quality assessing by Phred software (Phred-Phrap-Consed package). The low-quality sequences were trimmed off with Q13 (95% accuracy), and the vector sequences were screened with CROSS_MATCH program. All high-quality ESTs (sequences more than 100 bp after quality and vector trimming) were assembled by CAP3 software using default settings (overlap length cut-off >30 and overlap percent identity cut-off >75) (Huang and Madan 1999). Assembled clusters were manually revised by Consed software (Gordon et al. 1998) and then searched against UniProtKB/Swiss-Prot protein database in by BLASTX with e values 1e-5.

Clusters were considered as full length if they contained a complete ORF (open-reading frame), identified by BLASTX and start and stop codons as well as poly(A) tail (Min et al. 2005b). For skeletal muscle-specific or enriched transcripts without complete coding regions, appropriate clones were sequenced from 3′ ends again. Clones were subjected to further sequencing by primer walking to get the full insert of the clones after both 5′ and 3′ EST sequencing. All the sequences including 5′ and 3′ ESTs and extended sequences were processed and were finally assembled again using CAP3 as above.

Protein Analysis

Putative peptides were conceptually translated with OrfPredictor (Min et al. 2005a) and verified by searching against the protein database with BLSATP. Multiple sequence alignments were carried out using MUSCLE (Edgar 2004) or CLUSTAL_X (Larkin et al. 2007). The physiochemical properties of proteins were analyzed using ExPASy webserver.

Results and Discussions

Overview of ESTs from the S. chuatsi Skeletal Muscle cDNA Library

We sequenced at the 5′-terminus of each insert on 5,456 randomly selected clones from a cDNA library of the mandarin fish white muscle. After discarding the poor-quality and vector sequences, 5,191 readable sequences were produced, and 5,063 sequences were longer than 100 base pairs. The average length of these ESTs was 587 bp (Fig. 1). All the ESTs were deposited to dbEST [GenBank: GR473858–GR479048]. Subsequently, all high-quality 5′ ESTs were assembled into 1,691 clusters: 284 contigs (contigs refer to clusters consisting of more than one EST) and 1,407 singletons (singletons refer to clusters consisting of just one EST).
https://static-content.springer.com/image/art%3A10.1007%2Fs10126-010-9275-1/MediaObjects/10126_2010_9275_Fig1_HTML.gif
Fig. 1

Length distribution of 5′ ESTs from the Siniperca chuatsi skeletal muscle cDNA library. A total of 5,063 high quality 5′ ESTs were analyzed in the current study. Abscissa is the sequence length in 50-bp intervals, while the ordinate is the number of ESTs

After annotating the ESTs, clusters significantly matched to myofibrillar proteins, including myosin, actin, tropomyosin, and troponin complex, and they were abundant accounting about 24% of total ESTs (Fig. 2). More than one isoform existed in each of these myofibrillar proteins, and up to 25 isoforms were identified in these myofibrillar proteins, and eight of them were with complete ORFs (Table 1), as detailed later. Transcripts coding for proteins related to calcium binding/transporting such as parvalbumin and sarco/endoplasmic reticulum (SR) calcium transport ATPase (SERCa) were also rich, which accounted about 9% of total ESTs. All five isoforms identified in parvalbumin were with complete ORFs. Two isoforms were identified in SERCa, and one of them was with complete ORF (Table 1). In addition, other calcium-binding proteins such as calsequestrin, which helps hold calcium in the cisterna of the sarcoplasmic (Wang et al. 1998), were also identified.
https://static-content.springer.com/image/art%3A10.1007%2Fs10126-010-9275-1/MediaObjects/10126_2010_9275_Fig2_HTML.gif
Fig. 2

Functional annotation of ESTs annotated to muscle-related genes. The relative proportion of different types of transcripts: myosin (further divided into light chain, and heavy chain), actin, troponin (further divided into troponin C, I, and T), tropomyosin, parvalbumin, SERCa, other muscle proteins, other Swiss-Prot annotation, and no Swiss-Prot annotation

Table 1

Transcriptional isoforms of mandarin fish muscle filament proteins

Protein

Abbreviation

CDS

Length of propeptide

Clones

pI

Mw (Da)

