Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 8590
doi:10.1111/j.1745-7270.2008.00373.x
Expression patterns and
subcellular localization of porcine (Sus Scrofa) lectin,
galactose-binding, soluble 1 gene
Haifang Qiu, Shuhong Zhao, Mei
Yu, Bin Fan, and Bang Liu*
Laboratory of
Molecular Biology and Animal Breeding, Key Laboratory of Agricultural Animal
Genetics, Breeding and Reproduction of Ministry of Education, Huazhong
Agricultural University, Wuhan 430070, China
Received: July 4,
2007
Accepted: September
27, 2007
This work was
supported by the grants from the National Natural Science Foundation of China
(Nos. 30371029 and 30571007), the Technologies Research and Development Project
of Hubei Province (No. 2002AA201C27), and the Natural Science Foundation creative team projects
of Hubei Province (No. 2006ABC008)
*Corresponding
author: Tel, 86-27-87282680; fax,
86-27-87280408; E-mail, [email protected]
Lectin,
galactose-binding, soluble 1 (LGALS1) gene encodes galectin-1, an
atypical secretory protein that plays an important role during myoblast
proliferation and differentiation. In this study, the porcine LGALS1
gene was cloned and characterized from pig muscle. The predicted protein
sequence shared a high identity with its mammalian counterparts. Reverse
transcription-polymerase chain reaction revealed that porcine LGALS1
was expressed at 33 day post-coitus (dpc) and 65 dpc at a relatively high
level, and then decreased to 90 dpc during fetal skeletal muscle development,
suggesting that galectin-1 is a potent factor implicated in the formation of
myofibers. LGALS1 was found widely expressed in all tissues and
transient transfection indicated that galectin-1 locates both in cytoplasm and
nucleus. Genomic sequences and analysis predicted a promoter region at
approximately 1.279–1.529 kb, but dual-luciferase reporter assay
indicated that it has little promoter activity.
Keywords expression
pattern; subcellular localization; promoter activity; LGALS1
Galectins are structural proteins with at least one characteristic
carbohydrate recognition domain with an affinity for b-galactosides [1–2]. To date, 15
different galectins have been characterized and they are numbered according to
the chronology of discovery (galectin-1 to galectin-15). They are also widely
distributed from lower to higher vertebrates [3]. Galectin-1 was the first
discovered mammalian galectin [4] and it is secreted during differentiation and
accumulates with laminin in the basement membrane surrounding each myofiber
[5]. It is expressed in a wide range of vertebrate tissues, particularly in
developing cardiac, smooth, and skeletal muscle. Studies showed that galectin-1
plays a role during skeletal muscle development [6] and that peak galectin-1
expression in muscle coincides with formation of myofibers [7]. The
down-regulated expression of galectin-1 in migrating tumor cells could impair
malignancy development in different ways [8–10]. Pig is an important meat animal, and meat production is determined
by the number and size of myofibers. Meat quality is determined by the
proportions of muscle fiber type [11]. In porcine muscle development, there are
two major waves of fiber generation, a primary generation from 33 day
post-coitus (dpc) to approximately 65 dpc, and a secondary generation from
approximately 54 to 90 dpc [12]. Hence, 33 dpc, 65 dpc, and 90 dpc are key
stages during prenatal skeletal muscle development. The study of lectin,
galactose-binding, soluble 1 gene (LGLAS1) will contribute to the
understanding of myofiber development.Here, the sequences of porcine LGALS1 were characterized, and
its expression pattern, protein location, and activity of the predicted
promoter region in PK15 cells were investigated.
Materials and methods
Tissue sampling, RNA
isolation, and cDNA preparation
Chinese indigenous Tongcheng pigs were used in this study. The fetal
skeletal muscles from three ages, 33, 65, and 90 dpc, were harvested, frozen in
liquid nitrogen, and stored at –80 ?C. Ten different tissues (heart, liver, spleen, lung, kidney,
skeletal muscle, small intestine, lymph node, testis, and brain) were collected
for spatial expression studies.Total RNA was extracted by Trizol
reagent (Invitrogen, Carlsbad, USA). RNA concentration was measured by a
Beckman DU 640 spectrophotometer (Beckman, Fullerton, USA). Then cDNA was
synthesized using Moloney murine leukemia virus reverse transcriptase
(Promega, Madison, usa). Two
micrograms of total RNA were combined with 5 mM oligo(dT)15 and 8 ml diethyl pyrocarbonate water,
then incubated at 70 ?C for 5 min to denature secondary structures. After
cooling the mixture rapidly to 0 ?C, 10 ml of 5?reverse transcriptase buffer, 250 mM dNTPs, 40 U RNase inhibitor
(Promega), and 400 U Moloney murine leukemia virus reverse transcriptase were
added to a total volume of 50 ml. The mixture was incubated at 42 ?C for 60 min, then at 95 ?C for 5
min to destroy the RNase, and then treated with RNase-free DNase (Fermentas,
Vilnius, Lithuania).
