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Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats
Gene 503 (2012) 222–228
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Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats Xia Meng 1, Feihu Li, Shulin Chen ⁎, Caiyan Tang, Wenhua Zhang, Zhonghui Wang, Shanting Zhao College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi 712100, China
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Article history: Accepted 20 April 2012 Available online 8 May 2012 Keywords: Neuron-specific enolase Corpus luteum Diffuse neuroendocrine system Immunofluorescence staining Molecular cloning Coding sequence
a b s t r a c t Neuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by the immunofluorescence method and the ultra structural characteristics of luteal cells were detected by the electron microscopy to identify the existence of DNES cells in the pregnant CL of dairy goats. Besides, the coding sequence of dairy goats NSE gene was cloned and its biological information was analyzed. Results revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the whole pregnant CL. In addition, these cells showed typical characteristics of DNES cells in the electron microscopy. These results suggested that many DNES cells exist in the pregnant CL of dairy goats. Meanwhile, we identified the coding sequence of dairy goats NSE (GenBank Accession No. JN887466). Its nucleotide sequence homology was found to be 97.9%, 89.3%, 90% and 92.6%, respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These results first showed that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species. Our findings demonstrated the possible existence of DNES cells in pregnant CL, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. Characterization of gene sequence of dairy goats NSE will enable us to synthesize interference RNA for further study on the role of NSE on the formation, function and apoptosis of pregnant CL in dairy goats. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The dispersed or diffuse neuroendocrine system (DNES) was first suggested by Pearse in 1977; it consists of endocrine neurons and amine precursor uptake and decarboxylation (APUD) cells (Pearse, 1977). DNES regulates and controls the physiological course of the organism together with the nervous system. Therefore, it plays an important role in adjusting and controlling the activity of the organism. The concept of DNES can reflect the regulated meaning with movable organism, and it has led the endocrinology to a new scope which has Abbreviations: A, adenosine; APUD, amine precursor uptake and decarboxylation; C, cytidine; cDNA, DNA complementary to RNA; CL, corpus luteum; CDS, coding sequence; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; DNES, diffuse neuroendocrine system; FITC, fluorescein isothiocyanate; G, guanosine; Ig, immunoglobulin(s); NSE, neuron-specific enolase; PBS, phosphate buffer; PCR, polymerase chain reaction; PEP, phosphoenol-pyruvate; PGF2α, prostaglandin F2α; PI, isoelectric point; RNAi, RNA interference; SYN, synapsin; T, thymidine; TEM, transmission electron microscopy; TNF, tumor necrosis factor; 2PG, 2-phospho-D-glycerate; 5-HT, 5-hydroxy tryptamine. ⁎ Corresponding author at: Xinong Road No. 22, College of Veterinary Medicine, Northwest A & F, University, Yangling, Shaanxi 712100, China. Tel.: + 86 029 8709 1359; fax: + 86 029 8709 1032. E-mail addresses: [email protected] (X. Meng), [email protected] (S. Chen). 1 Xinong Road No. 22, College of Veterinary Medicine, Northwest A & F, University, Yangling, Shaanxi 712100, China. Tel.: + 86 029 87091359; fax: + 86 029 8709 1032. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.058
been divided into DNES and non-DNES. Most endocrine cells, tissues and organs belong to the DNES while cells such as adrenocortical cells and thyroid follicular cells that secret corticosteroids and thyroid hormones, respectively, were identified to be non-DNES. DNES cells secrete a variety of products, including low molecular weight substances, cytokines and chemokines. These substances act via neurocrine, endocrine, and paracrine mechanisms and maintain homeostasis by communication and reciprocal regulation between the nervous, endocrine, and immune systems (Polak and Bloom, 1986; Salzet, 2002). Studies have shown that DNES cells are distributed in many organs and tissues (Kaltsas et al., 2004), and have revealed that DNES cells also exist in the immune organs, cardiovascular and reproductive systems. The corpus luteum (CL) is a small, temporary endocrine structure in mammals that develops from the remains of a follicle following ovulation. If fertilization occurs, the CL enlarges and is maintained until the end of the pregnancy in most species except human and mares. It participates in a series of events during gestation by secreting hormones such as progesterone. If fertilization does not occur or in the end of the pregnancy (or postpartum), the CL undergoes a process of regression leading to its disappearance from the ovary, allowing the initiation of a new cycle. Thus the CL is a very important toll-gate in the mammalian reproduction. As an active Endocrine gland whose function and survival are limited in scope and time, it is yet unknown whether luteal cells belong to DNES