Best hit

% identity

Organism of best hit

MYL

MYL1_SC

Complete

192

37

4.62

20,752.5

MLE1_MUGCA

94

Liza ramada

MYL2_SC

Partial, lack C-terminal

120

7

MLRT_RABIT

83

Rabbit

MYL3_SC

Complete

150

257

4.42

16,652.82

|MLE3_MUGCA

95

Liza ramada

MYL4_SC

Partial, lack N-terminal

116

5

MYL6_RAT

82

Norway rat

MYL5_SC

Partial, with N-terminal

99

8

MYL9_PONAB

92

Sumatran orangutan

MYH

MYHhead1_SC

Partial, with N-terminal

932

46

MYSS_CYPCA

86

Common carp

MYHhead2_SC

Partial

192

2

MYSS_CYPCA

90

Common carp

MYHhead3_SC

Partial

433

1

MYSS_CYPCA

87

Common carp

MYHtail1_SC

Partial, with C-terminal

772

52

MYSS_CYPCA

84

Common carp

MYHtail2_SC

Partial

606

4

MYSS_CYPCA

83

Common carp

MYHtail3_SC

Partial

190

2

MYSS_CYPCA

78

Common carp

Actin

ACT1_SC

Complete

377

345

5.22

41,944.89

ACTS_OREMO

100

Mozambique tilapia

ACT2_SC

Partial, with N-terminal

190

3

ACTB_SALSA

99

Atlantic salmon

ACT3_SC

Partial, with N-terminal

107

2

ACTB_XENTR

100

Western clawed frog

Tropomyosin

TPM1_SC

Complete

284

75

4.69

32,683.62

TPM1_LIZAU

94

Golden gray mullet

TPM2_SC

Partial, lack N-terminal

150

19

TPM1_LIZAU

97

Golden gray mullet

TPM3_SC

Partial

132

1

TPM3_MOUSE

83

House mouse

Troponin T

TNNT1_SC

Complete

228

69

4.64

21,775.22

TNNT3_COTJA

49

Japanese quail

TNNT2_SC

partial, with N-terminal

188

2

TNNT3_RAT

48

Norway rat

Troponin I

TNNI1_SC

Complete

171

81

9.27

19,243.47

TNNI2_COTJA

54

Japanese quail

TNNI2_SC

Partial, lack N-terminal

154

2

TNNI2_COTJA

50

Japanese quail

TNNI3_SC

Partial, lack N-terminal

131

2

TNNI2_COTJA

57

Japanese quail

TNNI4_SC

Partial, lack C-terminal

175

1

TNNI1_HUMAN

72

Human

Troponin C

TNNC1_SC

Complete

160

84

3.96

18,188.1

TNNC2_ANGAN

79

European eel

TNNC2_SC

Complete

161

2

3.91

18,210.01

TNNC3_ANGAN

86

European eel

Parvalbumin

PRV1_SC

Complete

109

235

4.6

11,589.16

PRVA_CYPCA

75

Common carp

PRV2_SC

Complete

109

157

4.65

11,466.9

PRVB_SCOJP

85

Chub mackerel

PRV3_SC

Complete

109

15

4.46

11,495.91

PRVB_SCOJP

80

Chub mackerel

PRV4_SC

Complete

109

1

4.6

11,617.22

PRVA_CYPCA

74

Common carp

PRV5_SC

Complete

108

1

4.58

11,957.62

PRV7_DANRE

67

Zebrafish

SERCa

SERCa1_SC

Complete

957

30

5.09

10,4827.12

AT2A1_MAKNI

90

Atlantic blue marlin

SERCa2_SC

Partial, with N-terminal

353

9

AT2A2_MAKNI

82

Atlantic blue marlin

The abbreviations of mandarin fish muscle filament protein transcriptional isoforms, the completeness of their coding sequences (CDS), their lengths of the putatively deduced polypeptides, the number of clones comprising the isoforms, the predicted pI and Mw, and their best BLAST hits in Swiss-Prot database as well as the identity and the common names of the organism of best hits are shown

The integrity and maintenance of the muscle sarcomere require a complex interplay of so-called “glue” molecules that bind myosin/actin and link various filaments together or anchor filaments to membrane. Transcripts homologous to such “glue” molecules, up to 2% of total ESTs, also existed in S. chuatsi (Table 2). BLASTX searches against Swiss-Prot showed that 13 ESTs significantly matched to myosin-binding proteins, 3 matches to myomesin (another myosin-binding protein), and 10 ESTs homologous to alpha-actinin that cross-links actin filaments and titin molecules (Nwe et al. 1999; Atkinson et al. 2000). Other proteins regulating the length of actin filament were also identified, such as myozenin, tropomodulin, cofilin, profilin, twinfilins, coronin-1, and nebulin (Amali et al. 2004; Campinho et al. 2005).
Table 2

Functional distribution of the 5′ ESTs annotated to other muscle-related genes based on Swiss-Prot matches by BLASTX

Query name

EST counts

Length

Accession No.

