Download Cloning, Structural Analysis and Expression of Fast Twitch Skeletal Muscle TNNC2

2011 International Conference on Bioscience, Biochemistry and Bioinformatics
IPCBEE vol.5 (2011) © (2011) IACSIT Press, Singapore
Cloning, Structural Analysis and Expression of Fast Twitch Skeletal Muscle
TroponinC2 (TNNC2) Gene in Goat
Haolin Chen, Daihua Wang, *Gangyi Xu, Tintin Wu, Chengli Zheng
College of Animal Science and Technology, Sichuan Agricultural University, Ya’an, Sichuan, 625014 China
Haolin Chen E-mail: [email protected]
*
Corresponding author E-mail: [email protected]
a subunit of Troponin C (TNNC), fast skeletal troponinC2
(TNNC2) plays a critical role in skeletal muscle
contraction[5]. TNNC2 gene has expressed during the
myoblast differentiation and skeletal muscle development[6].
The allele T have positive effects on tenderness and marbling
score, and suggest that the allele could be used as a
molecular marker to study the meat quality in sheep[7]. Thus,
the gene involved in skeletal muscle growth, myoblast
differentiation and skeletal muscle contraction and was
considered usually as potential candidate gene for meat
quality[8], and identified to be an excellent model system for
studies of developmentally regulated gene expression in
skeletal muscle[9].
Tianfu goat is an emerging breed in China, which formed
by multiple crossbreeding with the local breeding Chengdu
Ma goat for maternal and imported England Saanen, Nubian,
Toggenburg and Boer for paternal. This goat breed is noted
for good quality meat, rapid growth, roughage resistance,
strong adaptability [10]. For this reason, we cloned the
TNNC2 gene in goat and analyzed its structural and the
tissue expression.
Abstract—TroponinC2 (TNNC2), a constituent of the troponin
complex located on the thin filament, plays a critical role in
skeletal muscle contraction and improving meat quality traits
of livestock and poultry. The TNNC2 gene has been identified
in many mammals, but very little known about goat. In the
study, we cloned TNNC2 gene in goat for the first time and
analyzed its tissue expression. Results indicated that TNNC2
contains 483bp CDS and encodes 160 amino acids, and it was
selectively expressed in the muscular tissues of goat. Its
nucleotide sequence was high similar with sheep (93.38%) and
alpaca (90.48%), and the identity of encoded amino acids
which was varied from 100% (sheep) to 70.81% (Atlantic
salmon) in animal. We found that the Ala113 and leu132 was
distributed very law in mammals. The amino acid residue of
Ca2+ binding sites Ⅱ was identical in mammals, Aves and
amphibian. This study may provide more insight into the
molecular structure and expression patterns of the vertebrate
TNNC2 gene.
Keywords-TroponinC2 gene; Molecular cloning; Structural
analysis; Expression; Goat
I.
INTRODUCTION
II.
The TNNC proteins belong to a member of multigene
family of Ca2+ binding proteins[1].There are two isoforms of
TNNC proteins found in vertebrate striated muscle,
troponinC2 (TNNC2) expressed in fast skeletal muscle and
troponinC1 (TNNC1) expressed in slow skeletal muscle and
cardiac muscle. The difference of the two isoforms is that
TNNC2 is activated by Ca2+ binding siteⅠandⅡon the
N-terminal, while TNNC1 is activated by Ca2+ binding
siteⅡ.
TroponinC2, which has four Ca2+ binding sites, is a
prominent member of the troponin complex. Each of these
Ca2+ binding sites is characterized by a 12-residue Ca2+
binding loop that is interposed between a pair of a-helices.
Each of the Ca2+ binding loops is rich in acidic amino acids
(Asp and Glu) that are responsible for the coordination of a
single Ca2+ [2]. The integral structure of TNNC2 consists of
a N-terminal and a C-terminal globular domains connected
by a flexible central helix, giving it a dumbbell-like shape,
and each globular domain occupies a pair of EF-hand motifs
which can bind to metal ions[3].
The diameter and type of muscle fiber will directly
influence on meat tenderness, in which the types of myofiber
can be classified according to their contractile nature[4]. As
MATERRIALS AND METHODS
A. Experimental animals and tissues preparation
Tianfu goats were slaughtered at six month, one year,
two years and adult. The tissue samples of the carcasses
(heart, liver, lung, kidney, abdominal muscle, longissimus
muscle, gluteus maximus and biceps brachii) were collected
immediately after slaughter and frozen in liquid nitrogen jar
for extracting total RNA.
