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Acta Biochim Biophys
Sin 2008, 40: 356-363
doi:10.1111/j.1745-7270.2008.00406.x
Cloning, expression, and
polymorphism of the porcine calpain10 gene
Xiuqin Yang1, Di
Liu1,2*, Hao Yu3, Lijuan Guo1, and Hui Liu1
1 College of Animal Science and Technology, Northeast
Agricultural University, Harbin 150030, China
2 Agricultural Academy of Heilongjiang Province,
Harbin 150086, China
3 Institute of Animal Husbandry and Veterinary
Medicine, Jilin University, Changchun 130062, China
Received: January
1, 2008
Accepted: February
29, 2008
This work was supported by a grant from
the Science Fund for Distinguished Young Scholars of Heilongjiang Province (No.
JC-05-19) and the Key Program Item for Science and Technology of Heilongjiang
Province (No. GB05B106)*Corresponding
author: Tel, 86-451-86677458; E-mail, [email protected]
Calpains are
calcium-regulated proteases involved in cellular functions that include muscle
proteolysis both ante- and post-mortem. This study was designed to clone the
complete coding sequence of the porcine calpain10 gene, CAPN10, to
analyze its expression characteristics and to investigate its polymorphism. Two
isoforms of the CAPN10 gene, CAPN10A and CAPN10B, were
obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid
amplification of cDNA ends methods combined with in silico cloning.
RT-PCR results indicated that CAPN10 mRNA was ubiquitously expressed in
all tissues examined and, with increasing age, the expression level increased
in muscles at six different growth points. In the same tissues, the expression
level of CAPN10A was higher than that of CAPN10B. In addition,
three single nucleotide polymorphisms were detected by the PCR-single-stranded
conformational polymorphism method and by comparing the sequences of Chinese
Min pigs with those of Yorkshire pigs. C527T mutation was a missense mutation
and led to transforming Pro into Leu at the 176th amino acid. The results of
the current study provided basic molecular information for further study of the
function of the porcine CAPN10 gene.
Keywords porcine; CAPN10 gene;
cloning; expression; polymorphism
Calpains, implicated in signal transduction, nerve development,
muscle growth, cell proliferation, apoptosis, and differentiation, constitute a
large family of intracellular Ca2+-dependent cysteine neutral
proteases. Calpains are believed to be related to physiological as well as
pathological conditions such as ischemia, senile dementia, arthrophlogosis, and
cancer [1–3]. In livestock animals, calpains play an important part in muscle
growth and development, myoblast fusion, and differentiation [4]. Calpains were
also shown to decompose key myofibrillar and associated proteins that maintain
the structure of skeletal muscle such as titin, nebulin, and desmin [5,6]. It
is the early post-mortem cleavage of these proteins that ultimately leads to
the tenderization of meat. So in post-mortem skeletal muscles of meat animals,
the proteolytic activity of the calpains has the primary influence on meat tenderness
[7,8].A number of calpains have been identified and their primary
structures determined by cDNA cloning in mammals. They can be classified on the
basis of structure into typical and atypical calpains. Typical calpains (calpain1,
2, 3, 8, 9, 11, and 12) have a four-domain structure in the catalytic subunit
that is comprised of domain I (autolytic activation), domain II (cysteine
catalytic site), domain III (“electrostatic switch”), and domain IV
(calmodulin-like calcium binding sites). Atypical calpains (calpain5, 6, 7, 10,
and 15) do not have calmodulin-like EF-hand sequences in their domain IV, and
some even lack domain IV. Calpain10 (CAPN10), whose calmodulin
domain was replaced by a divergent T domain containing no calcium-binding
EF-hand structure, was recently discovered. It is ubiquitously expressed in
many human and mouse tissues [9,10]. Genetic variation in this gene is
associated with an increased risk of type 2 diabetes mellitus in humans [9].