cDNA isolation, sequencing,
and analysis
The full-length cDNA sequence of porcine LGALS1 was
obtained using the rapid amplification of cDNA ends (RACE). Gene-specific
primers were designed using pig expressed sequence tag data from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/)
(Table 1). RACE was carried out according to manufacturer’s protocol of
the SMART RACE cDNA kit (Clontech, Palo Alto, USA). The polymerase chain
reaction (PCR) products were purified with a Gel Extraction Mini Kit (Takara,
Shiga, Japan) and cloned into plasmid pMD18-T (TaKaRa), then sequenced
commercially. Bioinformatics analysis was carried out using PROSITE and TargetP
(http://cn.expasy.org/tools/) [13].
Genomic sequences and analysis
Genomic DNA fragments were amplified by PCR in 20 ml of 1?PCR buffer (Fermentas) containing 50 ng porcine genomic DNA, 0.3 mM each primer,
75 mM
each dNTP, 1.5 mM MgCl2, and 2.0 U Taq DNA
polymerase (Fermentas). The PCR parameters were 5 min at 95 ?C followed by 30 s
at 94 ?C, 30 s at the annealing temperature (Table 1), and 30 s at 72
?C for 35 cycles, followed by a final extension of 5 min at 72 ?C. The PCR
products were purified, sequenced, and assembled.The genomic DNA sequences were analyzed by CpG Island Searcher (http://www.uscnorris.com/cpgislands2/cpg.aspx)
and Promoter Scan (http://www-bimas.cit.nih.gov/molbio/proscan/)
programs to find if there are some regulatory regions in this gene. Repetitive
elements were identified using RepeatMasker (http://www.repeatmasker.org).
Expression patterns of LGALS1
LGALS1 expression patterns were
determined by reverse transcription-PCR. One microliter of the resulting
single-stranded cDNA was amplified 27 cycles with LGALS1-specific
primers (Table 1). The housekeeping gene b-actin was used as an internal control. PCR products were separated by
electrophoresis on 2.0% agarose gels and visualized by ethidium bromide
staining. The PCR fragments were purified and directly sequenced to confirm
the correct amplification of the porcine LGALS1 gene.LGALS1 expression patterns were
determined by reverse transcription-PCR. One microliter of the resulting
single-stranded cDNA was amplified 27 cycles with LGALS1-specific
primers (Table 1). The housekeeping gene b-actin was used as an internal control. PCR products were separated by
electrophoresis on 2.0% agarose gels and visualized by ethidium bromide
staining. The PCR fragments were purified and directly sequenced to confirm
the correct amplification of the porcine LGALS1 gene.For tissue-specific expression analysis, total RNAs were isolated
from various tissues (heart, liver, spleen, lung, kidney, skeletal muscle,
small intestines, lymph node, testis, and brain). For temporal expression
analysis of LGALS1, total RNAs were isolated from skeletal muscle of
various fetal developmental stages (33, 65, and 90 dpc), and the total RNAs of
three individual fetuses were mixed in each stage in order to ensure
authenticity [14].
Subcellular localization in
PK15 cells
The open reading frame of porcine LGALS1 was amplified from
its cDNA clone and subcloned into the SalI-BamHI site of the
enhanced green fluorescent protein (EGFP) vector (pEGFP-N1; BD Biosciences
Clontech, Palo Alto, USA) to yield a mammalian expression plasmid, pEGFP-LGALS1.