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or not. In recent studies progesterone, androgen, estrogen, prostaglandin F2α (PGF2α), activin, inhibin and oxytocin have been detected in the CL (Davis et al., 1987; Ivell and Richter, 1984; McNatty et al., 1979). In addition, a series of cytokines and its specific receptors such as interferon (Neuvians et al., 2004), insulin-like growth factors, tumor necrosis factor (TNF), TNF receptor, plasminogen activator and its inhibitor, interleukin1, -6 (Sakumoto et al., 2006), monocyte chemoattractant protein-1 (Senturk et al., 1999) and leptin (Archanco et al., 2003) were detected in the CL of humans and rats. These results suggested that DNES cells may also exist in the CL and play a crucial role in the interactions between the neuroendocrine and reproductive systems. NSE is known as a glycolytic enzyme that catalyzes the interconversion between 2-phospho-D-glycerate (2PG) and phosphoenolpyruvate (PEP) (Tracy and Hedges, 2000). NSE has been considered to be localized in neurons, it is also present in peripheral and central neuroendocrine cells, also termed APUD cells, which belong to DNES now. The striking and consistent staining of APUD cells in rat and monkey by NSE antiserum coupled with quantitative evidence that in rat, monkey and man this represents actual NSE content make NSE a unique, generalized molecular marker for DNES cells (Schmechel et al., 1978). In practical terms, molecular markers are invaluable in defining DNES cells such as NSE, S-100 protein, SYN, 5-HT and so on. NSE was used as the key molecular marker to identify the DNES cells. The aim of the present study was to detect protein expression of NSE in pregnant CL of dairy goats in the gestation period by the immunofluorescence method, to clone the coding sequence (CDS) of dairy goats NSE gene and analyze its sequence homology with other species, and to detect
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the structural characteristic of luteal cells by electron microscopy to provide morphological data of DNES cells in the pregnant CL of dairy goats. The study demonstrated the existence of DNES cells in pregnant CL by using molecular marker NSE associated with morphological and ultrastructural characteristics of luteal cells, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. The study also provided valuable information on the gene sequence of NSE in dairy goats that is useful for future study on the function of NSE and research on the relation between NSE and the apoptosis of luteal cells during regression.
2. Materials and methods 2.1. Sampling All experimental procedures involving animals were conducted under the rules for experimental Animals of Northwest A & F University. Tissue samples of ovary from pregnant Guan Zhong Shaneng dairy goats (approximately 8 weeks of pregnancy) collected from a local abattoir were immediately removed and dissected longitudinally. One portion was used for specimen preparation for transmission electron microscopy; one portion was fixed in 4% buffered formalin for 24 h at 4 °C and the CL was separated from the ovaries, then the CL tissues were dehydrated using increasing ethanol concentrations 50%–70%–80%–90%–95%I– 95%II–100%I–100%II, embedded in paraffin; the other portion was immediately frozen in liquid nitrogen until it was used for RNA extraction.
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Fig. 1. Ultra-structure of luteal cells. (A) Nucleus and organelles of luteal cells (TEM ×26,000).1. Nucleus; 2. endoplasmic reticulum; 3. chondriosome; 4. lysosome. (B) Secretory granules in luteal cells (TEM × 26,000). 1. Nucleus; 2, 3 and 4. osmiophilic granule. (C) Osmiophilic granule structure and chondriosome in luteal cells (TEM ×26,000).1. Hollow-core construction of secretory granules; 2. halo of secretory granules; 3. chondriosome. (D) Halo of secretory granules in luteal cells (TEM ×26,000).1 and 2. Halo of secretory granules.