The investigations on the roles of calpains in meat tenderization have been
focused on the ubiquitous calpain1 and 2, and the muscle-specific calpain3 [11–15]. A few
studies have also shown that CAPN10 might also be a candidate gene for
meat tenderization. Ilian et al [16] studied the changes in CAPN10,
tenderization level, and desmin in sheep longissimi and found that calpain10
proteins were strongly correlated with the rate of tenderization. But the
number of published reports on CAPN10 is limited and further efforts should
be made to reveal its role in meat tenderization.The human CAPN10 gene has 15 exons spanning 31 kb. The
splicing mechanisms of CAPN10 are very complicated. The human CAPN10
has eight transcript variants (a–h), some of which also lack the protease domain
[9], and the mouse and rat CAPN10 each have two transcript variants. But
the pig CAPN10 has not been reported. In this study, two isoforms of pig
CAPN10, CAPN10A and CAPN10B, were cloned using
reverse transcription-polymerase chain reaction (RT-PCR) and rapid
amplification of cDNA ends (RACE) methods combined with in silico
cloning. Their expression was characterized, and the polymorphism in the coding
region was analyzed by PCR-single-stranded conformational polymorphism (SSCP)
and by comparing the sequences of Chinese Min pigs with those of Yorkshire
pigs. Such research is important for better understanding the gene’s function
in muscle growth and degradation.
Materials and Methods
Animals, tissue collection,
RNA isolation, and DNA extraction
Three Yorkshire pigs at each growth times (1-d-old, 21-d-old,
90-d-old, 180-d-old, 270-d-old, 360-d-old) with similar bodyweight were
purchased from Zhongzhi breeding station (Harbin, China) and slaughtered
by electrical stunning and severance of the carotid arteries for the cloning
and expression analysis of the CAPN10 gene. Stomach, kidney, spleen,
lung, heart, liver, large intestine, small intestine, gonad, uterus, fat, and
muscle were harvested, flash-frozen in liquid nitrogen and stored at –70 ?C. Total RNA
was extracted from various tissues using TRIzol reagent (Invitrogen, Carlsbad,
USA) according to the manufacturer’s instructions. The Chinese local breed Min pigs were used to investigate single
nucleotide polymorphisms (SNPs) of the CAPN10 gene by the
PCR-SSCP method. Genomic DNA was extracted from the ear using
phenol/chloroform.
Primers used for cloning and
expression
Using BLAST (http://www.ncbi.nlm.nih.gov/BLAST), electronic hybridization was carried out with human CAPN10
cDNA as a probe in the pig genome database. A few expressed sequence tags with
significant similarity were found and a contig was constructed using Seqman II
(Lasergene version 6; DNASTAR, Madison, USA). According to the contig, two
pairs of primers, A1 and A2, were designed. In order to obtain the whole coding
sequence of pig CAPN10, according to the sequences of PCR products using
A1 and A2 primers, three additional primers were designed for 3‘-RACE
and 5‘-RACE. For RT-PCR measurement of CAPN10 mRNA, primer pair
C1 was designed for CAPN10 and primer pair C2 was designed for b-actin (GenBank accession No. U07786). The primer sequences and their
positions in the cDNA sequence of pig CAPN10A or b-actin are listed in Table 1.
