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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 85�90 |
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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.
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 fluo�rescence 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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