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2.2. Transmission electron microscopy For ultra structural study, sections of CL tissues (1 mm) were fixed in 2.5% glutaraldehyde for 24 h. After fixation they were washed in 0.1 M phosphate buffer (PBS) and postfixed with OSO4 in 0.1 M PBS for 1 h. Slices were finally washed, dehydrated and flat-embedded in Epon812. Sections were stained with uranyl acetate and lead citrate. The sections were examined and photographed with JEM-100SX (Japan electron) transmission electron microscopy (TEM). The experiment has been repeated for three times.
information of primary structure of the NSE protein, including relative molecular mass and theoretical isoelectric point (PI), was predicted by ProtParam. The potential transmembrane domains were analyzed by Online Programs TMPRED. Protein domain in goat NSE was identified by using the SMART (Simple Modular Architecture Research Tool) server at http://smart.embl-heidelberg.de/. S.I.F.T. tools (sort intolerant from tolerant) were applied to classify as tolerated or deleterious the substitutions occurring in NSE. The program conducts numerous comparisons of loaded amino-acid sequences and subsequently calculates the probability of all possible substitutions for each amino-acid residue (Ng and Henikoff, 2001).
2.3. Immunofluorescence staining 3. Results Tissue sections (7 μm) were deparaffinized in xylene, and rehydrated in a graded series of ethanol 100%I–100%II–95%–80%–70%. Immunofluorescence staining was operated according to the manufacturer's instructions (Immunol Fluorence Staining Kit with FITC, Beyotime Biotechnology, China) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime Biotech, Jiangsu, China) solution was added to stain cell nucleus for 5 min after immunofluorescence staining. The sections were blocked with 2% normal goat serum, the primary antibody was antisera vs. NSE (1:400) (rabbit monoclonal antibody ERP3377, Epitomics Biotechnology, USA), and the secondary antibody was fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H+L) (1:75) (CW114A, CoWin Bioscience Co., Ltd., Beijing, China). NSE and cell nucleus were monitored with fluorescence microscope inspired by green and blue lights, respectively. Negative controls were produced by substituting the primary antibody with 0.1 M PBS. The experiment has been repeated for three times.
3.1. Ultra-structure of luteal cells The structural characteristics about nucleus and some organelles of luteal cells were shown in Fig. 1A. Electron microscopy data showed that some luteal cells had conspicuous, intracy-toplasmic, dense-core neurosecretory granules, which ranged from round to pleomorphic. Osmiophilic granule existed in luteal cells (Fig. 1B). The granules scattered in the cytoplasm, about 100–500 μm in diameter, are round or oval. There was a peri-theca around the granules and a nucleoid with different electron emission densities and shapes located centrally or leaning toward one side of the granules. Some secretory granules were hollow (Fig. 1C). The nuclei were round or slightly irregular, with small nucleoli. Halo, which has clearance in various widths existed between the nucleoid and the peri-theca (Figs. 1C, D). 3.2. Localization of NSE immunoreactive cells
2.4. RNA extraction and RT-PCR Total RNA was isolated using RNAiso Plus (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions. The first strand of cDNA was obtained using Reverted First Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). PCR primer pairs which cover the whole CDS of the NSE gene were designed using Primer Premier 5.0 based on the conserved sequence of NSE in Rattus norvegicus and Bos taurus. The forward and reverse primers, 5′ATGTCCATAGAG AAGATTTGGGCC-3′ and 5′-TCACAGCACACTGGGGT TGCG-3′, respectively, were synthesized and stored at −20 °C. A 1305 bp nucleotide of the goat NSE was