cDNA cloning of pig CAPN10
Total RNA was extracted from pig liver tissue. RT was carried out
using 1 mg total RNA as a template with Superscript II and oligo(dT) primers
(Invitrogen). PCR was carried out with 1 ml RT reaction mixture in a
25 ml
final volume including 1?PCR
reaction buffer, 200 mM each dNTP, 1 U Taq DNA polymerase, and 0.2 mM each forward
and reverse primer. The thermal profiles were 94 ?C for 5 min followed by 35
cycles of 94 ?C for 1 min, 60 ?C (primer A1)/58 ?C (primer A2) for 1 min, 72 ?C
for 1.5 min, and an extension at 72 ?C for 7 min.For cloning the ends of pig CAPN10 cDNA, 3‘-RACE was
carried out using 3‘-Full RACE core set (version 2.0; TaKaRa, Dalian,
China) according to the manufacturer’s instructions, and the specific outer and
inner primers were A2 and B1, respectively; the 5‘-RACE reaction was
carried out according to the protocol of the 5‘-RACE system for rapid
amplification for cDNA Ends (version 2.0; Invitrogen), and the specific outer
and inner primers were B2 and B3, respectively.For cloning the ends of pig CAPN10 cDNA, 3‘-RACE was
carried out using 3‘-Full RACE core set (version 2.0; TaKaRa, Dalian,
China) according to the manufacturer’s instructions, and the specific outer and
inner primers were A2 and B1, respectively; the 5‘-RACE reaction was
carried out according to the protocol of the 5‘-RACE system for rapid
amplification for cDNA Ends (version 2.0; Invitrogen), and the specific outer
and inner primers were B2 and B3, respectively.The products of RT-PCR and RACE were electrophoresed on a 1% agarose
gel with ethidium bromide staining and purified by a Gel extraction mini kit
(Watson Biotechnologies, Shanghai, China). Then the purified PCR products were
cloned into pMD18-T (TaKaRa) vector and sequenced by the Bioasia Co. (Shanghai,
China).
Sequence analysis
Overlapping fragments amplified by RT-PCR, 3‘-RACE, and 5‘-RACE
were assembled by the DNAMAN package (version 5.2.2; Lynnon Biosoft, Quebec,
Canada). The open reading frame was found using the ORF Finder (http://www.ncbi.nlm.nih.Gov/gorf/gorf.html).
The conserved domain was analyzed in the Prosite database (http://cn.expasy.org/prosite/). An
unrooted phylogenetic tree was constructed using the ClustalW program that
calculates distances based on progressive multiple alignment and uses the
neighbor-joining method for tree construction.
Measurement of CAPN10
mRNA
The expression profile of 12 tissues from 180-d-old Yorkshire pigs
and muscles from six growth stages were determined by RT-PCR assays. Primer
pair C1 (Table 1) was designed according to the pig CAPN10A cDNA
sequence and human CAPN10 genomic sequence to make the amplicon span
introns, preventing amplification of any contaminating genomic DNA. The gene
expression values were normalized using the pig b-actin gene amplified with primer pair C2 (Table 1). One
microgram of total RNA extracted from each tissue was reversely transcribed
into first-strand cDNA in a 20 ml volume with an oligo(dT) primer according to the specifications of
the BcaBEST RNA PCR kit (version 1.1; TaKaRa). One microliter of the resultant
cDNA product was subjected to PCR in a 25 ml volume with the same
components as for primer pair A1. The PCR conditions were 94 ?C for 3 min
followed by 34 cycles (CAPN10)/23 cycles (b-actin) of 94 ?C for 30 s, 59 ?C for 30 s, 72 ?C for 30 s, and primer
extension at 72 ?C for 7 min. In order to precisely compare the expression
level in muscles, the linear increasing experiment was carried out in muscle to
ensure that the PCR products for both CAPN10 and b-actin were evaluated during the exponential phase, and the 34/23 cycles
were selected. The images were scanned and quantified by the Laboratory Imaging
and Analysis System (UVP, Upland, usa).
The ratio of the intensities of CAPN10 versus b-actin in the same muscle represented the relative expression level of the
target gene. The experiments were repeated three times. SAS (version 8.02; SAS,
Cary, USA) and Excel (version 2003; Microsoft, Washington, USA) were used for
data analysis and histogram plotting.
Measurement of two isoforms of
CAPN10
To compare the mRNA level of two isoforms of pig CAPN10 in
the same tissue, competitive RT-PCR was carried out in a 25 ml volume using
primer pair A2 and 1 ml cDNA mixture synthesized by RT as carried out in semi-quantitative
RT-PCR. The PCR products were separated on 2% agarose gel.