Identity

Description

Contig65

3

715

sp|P20111|ACTN2_CHICK

86

Alpha-actinin-2

Contig187

6

1,532

sp|Q0III9|ACTN3_BOVIN

88

Alpha-actinin-3

gy_0035_D06.ab1

1

553

sp|Q0III9|ACTN3_BOVIN

76

Alpha-actinin-3

gy_0011_A02.ab1

1

627

sp|P31231|CASQ1_RANES

50

Calsequestrin-1

Contig209

2

798

sp|Q6B7M7|COF1_SHEEP

60

Cofilin-1

Contig84

2

644

sp|Q5G6V9|COF2_PIG

75

Cofilin-2

gy_0056_G02.ab1

1

667

sp|Q5G6V9|COF2_PIG

93

Cofilin-2

gy_0042_C10.ab1

1

521

sp|Q9XS70|COR1B_RABIT

57

Coronin-1B

Contig170

5

1,210

sp|Q9D119|DYSI1_MOUSE

68

Dysferlin-interacting protein 1

Contig255

8

1,322

sp|Q9D119|DYSI1_MOUSE

73

Dysferlin-interacting protein 1

gy_0057_F05.ab1

1

619

sp|Q9D2N4|DTNA_MOUSE

83

Dystrobrevin alpha

gy_0033_C02.ab1

1

486

sp|Q14315|FLNC_HUMAN

87

Filamin-C

Contig94

2

925

sp|P18520|ION3_CARAU

81

Intermediate filament protein ON3

gy_0019_F09.ab1

1

627

sp|Q99JW4|LIMS1_MOUSE

84

LIM and senescent cell antigen-like-containing domain protein 1

gy_0028_B10.ab1

1

629

sp|Q8QFP8|LIMK1_CHICK

72

LIM domain kinase 1

gy_0032_A08.ab1

1

548

sp|P53668|LIMK1_MOUSE

83

LIM domain kinase 1

gy_0008_C02.ab1

1

599

sp|P52179|MYOM1_HUMAN

60

Myomesin-1

gy_0006_C07.ab1

1

710

sp|Q62234|MYOM1_MOUSE

66

Myomesin-1

gy_0038_G04.ab1

1

600

sp|P54296|MYOM2_HUMAN

50

Myomesin-2

gy_0007_F08.ab1

1

675

sp|Q5XKE0|MYPC2_MOUSE

64

Myosin-binding protein C, fast-type

Contig158

7

832

sp|Q05623|MYBPH_CHICK

53

Myosin-binding protein H

Contig224

5

1,214

sp|Q05623|MYBPH_CHICK

63

Myosin-binding protein H

gy_0021_F01.ab1

1

681

sp|Q27966|MYO1C_BOVIN

63

Myosin-Ic

gy_0049_F01.ab1

1

281

sp|Q9UBF9|MYOTI_HUMAN

56

Myotilin

gy_0011_H12.ab1

1

613

sp|Q9Y217|MTMR6_HUMAN

72

Myotubularin-related protein 6

gy_0013_A12.ab1

1

557

sp|Q9Y216|MTMR7_HUMAN

62

Myotubularin-related protein 7

Contig101

5

1,681

sp|Q4PS85|MYOZ1_PIG

57

Myozenin-1

Contig184

2

794

sp|P20929|NEBU_HUMAN

57

Nebulin

gy_0037_B06.ab1

1

674

sp|P20929|NEBU_HUMAN

45

Nebulin

gy_0048_C05.ab1

1

667

sp|Q86VF7|NRAP_HUMAN

56

Nebulin-related-anchoring protein

gy_0041_E07.ab1

1

646

sp|Q62920|PDLI5_RAT

62

PDZ and LIM domain protein 5

Contig247

9

1,097

sp|Q3SX40|PDLI7_BOVIN

45

PDZ and LIM domain protein 7

gy_0008_E05.ab1

1

611

sp|Q6P7E4|PDLI7_DANRE

63

PDZ and LIM domain protein 7

gy_0014_H04.ab1

1

701

sp|Q15149|PLEC1_HUMAN

69

Plectin-1

gy_0021_C11.ab1

1

484

sp|Q9QXS1|PLEC1_MOUSE

55

Plectin-1

gy_0045_H06.ab1

1

646

sp|Q32PB1|PROF3_BOVIN

35

Profilin-3

Contig174

6

1,359

sp|Q6T8D8|TELT_BOVIN

41

Telethonin

gy_0048_A08.ab1

1

650

sp|Q8CGB6|TENC1_MOUSE

41

Tensin-like C1 domain-containing phosphatase

Contig58

2

834

sp|Q8WZ42|TITIN_HUMAN

57

Titin

gy_0025_C09.ab1

1

665

sp|Q8WZ42|TITIN_HUMAN

65

Titin

gy_0026_D03.ab1

1

608

sp|Q8WZ42|TITIN_HUMAN

74

Titin

gy_0007_G12.ab1

1

635

sp|A2ASS6|TITIN_MOUSE

58

Titin

gy_0008_H12.ab1

1

657

sp|A2ASS6|TITIN_MOUSE

67

Titin

gy_0011_G11.ab1

1

641

sp|A2ASS6|TITIN_MOUSE

64

Titin

gy_0039_D09.ab1

1

595

sp|A2ASS6|TITIN_MOUSE

63

Titin

Contig64

5

1,479

sp|A0JNC0|TMOD1_BOVIN

61

Tropomodulin-1

Contig114

2

763

sp|Q6P9T8|TBB2C_RAT

87

Tubulin beta-2C chain

gy_0024_B12.ab1

1

662

sp|Q6GMH3|TWF2_DANRE

80

Twinfilin-2

The clusters annotated to other muscle-related genes, the numbers of ESTs, their length, and the accession no. of their best BLAST hits in Swiss-Prot database as well as the identity and the description of the best hits are shown