amplified in a 50 μL reaction mixture which was made up according to the manufacturer's instructions (TaKaRa Biotechnology, Dalian, China). PCR reaction was performed in the Alpha Unit Block Assembly for DNA Engine Systems (Bio-Rad, USA) as follows: Touchdown PCR, denaturation at 94 °C for 3 min, 30 amplification cycles including denaturation at 94 °C for 30 s, annealing for 30 s (the annealing temperature of the reaction was decreased 0.5 °C every second cycle from 65 °C to a ‘touchdown’ at 50°C, at which temperature 30 cycles were carried out) and extension at 72 °C for 90 s, followed by a final 10 min extension at 72 °C. The amplified DNAs were confirmed by electrophoresis on a 1% agarose gel and then purified using a DNA fragment gel purification and extraction kit (BioDev-Tech, China). 2.5. Sequencing and genotyping The purified DNAs were ligated into the pMD18-T vector (TransGen, China) according to the manufacturer's instructions. The ligated vector was transformed into DH5α competent cells (Transgen Biotech, Beijing, China) and the plasmid DNA was confirmed by enzyme digestion with EcoR I and Hind III. The successfully constructed plasmid was purified and sequenced at both directions by GenScript (Nanjing, China). The NSE gene sequences and deduced amino acid alignment were analyzed using DNASTAR (Version 5.01), and were compared with NSE sequences of other mammals available in the GenBank database. The biological
The immunofluorescence study revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the entire CL (Fig. 2A). The background was black under the Laser scanning confocal microscope. Immunoreactive cells were clearly visible in green with dark green stained granules in the cytoplasm and a blue nucleus that is located centrally or laterally in the cell. Cells were in various shapes including round, oval, and triangular. The large luteal cells were about 22–25 μm in diameter and had a low nuclear to cytoplasmic ratio; the small luteal cells were about 12–20 μm in diameter and had a high nuclear to cytoplasmic ratio. The positive reaction occurred mainly in the large luteal cells; however, it was also found in the small luteal cells. No cytoplasmic stained cells were evident when the primary antibody was replaced by 0.1 M PBS (Fig. 2B). 3.3. Results of RT-PCR and restriction enzyme digestion The NSE CDS of dairy goats was amplified, and the target PCR product had an approximately 1305 bp nucleotide fragment as shown by electrophoresis on 1% agarose gel (Fig. 3A). Enzyme digestion of the constructed plasmid by ligating the amplified NSE fragment to the pMD18-T vector yielded the 1305 bp fragment (Fig. 3B). 3.4. Sequence analysis of the CDS of dairy goat NSE gene The NSE CDS sequence was analyzed by the DNASTAR software and aligned with the consensus sequence of other known mammals available in the GenBank database. The newly determined dairy goat NSE CDS sequence has been deposited in the GenBank database with accession number JN887466. When compared with other mammalian NSE, the nucleotide sequence alignments of caprine NSE (JN887466) showed high identity with that of B. taurus (NM_001101125, 97.9%), R. norvegicus (NM_139325.2, 89.3%), M. musculus (NM_013509.2, 90%), and H. sapiens (NM_001975.2, 92.6%) (Fig. 4A). When compared with other mammalian NSE, the amino acid sequence alignments of caprine NSE showed high identity with that
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Fig. 2. Localization of NSE protein in corpus luteum. (A) NSE immunoactive cell in corpus luteum (×100). Green fluorescent NSE in the cytoplasm of luteal cells was observed with FITC filter; nuclei were counterstained blue with DAPI. LLC: large luteal cells; SLC: small luteal cells. (B) Control for (A) without primary antibody (×100). No detectable cytoplasmic staining was observed; nuclei were counterstained blue with DAPI.