Development of PCR-SSCP assays
and screening the population
Primer pair PF (5‘-CCTGGTGGACCTCACTGG-3‘) and PR (5‘-ACCGAGCAGCTTATCAGACA-3‘)
was designed to amplify a 174 bp fragment in the coding sequences region
according to the pig CAPN10A cDNA. The PCR reactions were the same as
for primer pair A1 except that the annealing temperature was 57 ?C and the
template was 25 ng genomic DNA. The PCR products were used for SSCP to
investigate sequence polymorphisms of the CAPN10 gene. The SSCP
procedure were as follows: 3 ml PCR products were mixed with 8 ml loading buffer (98%
formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 10 mM EDTA, and 10%
glycerol) in a tube, denatured in 98 ?C for 10 min, placed on ice for 5 min,
then electrophoresed for 16 h at 10 V/cm on a 16% polyacrylamide gel. Silver
staining method was developed to display the bands [17]. The homozygous
individuals with different genotypes were cloned and sequenced by Bioasia Co..
Results
Sequence of two isoforms of
pig CAPN10 cDNA
Specific products were obtained from each primer pair. The PCR had
two different products using primer pair A2 and the sequence analysis showed
that they belonged to different transcript variants of pig CAPN10. After
assembling the sequence of RT-PCR and RACE products by the DNAMAN package
(version 5.2.2), two isoforms of pig CAPN10 were obtained. The cDNA of CAPN10A
is 2452 bp long containing a 116 bp 5‘-untranslated region (UTR), the
complete coding region of 2004 bp, and a 332 bp 3‘-UTR with a typical polyadenylation
signal (AATAAA). Conceptual translation predicted a protein of 667 amino acids
with a theoretical molecular mass of 74 kDa and an isoelectric point of 7.96.
The cDNA of CAPN10B results from a 110 bp deletion from the sequence of CAPN10A
and is identical to the remainder of CAPN10A throughout the coding
region and the UTR. The deletion sequence is from bases 1934 to 2043 in CAPN10A
cDNA. The open reading frame of CAPN10B is 1923 bp with a different stop
codon from CAPN10A, encoding a protein of 640 amino acids with a
theoretical molecular mass of 71 kDa and an isoelectric point of 7.89. These
sequence data have been submitted to the GenBank database under accession No.
DQ647669 for CAPN10A and DQ647668 for CAPN10B.Amino acid sequence analysis of the two isoforms of CAPN10
revealed that pig CAPN10 could be divided into the canonical four-domain
structure typical of calpains: I, II, III, and the divergent C-terminal domain
T containing no calcium-binding EF-hand structures. Domain II contained the
Cys, His, and Asn residues found in the active sites of other calpain catalytic
subunits. Domain T showed no significant similarity to the calmodulin-like Ca2+-binding domain IV of the traditional calpains (Fig. 1).
Molecular phylogenetic
analysis of CAPN10
The cDNA sequences of calpain10 for human (GenBank accession No.
NM023083), mouse (GenBank accession No. C005681), rat (GenBank accession No.
B13362), chicken (GenBank accession No. M422752), dog (GenBank accession No.
XM843215), and cattle (GenBank accession No. XM001256354), downloaded from
GenBank (http://www.ncbi.nlm.nih.gov/),
were used to determine the sequence identity among these vertebrates. The identity
was between 61.8% and 95.5% in the coding sequences, and between 60.2% and
96.8% in deduced amino acid sequences. Molecular genetic trees were constructed
using the methods of neighbor-joining for coding sequences (Fig. 2). The
tree shown in Fig. 2 suggests that pig is more closely related to cow
than dog, human, or mouse and the result is consistent with the phylogenetic
tree of the vertebrate using other molecular markers.
Expression analysis of CAPN10
As shown in Fig. 3, CAPN10 mRNA was expressed in all
tissues studied with abundant transcript in stomach, kidney, spleen, lung,
heart, liver, and large intestine, with the lowest level in fat. The expression
level in muscles from pigs at six different growth points was gradually
increased, with the exception of muscle from 1-d-old pigs (Fig. 4). The expression of the two isoforms of CAPN10 at the mRNA
level was compared by competitive RT-PCR and the results are shown in Fig. 5.
In all tissues examined, the expression level of CAPN10A was higher than
that of CAPN10B.