Identification and Analysis of Transcriptional Isoforms of the Major Myofibrillar Proteins

Myosin Structure

Conventional myosin contains two heavy chains (MYH) and four light chains (MYL) (Rodgers et al. 1987). The C-terminal of each MYH, which defines the maximal shortening velocity of muscle (Moss et al. 1996), folds into α-helical coiled-coil structure and joins two MYHs together (Weeds and McLachlan 1974). In contrast, its N-terminal is composed of two regions, the head domain that binds the filamentous actin and hydrolyzes ATP to generate force to “walk” along the filament, and the neck domain that acts as a linker binding two myosin light chains together and as a lever arm for transducing force generated by the head domain (Ruegg et al. 2002). Prior to this study, there were five myosin sequences of mandarin fish S. knerii skeletal muscle MYLs (myosin light chains) deposited in GenBank: four myosin light chain sequences (Zhang et al., unpublished): MYL1_SKr (gb|ACJ47229.1), MYL3_SKr (gb|ACJ12595.1), MYL3b_SKr (gb|ACJ47231.1), and MYL2_SKr (gb|ACJ47230.1), and one myosin heavy chain sequence MYH_SKr (gb|ABO31103.1) (Zhang et al. 2009a, b).

Myosin Light Chain

Five clusters were homologous to myosin light chains or polypeptides (MYLs) (Table 1). Two clusters contained complete open reading frames. One consisting of 37 ESTs that encoded a polypeptide of 192 amino acids (aa) and was designated MYL1_SC, which had the same sequence as MYL1_SKr. Another of 257 ESTs, designated MYL3_SC, encoded a polypeptide of 150 amino acids and was also identical to MYL3_SKr. The rest 3 clusters contained incomplete open reading frames. Thus, there appears to be at least five MYL isoforms in the mandarin fish skeletal muscles. Previous studies demonstrated that two essential light chain isoforms (MYL1 and MYL3) in birds and mammals were produced from a single gene by alternative splicing (Periasamy et al. 1984). In this study, MYL1_SC and MYL3_SC differed in many variances that scattered in the whole sequence and appeared to be produced by two different genes, consistent with other findings on MYL1 and MYL3 in other fishes (Dalla Libera et al. 1991). The MYL3_SC isoform, the major isoform, was 150 aa in length, which would lead to a polypeptide of 16.65 kDa with a theoretical isoelectric point (pI) of 4.42. As with other vertebrates, this MYL lacked the Pro-Ala-rich N-terminal extension of the long vertebrate essential light chain isoform. Comparative sequence analysis revealed that MYL3_SC shared 95% identity with MLE3_MUGCA, myosin light chain 3 of Liza ramada Liza ramado skeletal muscle isoform (P82160).

MYL1_SC isoform, the minor isoform, was 192 amino acids in length resulting in a polypeptide of 20.75 kDa with a theoretical pI of 4.62. In comparison with MYL3_SC, MYL1_SC isoform had an N-terminal extension of 41 aa and showed 94% identity with MLE1_MUGCA, myosin light chain 1 of Liza ramada L. ramado skeletal muscle isoform (P82159.1). This N-terminal extension consisted of a repetitive Ala-Pro-rich segment (20 aa) and a highly charged interactive “sticky” element (residues 1–15) at the most N-terminus (Hayashibara and Miyanishi 1994; Timson et al. 1999), which was bound to a cluster of C-terminal acidic residues 360–364 of actin (Trayer et al. 1987; Hirayama et al. 2000).

It was observed that the discrepant amino acids were scattered in the inter regions of two EF-hand region; the conserved amino acids were concentrated in EF-hand regions, and EF-hand II was more conserved than EF-hand I (Fig. 1A, Supplemental File). Essential light chain belongs to the EF-hand family of calcium-binding proteins. Therefore, EF-hand II was supposed to be the main calcium-binding sites, at least in MYL1_SC, because it was highly conservative and remote from the repetitive Ala-Pro-rich segment in MYL1_SC (Huriaux and Focant 1977).