B. Cloning of TNNC2 gene and construction of
recombinant plasmid
Total RNA was extracted from goat by using Trizol
(TaKaRa, Dalian, Chian). According to a procedure of the
synthesis kit, the first strand cDNA was synthesised from
total RNA. According to Bos taurus TNNC2 gene sequence
(GenBank Accession No. NM001076373), a pair of primer
was designed with software primer 5.0 in the conserved
region.
The
forward
primer
was
5′-TGATGACGGACCAGCAGG-3′, and the reverse primer
was 5′-CTTCTTACTGCACGCCCTC-3′. Then using the
primers, a part of cDNA fragment was amplified by RT-PCR
with first strand cDNA as templates. The PCR started with
pre-denature at 95 °C for 5 min, followed by 35 cycles
283
(94 °C, 40 s; 53 °C, 40 s; 72 °C, 50 s),and ended with final
extension at 72 °C for 10 min. Then the PCR products were
separated by electrophoresis on a 1.5% agarose gel, and
purified by using the PCR Rapid Purification Kit
(BioDev-Tech). Purified PCR products were cloned by
pMD18-T vector (TaKaRa, Dalian) and were sent to
TaKaRa Biotechnology (Dalian) Co. Ltd. for sequencing.
pairs:
5′-GTCACCAACTGGGACGACA-3′
and
5′-AGGCGTACAGGGACAGCA-3′. Total RNA was
extracted from the heart, liver, lung, kidney, abdominal
muscle, longissimus muscle, gluteus maximus and biceps
brachii respectively. The amplifications were performed in a
20μL reaction volume containing 12.5μL of 2× SYBR
Premix ExTaq (TaKaRa, Dalian), 5μL of each primer, 2μL
of diluted cDNA, 4.5μL ddH2O. The PCR amplification was
carried out as follows: 95℃ for 10 s followed by 40 cycles
of 95℃ for 5 s and 60℃ for 34 s. The 2 –△△Ct method
was used to analyze the expression level of the gene in
goat[12]
C. Sequence analysis
Using BLAST software, we performed a sequence
similarity analysis for the TNNC2 nucleotide and amino acid
sequences (http://www.ncbi.nlm.nih.gov/BLAST). Also, we
determined a theoretical molecular weight and isoelectric
point
by
using
Peptide
Mass
(http://us.
expasy.org/tools/peptide-mass.html).The
phosphorylation
site
was
estimated
by
the
neural
network
(http://www.cbs.dtu.dk/services/NetPhos).
Multiple
alignment analysis was conducted by the program
CLUSTAL X and Bio-Edit software, and the phylogenetic
analysis was performed by MEGA version 4.0 with
Neighbor-Joining
method.
The
SWISS-MODEL(http://swissmodel.expasy.org) was used to
model the protein conformation, which was viewed in the
Swiss Pdb Viewer[11]
III.
RESULT
A.
cDNA cloning and sequence analysis of TNNC2
A 530bp sequence of TNNC2 gene was obtained by
homologous cloning using the cDNA from the longissimus
of Tianfu goat (GenBank accession No. HQ640745). The
sequence analysis showed that CDS of TNNC2 was 483bp
with 160 encode amino acids residues (Fig. 1) and 18.1kDa
predicted molecular weight and 4.06 isoelectric points. The
total number of negatively charged residues (Asp + Glu) was
45 and positively charged residues (Arg+ Lys) was 16, and it
indicated the protein took negative electricity. There were
seven phosphoric sites successfully predicted by the neural
network (Fig. 1). There was no signal peptide in the protein.
Hydrophobic correlation analysis showed that the minimum
was -3.12 and the maximum was 1.18 (Fig. 2). The result
indicated that the protein hydrophobicity ability was strong.
The secondary structure of the protein was mainly with
α-helix, random coil and β-sheet, and the β-sheet only exists
in the area of EF-Hand SiteⅠ(Fig. 3)
D. RT-PCR expression analysis
Tissue-specific expression pattern of TNNC2 was
performed by semi-quantitative (RT)-PCR using goat β-actin
gene as an internal expression control. The primers of
β-actin, which were used to adjust the concentration of
cDNA, was designed based on the nucleotide sequence
deposited in the Gene Bank (Accession No. U39357).