of B. taurus (NP_001094595, 99.1%), R. norvegicus (NP_647541, 97%), M. musculus (NP_038537, 97.2%), and H. sapiens (NP_001966, 98.2%) (Fig. 4B). The deduced amino acid alignment of 5 mammalian NSE proteins was shown in Fig. 5A. The amino acid sequence of caprine NSE started with the consensus sequence MSIEK, which is the characteristic of the animals analyzed in this study. The starting sequence was different from that of R. norvegicus. In both the C-terminal domain and the N-terminal domain, there were some highly stable regions, showing high conservation. In the N-terminal domain, a fragment with the sequence Asp-Ser-ArgGly-Asn-Pro-Thr-Val-Glu (approximately residues 13–21) was one such region. Another highly stable region in the N-terminal domain was ProSer-Gly-Ala-Ser-Thr-Gly (residues 36–42). The N-terminal domain ended with highly nonvariable elements Gly-Ala-Asn-Ala-Ile-Leu-GlyVal-Ser-Leu-Ala-Val (residues 107–118). The C-terminal region started with highly nonvariable fragment Leu-Pro-Val-Pro (residues 145–148). The C-terminal domain (~290 amino-acid residues), was relatively rich in conservative elements, such as Gly-Asp-Glu-Gly-Gly-Phe-Ala-Pro (residues 208–215), Val/Leu-Ser-His-Arg (residues 369–372), and Gly-GlnIle-Lys-Thr (residues 391–395), divided by weakly variable fragments. In the 5 mammalian NSE molecules 18 sites of tolerated substitutions were present, eleven of which lay in the N-terminal domain. Meanwhile, caprine NSE had 6 sites of tolerated substitutions, five of which lay in the N-terminal domain. The phylogenetic tree of 5 mammalian NSE proteins is presented in Fig. 5B. Caprine and B. taurus reside in the same group with small
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variations between them, and are phylogenetically distant from other mammals including H. sapiens, R. norvegicus and M. musculus. 4. Discussion As NSE is a unique, generalized molecular marker for DNES cells, NSE was used as the key molecular marker to identify the DNES cells. Previous classification of DNES cells has had to rely on staining and physiological properties which occasionally proved difficult to document for a given cell class, the presence of NSE provides a simple and useful method for identifying members of this group (Schmechel et al., 1978). The present study provided the distribution of NSE in the caprine CL. As NSE antiserum reacts specifically and uniformly with DNES cells, NSE positive cells were DNES cells. Additionally, we found that the luteal cells bore physical signs of DNES cells according to their morphological and ultra-structural characteristics (Barakat et al., 2004). Considering the above results, it is certain that DNES cells exist in CL of goats. We identified the DNES cells in CL by using molecular marker associated with morphological and ultra-structural characteristics of luteal cells. NSE is known as a glycolytic enzyme that catalyzes the interconversion between 2-phospho-D-glycerate (2PG) and phosphoenolpyruvate (PEP) (Tracy and Hedges, 2000). NSE is highly expressed in the neuronal neuroendocrine tissues and in lung cancer tissues (Ferrigno et al., 2003). Many reports are available demonstrating the direct correlation between increased expression of enolase and progression of neuroendocrine tumors, neuroblastoma and lung cancer (Eriksson et
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Fig. 3. Results of RT-PCR and restriction enzyme digestion. (A) Analysis of goat NSE PCR product by agarose gel electrophoresis.M. DNA marker DL1000; 1. dairy goat NSE CDS. (B) Identification of recombinant plasmids by double-enzyme cleavage method. M. DNA marker DL5000; 1. Restriction enzyme cutting products.
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(B) Fig. 4. Sequence identity of 5 mammalian NSE protein genes. (A) Nucleotide sequence identity of 5 mammalian NSE protein genes.1. Caprine (JN_887466). 2. Bos taurus (NM_001101125). 3. Rattus norvegicus (NM_139325.2). 4. Mus musculus (NM_013509.2). 5. Homo sapiens (NM_001975.2). (B) Amino acid sequence identity of 5 mammalian NSE protein genes. 1. Caprine (AEV_53911). 2. B. taurus (NP_001094595). 3. R. norvegicus (NP_647541). 4. M. musculus (NP_038537). 5. H. sapiens (NP_001966).