Myosin Heavy Chain

The conventional myosin heavy chain (MYH) generally has a MW of about 220 kDa encoding more than 1,900 aa. It can be subdivided into three fragments: S1 (or subfragment 1, the head), S2 (subfragment 2, the proximal one third of the tail), and light meromyosin (LMM, the distal two thirds of the tail) (Kikuchi et al. 1999). Six clusters were annotated to different parts of the common carp Cyprinus carpio skeletal muscle isoform of myosin heavy chain (MYSS_CYPCA: Q90339) (Fig. 3). Three clusters were homologous to the head of MYSS_CYPCA and other three clusters to the tail of MYSS_CYPCA. So far, one cluster encoding complete globular head region and another complete LMM fragment were obtained, although none of them were complete MYH.
https://static-content.springer.com/image/art%3A10.1007%2Fs10126-010-9275-1/MediaObjects/10126_2010_9275_Fig3_HTML.gif
Fig. 3

Schematic of the mandarin fish myosin heavy chain fragments. The mandarin fish myosin heavy chain fragments obtained from EST assembly are placed immediately below the relevant portions of the linear map of Cyprinus carpio fast skeletal myosin heavy chain. The numbers under each myosin heavy chain fragment indicate the locations of the start and end positions of its best match in Cyprinus carpio fast skeletal myosin heavy chain. The identity between each myosin II heavy chain fragment and Cyprinus carpio fast skeletal myosin heavy chain is shown in parentheses. Abbreviations: Q90339|MYSS_CYPCA, Cyprinus carpio fast skeletal myosin heavy chain; S1, myosin subfragment 1; S2, myosin subfragment 2; LMM, L-meromyosin

Three clusters matched the head region of MYH. The largest was composed of 46 ESTs and designated as MYHHead1_SC. It deduced a 932 aa of MYSS_CYPCA and possessed the start codon and 5′ UTR. Another cluster deduced a 192 aa polypeptide and was designated as MYHHead2_SC. The other deduced a 433 aa polypeptide and was designated as MYHHead3_SC. All these three clusters showed highest homology to MYSS_CYPCA, with 86% identity for MYHHead1_SC, 90% for MYHHead2_SC, and 87% for MYHHead3_SC (Table 1).

In the multi-sequences alignment, MYHHead1_SC was with the same sequence as the MYHHead_SKr (Zhang et al., unpublished) and MYHHead3_SC was more different to MYHHead1_SC from MYHHead2_SC to MYHHead1_SC. The different amino acids were scarcely located in the ATP-binding and actin-binding domain (Fig. 2, Supplemental File), which produce force and transduces it along the filament (Ruegg et al. 2002).They were concentrated in the less-conserved regions such as the Loop I region, which was near the nucleotide-binding pocket and likely to tune the rate constant for ADP release (Goodson et al. 1999).

Three clusters significantly matched different tail regions of MYSS_CYPCA. The most abundant transcript was composed of 52 ESTs and designated as MYHTail1_SC. It deduced a 772 aa polypeptide sharing 84% identity with the C-terminal of MYSS_CYPCA and possessed the stop codon and 3′ UTR. Other two clusters encoded the polypeptides of 606 aa and 190 aa long, and they were designated as MYHTail2_SC and MYHTail3_SC, respectively. MYHTail2_SC shared 83% identity with the counterparts of MYSS_CYPCA and MYHTail2_SC 78% identity.

In alignment with MYHTail_SKr, MYHTail1_SC differed six amino acids, and two of them (T507R and N509Q) located in the S2 region, the remaining four existed in the LMM region. MYHTail2_SC had the same replacement in the S2 region of MYHTail_SKr and the third different amino acid (A792T) occurred in LMM region (Fig. 3, Supplement file). With respect to MYHTail3_SC, there were apparent differences in the counterparts of other isoforms (Fig. 4, Supplement File). These differences in LMM region over mandarin fish MYH isoforms may be responsible for its thermo-stability as indicated by other studies (Kakinuma et al. 2000a).

Actin

Actins are highly conserved proteins that are involved in various types of cell motility and ubiquitously expressed in all eukaryotic cells. Three main groups of actins isoforms (alpha, beta, and gamma) exist in vertebrates. The alpha actin exists in muscle tissues and is a major constituent of the contractile apparatus, while the beta and gamma actins coexist in most cell types as components of the cytoskeleton and as mediators of internal cell motility (Venkatesh et al. 1996). There were three clusters significantly matching to actins. The major cluster, designated as ACT1_SC, included 345 ESTs and possessed the start codon, 5′ and 3′ UTR. ACT1_SC encoded a 377 aa-long polypeptide, which was identical to actin, alpha skeletal muscle of Mozambique tilapia Oreochromis mossambicus (ACTS_OREMO: P68264). The cluster of three ESTs, designated as ACT2_SC, deduced a polypeptide of 190 aa sharing 99% identity with the N-terminal of Atlantic salmon Salmo salar cytoplasmic actin (ACTB_SALSA: O4216). The cluster of two ESTs deduced the polypeptide identical to a 107 aa-long region in the C-terminal of western clawed frog Xenopus (Silurana) tropicalis cytoplasmic actin 1 (ACTB_XENTR: Q6NVA9) and was designated as ACT2_SC. The results suggested that in S. chuatsi white muscle, ACT1_SC is the muscle isoform, and ACT2_SC and ACT3_SC are cytoplasmic isoforms.