PCR-SSCP analysis of CAPN10
The PCR-SSCP method was developed successfully for screening
individual Min pigs. The polymorphism resulted in three genotypes, defined as
AA, AB, and BB (Fig. 6). Sequencing results showed that there were two
silent mutations, T567C and G573A, in the coding sequences region of the CAPN10
gene (GenBank accession No. DQ647668) between the sequences of AA and BB.
Comparison with the sequences submitted to GenBank revealed the third mutation,
C527T (Fig. 7). In the cloning of the CAPN10 gene, several
Yorkshire pigs were used and cytosine at the position of 527 bp was verified,
so there were three mutations in the region. The C527T mutation was a missense
mutation and led to transforming Pro into Leu at the 176th amino acid of the
mutant protein.
Discussion
In this study, using molecular biology techniques and in silico
cloning, two isoforms of pig CAPN10 were cloned. This is the first
report on the CAPN10 gene in pig. Sequence analysis showed that pig
CAPN10A has a similar splicing pattern to human CAPN10A and one of
the mouse/rat isoforms, whereas CAPN10B is distinct from any
human/rat/mouse isoforms. This further showed that CAPN10 is extremely
intricate terms of structure and function. The domain architecture of CAPN10
is unique compared to the conventional calpain1 and 2 in that the
calmodulin-like domain is replaced by a divergent T domain with no EF-hand
structures. This implies that the mechanism of CAPN10 regulation by
calcium is distinct from that of calpain1 and 2. Homology analysis showed that CAPN10
was highly conserved among animals, supporting the hypothesis of extensive
conservation between the CAPN10 genes among vertebrate species, and
implied that CAPN10 could exert many functions during animal growth and
development.As expected, based on its ubiquitous distribution in rat tissues
[10], RT-PCR revealed that CAPN10 transcripts were present in all
tissues studied, confirming the essential roles of the calpains in pig and
indicating their necessity for housekeeping functions [18]. However, their
relative amounts in different tissues from 180-d-old pig were unevenly
distributed, supporting the notion that calpains are highly regulated genes.
Our results also showed that the highest level of expression was in large
intestine (Fig. 3), whereas in the adult human, heart showed the highest
level of expression [9], and in young rat, the highest level of expression was
in brain [10]. Considering these facts, the expression pattern of CAPN10
at the mRNA level appears to be different between species.In mammals, calpains are known to play a significant role in muscle
protein turnover by mediating the degradation of myofibrillar proteins: desmin,
filament, C-protein, tropomyosin, troponin T, troponin I, titin, nebulin,
vimentin, gelsolin, and vinculin [19–21]. Proteolysis of these proteins affects
muscle deposition in the living body, and impacts whole-muscle food texture,
leading to meat tenderization in the early post-mortem period. The expression
analysis at the mRNA level in muscles at six different growth points showed
that the relative amount of the CAPN10 gene was present at its lowest
level in muscle from 21-d-old pig and gradually increased to its highest level
in 360-d-old pig. With increasing age, the capability of muscle deposition
decreases, whereas the relative expression level of CAPN10 was
increased, further supporting the fact that CAPN10 decomposes
myofibrillar proteins. The high expression level in muscle of 1-d-old pigs
might be due to maternal influence. When a gene is identified, there may be
more than one polymorphism within it. An SNP occurring in the coding region
(cSNP) could affect the expression level and protein structure, whereas a
non-synonymous mutation that changes the amino acid is likely to have an effect
on phenotype, making the cSNP more functional for marker-assisted selection
[22,23]. In this study, three cSNPs were detected by PCR-SSCP and aligning
sequences of Yorkshire and Min pigs, which laid the foundation for further study
on the association of polymorphism with traits. The Pro176Leu mutation existed
in a relatively conservative region of the catalytic domain and sequence
analysis showed that, by aligning the sequence with that of chicken, human,
mouse, rat, cattle, and dog from GenBank, Pro did not appear in that site in
other species (Fig. 8). It will be interesting to investigate whether
the mutation was specific to pig and influenced the function of CAPN10.
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