The primers are listed in Table 1 and the vector was sequenced for
accuracy. PK15 cells were cultured in RPMI 1640 medium containing 10% fetal
bovine serum, 4 mM glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin
under humidified air containing 5% CO2 at 37 ?C and seeded onto
cover slips. Transient transfection was carried out using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s instructions. After 48 h, the cells in a 6-well plate were washed using phosphate-buffered saline, then fixed for 15 min with 4% para-formaldehyde. After the
washing steps and incubation with 10 mM Hoechst 33342 for 10 min, the slides were
mounted, sealed, and analyzed by confocal microscopy (TCS-SP2; Leica,
Heideberg, Germany). Leica IM500 confocal software was used to generate images
of individual fluorescent markers as well as overlay pictures that showed the
relative distribution of the fusion protein.
Transient transfection and
dual-luciferase reporter assay
A 1.268 kb predicted regulatory region was amplified with the
primers shown in Table 1. The amplified fragment was inserted into the NheI-HindIII
site of pGL3-Basic (Promega) to construct the pGL3-1.268 kb vector. The
pGL3-1.268 kb vector was co-transfected into PK15 cells in triplicate with an
internal control pRL-TK (Promega). The cells were transfected with
Lipofectamine 2000 in 24-well plates. Each well included 1.5105 cells, 0.8 mg pGL3-1.268 kb, 0.08 mg pRL-TK, 2 ml Lipofectamine
2000, and 500 ml RPMI 1640 medium without serum or antibiotics. Empty pGL3-Basic
(promoter-less) with pRL-TK was also transfected in triplicate in parallel as a
control. All cells were analyzed for dual-luciferase reporter gene expression
48 h after completion of the transfection procedure. The activities of firefly
luciferase in pGL3 and Renilla luciferase in pRL-TK were determined following
the dual-luciferase reporter assay protocol recommended by Promega. The cells
were rinsed with phosphate-buffered saline after harvest and cell lysates were
prepared by manually scraping the cells from culture plates in the presence
of 1?passive lysis buffer. Twenty
milliliters of cell lysate was transferred into the luminometer tube containing
100 ml Luciferase Assay Reagent II (Promega). Firefly luciferase activity (M1)
was measured, then Renilla luciferase activity (M2) was measured after adding
100 ml Stop & Glo reagent (Promega). The program of the luminometer was a 2
s pre-measurement delay followed by a 10 s measurement period for each assay.
Results
Molecular cloning and sequence
analysis of porcine LGALS1 gene
Analysis of the cDNA sequence of porcine LGALS1 revealed the
following results. The full-length cDNA of porcine LGALS1 is 559 bp and
contains an open reading frame of 408 bp encoding a protein of 135 residues
with a calculated molecular mass of 14.72 kDa and an isoelectric point of 4.86.
It contains a 5‘-untranslated region of 71 bp and a 3‘-untranslated
region of 80 bp with a consensus AATAAA polyadenylation signal 21 bp before the
poly(A) stretch. The sequence of porcine LGALS1 had been submitted to
GenBank (GenBank accession no.
DQ367936). There are several phosphorylation, N-myristoylation
and galaptin signature sites, but no protein-binding motifs, signal peptide, or
transmembrane regions are common to any other known protein family predicted by
ExPASy. A BLAST search (http://www.ncbi.nlm.nih.gov/blast) in the
GenBank database indicated that the predicted protein shared high similarity
with other mammals, 85% identity to human and rat, and 83% identity to mouse.The genomic DNA sequences of porcine LGALS1 (GenBank
accession no. DQ367937) were
obtained by PCR amplification. It is interesting that there is a CpG island
predicted by CpG Island Searcher, from 1.168 kb to 2.281 kb, the GC level is
61.4%, and ObsCpG/ExpCpG is 0.705. A promoter is also found in this region,
from 1.279 to 1.529 kb, the score is 78.02%, and there are some important
transcription factors such as Sp1, myosin-specific factor, and UCE-2. In human
and mouse, there are also similar structures analyzed by the same method. Three
short interspersed sequence nucleotide elements (124–296, 729–835, and 2555–2671), one long
interspersed sequence nucleotide elements (2391–2495), and one simple
repeat (2279–2309) were also discovered in this gene.
Spatial and temporal
expression patterns of LGALS1
Porcine LGALS1 was expressed at the highest level in the
skeletal muscle with prominent expressions detected in the lung, lymph node,
and testis, and lower levels detected in heart, liver, spleen, kidney, small
intestine, and brain (Fig. 1). These data are generally in agreement
with the expression pattern of LGALS1 in both human and mouse [15].As shown in Fig. 2, porcine LGALS1 was expressed at 33
and 65 dpc at a relatively high level, then decreased at 90 dpc.