The binding sites of ATP (Ser16, Asp156, His163, Glu216, Gly304, and Lys338), gelsolin (Tyr145, Gly148, Glu169, Gly344, Ile347, Leu348, Leu351, Ser352, and Thr353), and profilin (Tyr168, Glu169, Tyr171, Leu173, His175, Asp288, Asp290, Thr353, Glu363, Arg374, and Lys375) to actin were identical in both of the mandarin fish actin isoforms compared with other species (Fig. 5, Supplement File). Profilin enhances actin filament growth, while gelsolin severes actin filament (Okano et al. 1998; Witke et al. 1998). These conserved positions were essential for the dynamics of actin filament.

Tropomyosin/Troponin Complex

Tropomyosin/troponin complex play a central role in the calcium-dependent regulation of vertebrate striated muscle contraction (Kakinuma et al. 2000b; Paul et al. 2009). In striated muscle cells, tropomyosin wraps itself around actin filaments and interacts with troponin through troponin T subunit. Troponin consists of three components: troponin I, which inhibits ATPase activity of actomyosin; troponin T, which contains the binding site for tropomyosin; and troponin C, which binds calcium to abolish the inhibitory action of troponin on actin filaments (Murakami et al. 2007). Different isoforms of each component in tropomyosin/troponin complex can modulate sarcomeric performance by either increasing or decreasing Ca2+ sensitivity that regulates muscle contraction and relaxation (Gillis et al. 2007).

Tropomyosin

Tropomyosin (TPM) widely distributes in eukaryotes and associates with actin. Existence of tropomyosin isoforms provides a powerful mechanism to accommodate the diverse functions associated with actin filaments in many cell types. In vertebrates, four genes encoded more than 20 tropomyosin isoforms (Johnson et al. 1967). Three clusters significantly matched to Tropomyosin alpha-1 of golden gray mullet (Liza aurata) (TPM1_LIZAU: P84335) were identified. One consisting of 75 ESTs contained a complete open-reading frame coding for a 284 aa-long polypeptide and was designated as the major tropomyosin isoform, TPM1_SC (Zhang et al., unpublished data). It was with theoretical pI of 4.69 with molecular weight of 32.68 kDa (Table 1). Another of 19 ESTs, designated as TPM2_SC, encoded the C-terminal 144 amino acid residues region. There was one difference located in the 284th position between TPM1_SC and TPM2_SC. The other cluster of one EST deduced a 132 aa-long polypeptide designated as TPM3_SC. Comparison of putatively deduced polypeptides of TPM1_SC and TPM3_SC revealed that the 12 differences scattered over the region from 135 to 199 aa (Fig. 6, Supplement File). This suggested that the TPM1_SC and TPM3_SC might be produced by two different genes, and there are at least three tropomyosin isoforms in the S. chuatsi skeletal muscle.

Troponin T

Two clusters were annotated to troponin T homologues. The most abundant cluster consisted of 69 ESTs and contained a complete open-reading frame for 228 amino acid residues, designated as TNNT1_SC. It showed 49% identity with troponin T of Japanese quail Coturnix coturnix japonica (TNNT3_COTJA: P06398). Another cluster of two ESTs, designated as TNNT2_SC, encoded a peptide of 188-long amino acids lacking the C-terminal. TNNT2_SC shared 48% identity with troponin T of Norway rat Rattus norvegicus (TNNT3_RAT: P09739) (Table 1). In S. chuatsi fast skeletal muscle, the major isoform TNNT1_SC was less conserved than troponin C and troponin I. It showed the highest homology (49% identity) to troponin T in Japanese quail C. coturnix japonica, 48% identity to Norway rat R. norvegicus and human Homo sapiens, 47% identity to house mouse Mus musculus and chicken Gallus gallus.

In alignment with troponins from other vertebrates, the N-terminal region was highly variable due to the Glu-rich insertion. The major isoform TNNT1_SC lacked the Glu-rich (23 Es in sum) segment in other vertebrates. In comparison with TNNT1_SC, the minor isoform TNNT2_SC had the Glu-rich insertion of 51 amino acids in length and up to 16 Pro residues, which is aliphatic, has cyclic structure, and plays an important role of stabilizing the protein structure, occurred in this insertion (Fig. 7, Supplement File). The structural alterations of highly variable N-terminal region, caused by its variances of amino acid sequences, length, and charge, affected the binding affinity to troponin I, troponin C and tropomyosin (Wang and Jin 1998; Jin et al. 2000; Jin and Root 2000), and Ca2+ activation of ATPase activity (Biesiadecki and Jin 2002). Besides the highly variable N-terminal region, there was one difference between these two troponin T in S. chuatsi fast skeletal muscle isoforms in the conserved central region, which contained a second tropomyosin-binding site (Johnson et al. 1967). These differences were supposed to modulate the muscle contractility by affecting the interacting of troponin T with troponin C, troponin I, and tropomyosin directly. The influences of these differences over troponin T isoforms on the contractility of mandarin fish muscle needed further investigation.