al., 2000). The expression level of enolase is too high for its contribution to glycolysis alone (Chai et al., 2004). Recent studies have demonstrated that several glycolytic enzymes are capable of functioning in different biological and physiological processes (Pancholi, 2001). Enolase is a multifunctional protein (Kim and Dang, 2005). The p19ras inhibits lung cancer cell proliferation by blocking of energy supply through the repression of NSE-enzyme activity (Jang et al., 2010), NSE gene silencing suppresses proliferation and promotes apoptosis of lung cancer cells in vitro (Zhou et al., 2011), all these results suggest that NSE gene expression promotes the cell proliferation and inhibits cell apoptosis. As NSE was located in the caprine CL, we speculate that NSE may play some certain role in the formation, function and apoptosis of CL, but its specific function in CL is still unclear and further study is needed to elucidate the NSE function in the CL. RNA interference (RNAi) is a valuable tool for the gene function analysis. We plan to use siRNA to silence the NSE gene in order to study the function of NSE in the CL. However, little is known about the sequence and molecular characteristics of the caprine NSE gene. In the present study, the CDS of caprine NSE gene was firstly isolated. The nucleotide sequence homology of NSE CDS of dairy goats was found to be 97.9%, 89.3%, 90% and 92.6% compared with that of B. taurus, R. norvegicus, M. musculus and H. sapiens, respectively, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These comparisons, similar to those of earlier work (Hannaert et al., 2000; Piast et al., 2005) demonstrated a high degree of nucleotide sequence and amino acid sequence conservation in NSE. The amino-acids critical for enzyme function are situated in especially well conserved fragments of the molecule. The active sites His-158, His190, Arg-372, Lys-394, Asp-318 and Lys-343, as well as the magnesium ion ligand binding sites Asp-245, Glu-293 and Asp-318 are conserved in
caprine NSE protein as they are in other mammalian NSE proteins (Lebioda and Stec, 1988; Mcaleese et al., 1988; Piast et al., 2005). Thiol groups may play a critical role in glycolytic enzyme activity and function (Banaś et al., 1988). It is worth mentioning that α and β enolase isoforms contain six cysteine residues (119, 337, 339, 357, 389 and 399). Besides, the NSE of B. taurus, R. norvegicus, M. musculus and H. sapiens contains all those mentioned above plus one additional Cys-270 (Piast et al., 2005). The present findings indicated that the goat NSE also contains all those six cysteine residues mentioned above. It might have important implications on structure and function of NSE. The above results proved that many DNES cells exist in the CL of dairy goats and the functional amino acids coded by the NSE gene were highly conserved in this species, as NSE gene expression promotes the cell proliferation and inhibits cell apoptosis in lung cancer cells of human with neuroendocrine differentiation (Zhou et al., 2011), we speculate that the gene NSE in caprine CL showed the same function as in human, that is, NSE gene expression may promote cell proliferation and inhibit cell apoptosis in caprine CL. 5. Conclusions In summary, the present study provided the distribution of NSE in the pregnant CL of dairy goats and proved that many DNES cells exist in the pregnant CL of dairy goats. It provided a morphological basis for further investigation on the role of pregnant CL in neuroendocrine and reproductive systems in dairy goats. In addition, the dairy goat NSE gene CDS was firstly identified in the present study. We found that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens, it was implied that the gene NSE in dairy
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Fig. 5. Protein sequence comparison of 5 mammalian NSE proteins. (A) Amino acid alignment of 5 mammalian NSE proteins, including Caprine (AEV_53911), Bos taurus (NP_001094595), Rattus norvegicus (NP_647541), Mus musculus (NP_038537) and Homo sapiens (NP_001966). The exclamation marks indicate the sites of tolerated substitutions. (B) The phylogenetic tree of 5 mammalian NSE proteins.
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Fig. 5 (continued).
goats had close homology to that of NSE of other species. These results may provide some new clue to reveal the function of NSE in the formation, function and apoptosis of pregnant CL of dairy goats. The revelation of the CDS of the dairy goats NSE gene will help construct siRNA to discover the relationship between NSE and the physiology of the pregnant CL during its formation and regression.
Acknowledgment This work was supported by the Natural Science Foundation of China (Project no. 30972151). We thank professor Walter H. Hsu for the critical proof reading of the manuscript and helpful suggestion.