Cellular localization of
porcine LGALS1 in PK15 cells
The cellular location of LGALS1 was studied by fluorescence
and confocal analysis of PK15 cells transiently transfected with pEGFP-LGALS1.
Hoechst 33342 was used to label nuclei. LGALS1 fusion protein was found to
localize both in cytoplasm and nuclei (Fig. 3). Green fluorescence was
detected through control cells, transfected with GFP vector alone.
Promoter activity of the
cloned 1.268 kb fragment
The firefly luciferase expression driven by the 1.268 kb predicted
promoter of LGALS1 was examined to evaluate the promoter activity. The
relative luciferase activity of the experimental sample is presented by the
ratio of the activities of firefly luciferase and Renilla luciferase (M1/M2).
The result showed that the relative activity (M1/M2) was 0.240, 48 h after
pGL3-1.268 kb was co-transfected into PK15 cells with pRL-TK. This was only
12-fold higher than that of pGL3-Basic co-transfection with pRL-TK (Fig. 4).
Discussion
LGALS1 is secreted during
differentiation and binds to lamin [16], and it inhibits cell-matrix
interaction. The inhibition of cell-matrix adhesion has been proposed to play
an important role in muscle formation in mouse [5,16]. Muscle mass is largely
determined by the number of muscle fibers and the size of those fibers. In this
study, the full-length cDNA of porcine LGALS1 gene was obtained. The
porcine LGALS1 gene was mapped to SSC5p11-p15 [17]. In the latest
released Pig QTL Database (http://www.animalgenome.org/QTLdb/pig.html)
[18], several QTLs for the proportion of muscle fiber types and their size,
which affects muscularity as well as functional properties of the musculature
and meat quality [19], were mapped to this small chromosomal region, indicating
that this gene might be a positional candidate gene for these traits. The temporal expression data indicate that porcine LGALS1 was
expressed at 33 and 65 dpc at a relatively high level, then decreased at 90
dpc, results similar to those of Tang et al [20]. In mouse, myoblasts
release galectin-1 while undergoing differentiation, but not while
proliferating. It is expressed at a high level when maximum cell fusion is
occurring during muscle development [6,7,21]. It was reported that the numbers
of pig skeletal muscle fiber stopped increasing at approximately 90 dpc [12],
and then the fiber began to hypertrophy; our results are in agreement with
these reports. Therefore, it could be inferred that LGALS1 acts as an
enhancing factor of myofiber formation during muscle development and it is
more important for increasing muscle fiber numbers than for fiber hypertrophy. We further examined LGALS1 expression in porcine tissues by
reverse transcription-PCR. Porcine LGALS1 showed a wide distribution in
tissues. Then we detected its protein, galectin-1, distribution in PK15 cells,
and we found it localized both in cytoplasm and nuclei. This might be relative
to its various biological functions. There is no predicted signal peptide in
this protein, so we deduce that galectin-1 might be secreted by non-classical
mechanisms, not through classical vesicle-mediated exocytosis. And there are
reports suggesting that galectin-1 is secreted by non-classical mechanisms in
rat and mouse [7,22]. However, the real mechanisms are not clear and need
further study.Introns are non-coding DNA sequences that widely reside in the
genome of eukaryote. The regulatory elements of introns often affect the
efficiency of gene expression [23], so we tried to test if there are regulatory
elements within LGALS1. Through prediction, we found a CpG island and a
possible promoter region. But the luciferase activity of the predicted promoter
region is rather weak, only 12-fold higher than that of pGL3-Basic. In general,
an over 50-fold increase in luciferase activity characterizes a typical
promoter region [24,25], so the result indicated that the predicted promoter
region has little promoter activity. There should be other regions that
regulate the gene expression of LGALS1. The result also indicated that
not all regulatory regions are within or nearby CpG islands.In summary, we have isolated and characterized the porcine LGALS1
gene. Data presented here provides biochemical and structural bases for future
studies of porcine LGALS1 function. It will potentially lead to a
better understanding of the mechanism of LGALS1 function in muscle
fiber formation, thereby influencing muscle development.
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