Troponin I

Four clusters were homologous to troponin I in S. chuatsi. The largest consisted of 81 ESTs and contained a complete ORF encoding a polypeptide of 171 aa in length. Each of the other two clusters consisted of two ESTs. They yielded polypeptides, lacking N-terminal, of 154 and 131 amino acids in length, respectively. The other cluster of one clone encoded another 175 aa-long polypeptide lacking C-terminal (Table 1). The putatively deduced polypeptides were different (Fig. 8, Supplement File). Therefore, at least four troponin I isoforms existed in S. chuatsi white muscle and, we designated 171 aa putative full length polypeptide as TNNI1_SC, 154 aa putative peptide without N-terminal as TNNI2_SC, 131 aa putative peptide as TNNI3_SC and 175 aa putative peptide without C-terminal as TNNI4_SC.

When searching against Swiss-Prot database, four TNNI isoforms could be classified into two groups: TNNI1_SC, TNNI2_SC and TNNI3_SC belong to the first group and TNNI4_SC belong to second. The first group was significantly matched to Japanese quail C. coturnix japonica troponin I (TNNI2_COTJA: P68247) with identity from 50% to 57%, while TNNI4_SC was highly homologous to human H. sapiens troponin I with 72% identity (TNNI1_HUMAN: P19237) and rabbit Oryctolagus cuniculus troponin I (TNNI2_RABIT: P02643).

Because sequences between two groups were too flexible and within the first group too conserved, TNNI4_SC was aligned with other four vertebrates further. The further alignment identified the highly conserved minimal/main inhibitory peptide region, which reportedly interacted with actin and troponin C, and retained the important properties of intact troponin I such as inhibiting actomyosin ATPase activity (Syska et al. 1976; Zot and Potter 1987; Farah and Reinach 1995). The C-terminal over species was conserved too (Fig. 9, Supplement File). The C-terminal was also conserved among the sequences of the two groups. However, the corresponding minimal/main inhibitory peptide region in group 2 of troponin I was much dissimilar to that in group 1. The impacts of these differences on the inhibition of actomyosin ATPase activity were worthy of further exploration.

Troponin C

Three clusters were with complete ORFs encoding troponin C homologs in S. chuatsi. The most abundant cluster consisted of 84 ESTs, and the other two were composed of two ESTs each. The former cluster contained a complete ORF encoding a polypeptide of 160 aa long, and it was designated as TNNC1_SC. The second cluster of two ESTs also had a complete ORF encoding putatively yielded a polypeptide of 161 aa in length designated as TNNC2_SC. It was supposed that TNNC2_SC was the cardiac type because it was a minor isoform, which was one amino acid longer than skeletal troponin in mammals (Gillis and Tibbits 2002). The third cluster of two ESTs, designated as TNNC3_SC, encoded the same full length polypeptide as TNNC1_SC.

As the major isoforms of troponin C in S. chuatsi, TNNC1_SC showed 79% identity with European eel Anguilla anguilla skeletal muscle troponin C, and relatively lower homology to troponin C in other vertebrates (72∼74%), such as in edible frog Rana esculenta (74%), house mouse M. musculus (73%), rabbit O. cuniculus, and human H. sapiens (72%). However, the minor isoform TNNC2_SC was more similar, with identity ranging from 75% to 86%, to other vertebrates than TNNC1_SC (Seguchi et al. 2007).

Troponin C belongs to a Ca2+-binding protein family that had four EF hands (Ca2+-binding motif) labeled sequentially as I–IV. Each EF-hand comprised of a helix–loop–helix motif where Ca2+ could be potentially bound by oxygen ligands of six amino acids in the loop region at positions 1, 3, 5, 7, 9, and 12 (Dalla Libera et al. 1991). TNNC1_SC and TNNC2_SC were aligned with Ca2+ coordination sequences in other species. Multi-sequence analysis showed that EF-hands I and II were much more conserved than EF-hands III and IV. Ca2+ binding sites in EF-hands I and II motif over species were identical, while the replacement of F with Y took place in EF-hand III and K with M in EF-hand IV (Fig. 10, Supplement File). This was consistent with their functions. In skeletal troponin C, EF-hands I and II, of low affinity Ca2+ sites, regulated the interaction of troponin C with troponin I by binding Ca2+ to open a hydrophobic cavity where troponin C interacted with troponin I. However, C-terminal region, with EF-hands III and IV of high affinity Ca2+ sites, primarily served as binding domain for troponin I and provided free energy for binding troponin I (Grabarek et al. 1992).