TNNC2 primer pairs: 5′-GCCAGACACCCACCAAAG-3′
and 5′-CGAAGATGCGGAAACACT-3′. β-actin primer
Figure 1. The Nucleotide and deduced amino acid sequences of TNNC2 from goat. The emerald green indicated initiation codon; *indicated terminal
codon; the red indicated phosphoric sites (Ser, Thr, Tyr)
284
Figure 2. The hydrophobicity profile of TNNC2. The horizontal scale indicated the number of amino acid residues and the vertical one was the relative
hydrophobic scale. Points below the zero horizontal line corresponded to hydrophobic region, and points above the line were hydrophilic.
Figure 3. The secondary structure of TNNC2 of goat .The blue line represents alpha helix, the red line represents extended strand, and the purple line
represents random coil.
(sheep) to 90.48% (alpaca), 85.37%(aves), 79.42%(fish) to
78.66%(amphibian,).The amino acid sequence of goat
TNNC2, had an 100% identity with sheep, giant panda and
mustang, and 87.73%, 88.34%, 70.81% to aves, amphibian,
fish respectively (Table. I).
There were eight missense mutation in mammals,
Residue 113 is a Pro in primates (human gorilla and monkey)
B.
Characteristics of deduced protein and phylogenetic
analysis of TNNC2
Comparison of TNNC2 protein with fifteen mammals
and three other vertebrates (aves, amphibian, fish) revealed
that caprine TNNC2 had a high degree of similarity with
these reported animals (Fig. 4). The nucleotide similarities of
caprine TNNC2 with these animals was varied from 93.38%
285
but other mammals is Ala. Residue 132 of perissodactyla and
artiodactyla is Leu but primates, lagomorpha and rodentia is
Ile.
Figure 4. Sequence comparison of representative species of TNNC2. The Ca2+-coordinating positions in each EF-hand site are shown above the sequences,
and the helices are labeled. The two interesting amino acid residue were highlighted by blue.
TABLE I.
LIST OF TNNC2 SEQUENCES USED IN THE ANALYSES AND IDENTITY
Organism
Common
Name
Ribonucleotide
identity
AA
identity
Capra hircus
Ovis aries
bos Taurus
Sus scrofa
Lama pacos
Equus caballus
Canis familiaris
goat
sheep
cow
pig
alpaca
horse
dog
99.38%
92.18%
93.58%
90.48%
94.20%
92.96%
Ailuropoda melanoleuca
panda
Homo sapiens
Callithrix jacchus
Pan troglodytes
human
Callithrix aurita
gorilla
Taxonomy
Identifier
Database
100%
98.14%
99.38%
98.13%
100%
99.38%
EM
EM
EM
EM
EM
EM
EM
HQ640745
NM001112821.1
NM001076373.1
EU131524.1
DQ646403.1
XM002928049.1
XM543023.2
Genbank
Genbank
Genbank
Genbank
Genbank
Genbank
Genbank
94.00%
100%
EM
XM002928049.1
Genbank
92.96%
91.93%
92.96%
98.75%
99.38%
98.75%
EM
EM
EM
BC005323.1
XM002747597.1
XM001157246.1
Genbank
Genbank
Genbank
286
Macaca mulatta
monkey
92.75%
99.38%
EM
XM001108623.2
Genbank
Rabbit
hare
95.45%
99.38%
EM
Y00760.1
Genbank
Oryctolagus cuniculus
Rattus norvegicus
Mus musculus
Gallus gallus
rabbit
rat
mouse
chicken
95.03%
90.83%
89.44%
85.37%
99.38%
99.38%
98.75%
87.73%
EM
EM
EM
bird
NM001082645.1
NM001037351.1
BC024390.1
NM205450.1
Genbank
Genbank
Genbank
Genbank
Xenopus tropicalis
Western clawed frog
79.42%
88.82%
AM
BC061286.1
Genbank
Xenopus laevis
African clawed frog
78.66%
88.34%
AM
NM001085939.1
Genbank
Salmo salar
Atlantic salmon
79.42%
70.81%
fish
BT049981.2
Genbank
74
Homo-sapiens
Bos-taurus
98
99
Pan-troglodytes
84
Macac-amulatta
Callithrix-jacchus
14
Equus-cab allus
Ailuropoda-melanoleuca
30
49
89
Canis-familiaris
Sus-scrofa
100
80
35
Rab b it
Oryctolagus-cuniculus
Ovis-aries
48
100
Capra-hircus
Mus-musculus
Rattus-norvegicus
99
100
Lama-pacos
Gallus-gallus
Xenopus-laevis
Salmo-salar
100
100
Xenopus-tropicalis
Figure 5. Phylogenic tree of TNNC2. The GenBank accession Nos of the sequences were listed in the table I.
accession, 1a2x) (Fig. 6A). And the Ca2+ binding sites were
marked out by the SPDBV 4.01 molecular-graphics program
(Fig. 6B).