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Cloning and expression of neuron-specific enolase in the corpus luteum of dairy goats Xia Meng 1, Feihu Li, Shulin Chen ⁎, Caiyan Tang, Wenhua Zhang, Zhonghui Wang, Shanting Zhao College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi 712100, China
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Article history: Accepted 20 April 2012 Available online 8 May 2012 Keywords: Neuron-specific enolase Corpus luteum Diffuse neuroendocrine system Immunofluorescence staining Molecular cloning Coding sequence
a b s t r a c t Neuron-specific enolase (NSE) is the key molecular marker for diffuse neuroendocrine system (DNES) cells, its expression in the pregnant corpus luteum (CL) of dairy goats was studied by the immunofluorescence method and the ultra structural characteristics of luteal cells were detected by the electron microscopy to identify the existence of DNES cells in the pregnant CL of dairy goats. Besides, the coding sequence of dairy goats NSE gene was cloned and its biological information was analyzed. Results revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the whole pregnant CL. In addition, these cells showed typical characteristics of DNES cells in the electron microscopy. These results suggested that many DNES cells exist in the pregnant CL of dairy goats. Meanwhile, we identified the coding sequence of dairy goats NSE (GenBank Accession No. JN887466). Its nucleotide sequence homology was found to be 97.9%, 89.3%, 90% and 92.6%, respectively, compared with that of Bos taurus, Rattus norvegicus, Mus musculus and Homo sapiens, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These results first showed that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens. It was implied that the gene NSE in dairy goats had close homology to that of NSE of other species. Our findings demonstrated the possible existence of DNES cells in pregnant CL, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. Characterization of gene sequence of dairy goats NSE will enable us to synthesize interference RNA for further study on the role of NSE on the formation, function and apoptosis of pregnant CL in dairy goats. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The dispersed or diffuse neuroendocrine system (DNES) was first suggested by Pearse in 1977; it consists of endocrine neurons and amine precursor uptake and decarboxylation (APUD) cells (Pearse, 1977). DNES regulates and controls the physiological course of the organism together with the nervous system. Therefore, it plays an important role in adjusting and controlling the activity of the organism. The concept of DNES can reflect the regulated meaning with movable organism, and it has led the endocrinology to a new scope which has Abbreviations: A, adenosine; APUD, amine precursor uptake and decarboxylation; C, cytidine; cDNA, DNA complementary to RNA; CL, corpus luteum; CDS, coding sequence; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; DNES, diffuse neuroendocrine system; FITC, fluorescein isothiocyanate; G, guanosine; Ig, immunoglobulin(s); NSE, neuron-specific enolase; PBS, phosphate buffer; PCR, polymerase chain reaction; PEP, phosphoenol-pyruvate; PGF2α, prostaglandin F2α; PI, isoelectric point; RNAi, RNA interference; SYN, synapsin; T, thymidine; TEM, transmission electron microscopy; TNF, tumor necrosis factor; 2PG, 2-phospho-D-glycerate; 5-HT, 5-hydroxy tryptamine. ⁎ Corresponding author at: Xinong Road No. 22, College of Veterinary Medicine, Northwest A & F, University, Yangling, Shaanxi 712100, China. Tel.: + 86 029 8709 1359; fax: + 86 029 8709 1032. E-mail addresses: [email protected] (X. Meng), [email protected] (S. Chen). 1 Xinong Road No. 22, College of Veterinary Medicine, Northwest A & F, University, Yangling, Shaanxi 712100, China. Tel.: + 86 029 87091359; fax: + 86 029 8709 1032. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.058
been divided into DNES and non-DNES. Most endocrine cells, tissues and organs belong to the DNES while cells such as adrenocortical cells and thyroid follicular cells that secret corticosteroids and thyroid hormones, respectively, were identified to be non-DNES. DNES cells secrete a variety of products, including low molecular weight substances, cytokines and chemokines. These substances act via neurocrine, endocrine, and paracrine mechanisms and maintain homeostasis by communication and reciprocal regulation between the nervous, endocrine, and immune systems (Polak and Bloom, 1986; Salzet, 2002). Studies have shown that DNES cells are distributed in many organs and tissues (Kaltsas et al., 2004), and have revealed that DNES cells also exist in the immune organs, cardiovascular and reproductive systems. The