Parvalbumin

Parvalbumins are soluble calcium-binding proteins that are especially abundant in fast-twitch muscles of fish. They aid muscle relaxation by competing with troponin C to bind the sarcoplasmic Ca2+ when Ca2+ is uptaken into the sarcoplasmic reticulum (SR) via SERCa (Berchtold et al. 2000). There were five clusters homologous to parvalbumin in the present study, each composed of 235, 157, 15, 1, and 1 ESTs. The former four clusters all had a complete ORF encoding 109 aa-long polypeptides with some variances in sequences, and they were designated as PRV1_SC, PRV2_SC, PRV3_SC, and PRV4_SC in sequence (GenBank accession number GU254023, GU254024, GU254025, and GU4026). The fifth cluster with a complete ORF encoding 108 aa-long polypeptides was designated as PRV5_SC. These five parvalbumin isoforms varied slightly from 4.46 to 4.65 in theoretical pI and from 11.47 to 11.96 kD in predicted molecule weight (Table 1). When searching against the Swiss-Prot database, the major isoform PRV1_SC showed the highest (75%) homology to common carp C. carpio parvalbumin alpha (PRVA_CYPCA: P09227). The other isoforms PRV2_SC and PRV3_SC shared 85% and 80% identity with parvalbumin beta (P59747) of chub mackerel Scomber japonicus, respectively. The minor isoforms PRV4_SC also significantly matched to PRVA_CYPCA with 74% identity, while another isoform PRV3_SC showed 67% identity with zebra fish Danio rerio parvalbumin-7 (PRV7_DANRE: Q804W2).

Each parvalbumin molecule has two EF-hands, which showed high affinity for Ca2+ and moderate affinity for Mg2+ (Declercq et al. 1988). In alignment with other isoforms of parvalbumin, Ca2+-binding regions were highly conserved. Ca2+-binding region I was identical among all parvalbumin isoforms, but PRV3_SC and PRV5_SC were different between S. chuatsi and carp (Fig. 11, Supplement File). These differences would influence the flexibility of loops in EF-hands and the Ca2+ binding affinity in terms of Mg2+ binding affinity and Mg2+ dissociation rates, as demonstrated in other studies (Schneider et al. 2006). However, how these differences affected the Ca2+ binding affinity of parvalbumin was unknown and needed further biochemical studies.

Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase1

SERCa is an ATP-dependent Ca2+ pump that actively translocates Ca2+ into the SR prior to muscle relaxation (Jayantha Gunaratne and Vacquier 2007). Two clusters significantly matched to AT2A1_MAKNI in Atlantic blue marlin Makaira nigricans (AT2A1_MAKNI: P70083.2). The larger cluster of 30 ESTs was 3821 bp long termed as the major isoform SERCa1_SC. The region of 584–3025 bp of SERCa1_SC was significantly matched to that of 177–990 aa in AT2A1_MAKNI with 90% identity and the region of 159–584 bp to that of 1–142 aa in AT2A1_MAKNI with 81% identity. SERCa1_SC also had the 5′ UTR, start codon, stop codon, and 3′ UTR. The deduced polypeptide had 957 amino acid residues with a predicted mass of 105 kDa and showed the highest homology to AT2A1_MAKNI with 90% identity. However, the region of 143–176 in AT2A1_MAKNI did not exist in SERCa1_SC. The second cluster composed of nine ESTs significantly matched the N-terminal 343 amino acid residues in AT2A1_MAKNI with 82% identity, designated as SERCa2_SC. The lost region in SERCa1_SC was identified in SERCa2_SC (Fig. 12, Supplement File). The alignment of nucleotide sequences of SERCa1_SC and SERCa2_SC demonstrated that the deletion of 143–176 aa region may result from the alternative splicing (Fig. 13, Supplement file).

Multi-sequence alignment revealed that putative cytoplasmic domain (1–48 aa) was less conserved, and the deletion of 143–176 aa was located in another putative cytoplasmic domain (111–253 aa). There was little information on the relationship between the sequences and the abilities of various SERCa isoforms to take back into sarcoplasmic reticulum. The impacts of the deletion of 143 to 176 aa fragment and other substitutions on the ability of SERCa to pump Ca2+ into the sarcoplasmic reticulum deserved further biochemical studies.

Conclusions

By analyzing the ESTs from the mandarin fish white muscle, we identified major isoforms of myofibrillar proteins and Ca2+-handling proteins (SERCa and parvalbumin). We also found the homologs of muscle “glue” molecules and regulators of actin filament such as alpha-actinins, myosin-binding proteins, myomesin, tropomodulin, cofilin, profilin, twinfilins, coronin-1, and nebulin. The sequence data and its analysis would provide a basis for our systematic understanding of the biochemical, bioenergetic, and physiological diversity of the mandarin fish white muscle at molecular level and expand the utility of fish systems as models for the muscle function.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 30771644; 30972263) and the Nature and Science Foundation of Hunan (09JJ6037). We thank Ying Liu for the technical assistance.

Supplementary material

10126_2010_9275_MOESM1_ESM.doc (212 kb)
Supplemental 1Supplementary figures (sequence alignments). This file containing alignments of polypeptides putatively deduced from mandrin fish muscle-related genes with other species. (DOC 211 kb)

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