C.
The structural model of TNNC2
SWISS-MODEL server, a fully automatic procedure,
was used to construct a 3D structural model. It was
constructed using bovine TNNC2 as the template (PDB
287
(A)
(B)
Figure 6. The 3D-structural model of TNNC2. A. The structure of alpha-helix, beta-strand and random-coils. B. The structure was shown in backbone, The
Ca2+ binding sitesⅠ, Ⅱ, Ⅲ, Ⅳ were highlighted by white, blue, red and yellow respectively.
transcription level of TNNC2 was highest in six-month old
goats. The expression amount of TNNC2 was highest in
longissimus muscle. There were no expression of TNNC2 in
the heart and other Non-muscle tissue (Fig. 7B).
D.
Relative Expression pattern of TNNC2 in goat
We performed semi-quantitative (RT)-PCR to analyze its
mRNA transcription levels. Results indicated there were
some differences in TNNC2 transcription levels between the
four developmental stages of the goat (Fig. 7A) The
288
(A)
(B)
Figure 7. Expression of TNNC2 gene in goat. The vertica1 axis indicated the value of the TNNC2 mRNA. A. The horizontal axis indicated the various
growth stages of TNNC2 in longissimus muscle. B. The horizonta1 axis indicated the various tissues of yearling. Line1-8 was heart, liver, lung,
kidney, gluteus maximus, longissimus muscle, abdominal muscle and biceps brachii, respectively.
IV.
acid sequences of various species indicated that TNNC2 of
goat is a member of the EF-hand superfamily (Fig. 2)
The analysis of encoded amino acids revealed that the
caprine TNNC2 was highly homologous with that of other
species, such as sheep (100%), human (92.96%), aves
(87.73%) (table.1). It suggests that there are rigid
structure-functional requirements for TNNC2 operating
within the mammalian striated muscle.
There were eight amino acids missense mutation in 16
species of mammals. All of this missense mutation is
conservative substitution except Thr18 in alpaca. Alpaca live
on in highlands of South America, with an average altitude
above 4, 000 meters[16]. As Thr is a polar, hydrophilic
residue and Ala is a nonpolar, hydrophobic residue, this
represents a non-conservative properties substitution. The
replacement of a hydrophilic residue (Thr) with a
hydrophobic residue (Ala) at position 18，and the presence
of a hydrophobic residue at position 17 (Ile)，represent an
adaptation of TNNC2 functioning of alpaca to
circumstances.
DISCUSSION
In the study, the cloning, structural analysis and
expression of troponinC2 (TNNC2) gene were completed for
the first in fast twitch skeletal muscle of goat. In ruminants,
meat tenderness was influenced by the pattern of the
animal’s growth, Carcass weight of slow-growing ruminants
is lower and more tenderness than that of faster-growing
[13].The TNNC2 was considered as a candidate gene with
meat quality in our study.
TNNC2 consists of two globular domains connected by a
α-helixes linker (fig. 6). Both N-terminal and C-terminal
domains contain two EF-hand Ca2+ binding sites. It is similar
to other Ca2+ binding proteins, EF-hands consist of two
α-helixes which connected by a flexible loop, forming what
is known as a helix-loop-helix. The helices and loop in the
motif each contain 12 amino acid residues[14-15] This motif
is also present in the deduced amino acid sequence of the
TNNC2 in goat (Fig. 1) Therefore, we consider that the
protein of TNNC2 of goat include four Ca2+ binding sites.
Comparison of calcium-binding site residues in the amino
289
The Pro113 present only to primates (human，gorilla and
monkey). The replacement may play a key role in the
evolution of primates. The Leu or Ile at 132 locus distribute
regularly in the 6 types of mammals. Moreover, Avian and
ectothermic species (Xenopus laevis and Xenopus tropicalis)
is also Ile at 132 locus. In other words, Leu exist only in
perissodactyla and Artiodactyla animals, and these may
represent an adaptation to the characteristics of horned
animals.
Binding of Ca2+ to the low-affinity sites of TNNC
(particularly Ca2+binding site II, which is conserved in
TNNC1 and TNNC2) is the trigger event for contraction[17].
This hypothesis is supported by the finding that TNNC1,
which contains only a single low-affinity Ca2+ binding site
(site II), can regulate fast skeletal muscle contraction in
response to Ca2+ after substitution of TNNC1 for TNNC2 in
permeabilized fast skeletal muscle fibers[18]. Compared to
siteⅠ, Ⅲ and Ⅳ, the sequence of site II of TNNC2 is the
most conservative, only a conservative substitution Ser70in
fish in the 20 species(fig. 4) as Thr and Ser both belong to
polar and hydrophilic amino acid residues. Therefore, it will
specify the importance of site II further.
We know that there are three distinct sites of the TNNC2
molecule and make important contacts with TNI [19-26].
The regions of TNNC2 (AA 50-60 and AA 90-100) interacts
with TNI (AA 104-115). This binding requires Ca2+ or Mg2+
occupancy of the high-affinity COOH-terminal Ca2+ binding
sites. and a peptide (comprising AA 126-136) of TNNC2
interacts with TNI in a Ca2+ independent manner[17]. We
found the amino acid is identical of TNNC2 (AA 50-60 and
AA 90-100) in mammals, Aves and amphibian. For all
mammalian, amphibian and fish, amino acid residue at 128
locus is a Thr, but that is Ile in Aves. As Ile is a nonpolar,
hydrophobicity residue and Thr is a polar, hydrophilic
residue, this represents a non-conservative properties
substitution. This substitution may play a key role in the
evolution of birds flying.
The cross-linking studies have also examined the
interaction of TNNC2 with TNT in either heterodimeric
complexes or trimeric complexes that also contained
TNI[27]. These studies demonstrated an interaction between
Cys98 of TNNC (located in the COOH-terminal half of the
molecule)
and
residues
175-178
of
TNT
(Met-Lys-Lys-Lys)[28]. Residue 98 is a Cys in all
mammalian, amphibian and avian TNNC2 but is a Val in
fish (Fig. 6). This may be the key that the terrestrial animal
adapt to the movement on land，what is more, it illustrate
the importance of touch the residue 98 with TNT.
Initial immunohistochemical studies showed that TNNC1
only in cardiac and slow skeletal muscle, whereas, TNNC2
was found exclusively in fast skeletal myocytes[9, 24, 29].
Research suggested that a TNC-like protein was present in
chick embryo brain[30]. Normally, the TNNC2 gene is not
expressed in ventricular myocytes. But inappropriate
expression of TNNC2 gene may be appeared in cardiac
muscle in some pathological state, and might be related to
the clinical[31]. In invertebrates, TNNC is expressed in a
variety of non-muscle tissues[32]. Semi-(RT)-PCR analysis
has indicated that expression of the porcine gene is restricted
to skeletal muscle (biceps brachii, quadriceps femoris,
longissimus dorsi)[33]. In our study, we have also found that
TNNC2 of goat was expressed in gluteus maximus,
longissimus muscle, abdominal muscle and biceps brachii.
This result implied that the TNC gene may be an excellent
model system for elucidating the molecular mechanisms that
regulate skeletal versus cardiac muscle-specific gene
expression.
V.
CONCLUSION
In the present study, the full-length cDNA of TNNC2
gene was cloned with analyzed the amino acids sequence
and space structure in goats. We found that the TNNC2 gene
of goat had high degree of identity with other mammals. By
comparing the sequences of TNNC2, we have discovered a
number of amino acid residues which may have a critical
impact for their functional studies. The TNNC2 showed a
high level of expression in skeletal muscle of goat. The
results of expression profiles of TNNC2 gene will help to
explain molecular mechanisms of regulating skeletal and
development.
ACKNOWLEDGMENT
This work was financially supported by the National
Modern Industry Technology System on Sheep and Goat for
Meat (NYCYTX-39) and the Projects of Selecting New
Breed on Livestock in Sichuan Province (01NG002-06).
Research Instistute of Animal Genetics＆Breeding, College
of Animal Science & Technology of Sichuan Agricultural
University, 46# Xinkang Road, Yucheng district, Yaan
625014, Sichuan province of China. Tel: +86 (0835)
288-6096; Fax: +86 (0835) 288-6080; E-mail: goatsxgy @
263.net.
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