corpus luteum (CL) is a small, temporary endocrine structure in mammals that develops from the remains of a follicle following ovulation. If fertilization occurs, the CL enlarges and is maintained until the end of the pregnancy in most species except human and mares. It participates in a series of events during gestation by secreting hormones such as progesterone. If fertilization does not occur or in the end of the pregnancy (or postpartum), the CL undergoes a process of regression leading to its disappearance from the ovary, allowing the initiation of a new cycle. Thus the CL is a very important toll-gate in the mammalian reproduction. As an active Endocrine gland whose function and survival are limited in scope and time, it is yet unknown whether luteal cells belong to DNES
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or not. In recent studies progesterone, androgen, estrogen, prostaglandin F2α (PGF2α), activin, inhibin and oxytocin have been detected in the CL (Davis et al., 1987; Ivell and Richter, 1984; McNatty et al., 1979). In addition, a series of cytokines and its specific receptors such as interferon (Neuvians et al., 2004), insulin-like growth factors, tumor necrosis factor (TNF), TNF receptor, plasminogen activator and its inhibitor, interleukin1, -6 (Sakumoto et al., 2006), monocyte chemoattractant protein-1 (Senturk et al., 1999) and leptin (Archanco et al., 2003) were detected in the CL of humans and rats. These results suggested that DNES cells may also exist in the CL and play a crucial role in the interactions between the neuroendocrine and reproductive systems. NSE is known as a glycolytic enzyme that catalyzes the interconversion between 2-phospho-D-glycerate (2PG) and phosphoenolpyruvate (PEP) (Tracy and Hedges, 2000). NSE has been considered to be localized in neurons, it is also present in peripheral and central neuroendocrine cells, also termed APUD cells, which belong to DNES now. The striking and consistent staining of APUD cells in rat and monkey by NSE antiserum coupled with quantitative evidence that in rat, monkey and man this represents actual NSE content make NSE a unique, generalized molecular marker for DNES cells (Schmechel et al., 1978). In practical terms, molecular markers are invaluable in defining DNES cells such as NSE, S-100 protein, SYN, 5-HT and so on. NSE was used as the key molecular marker to identify the DNES cells. The aim of the present study was to detect protein expression of NSE in pregnant CL of dairy goats in the gestation period by the immunofluorescence method, to clone the coding sequence (CDS) of dairy goats NSE gene and analyze its sequence homology with other species, and to detect
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the structural characteristic of luteal cells by electron microscopy to provide morphological data of DNES cells in the pregnant CL of dairy goats. The study demonstrated the existence of DNES cells in pregnant CL by using molecular marker NSE associated with morphological and ultrastructural characteristics of luteal cells, providing new clue for further understanding of interactions between the neuroendocrine and reproductive systems. The study also provided valuable information on the gene sequence of NSE in dairy goats that is useful for future study on the function of NSE and research on the relation between NSE and the apoptosis of luteal cells during regression.
2. Materials and methods 2.1. Sampling All experimental procedures involving animals were conducted under the rules for experimental Animals of Northwest A & F University. Tissue samples of ovary from pregnant Guan Zhong Shaneng dairy goats (approximately 8 weeks of pregnancy) collected from a local abattoir were immediately removed and dissected longitudinally. One portion was used for specimen preparation for transmission electron microscopy; one portion was fixed in 4% buffered formalin for 24 h at 4 °C and the CL was separated from the ovaries, then the CL tissues were dehydrated using increasing ethanol concentrations 50%–70%–80%–90%–95%I– 95%II–100%I–100%II, embedded in paraffin; the other portion was immediately frozen in liquid nitrogen until it was used for RNA extraction.
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Fig. 1. Ultra-structure of luteal cells. (A) Nucleus and organelles of luteal cells (TEM ×26,000).1. Nucleus; 2. endoplasmic reticulum; 3. chondriosome; 4. lysosome. (B) Secretory granules in luteal cells (TEM × 26,000). 1. Nucleus; 2, 3 and 4. osmiophilic granule. (C) Osmiophilic granule structure and chondriosome in luteal cells (TEM ×26,000).1. Hollow-core construction of secretory granules; 2. halo of secretory granules; 3. chondriosome. (D) Halo of secretory granules in luteal cells (TEM ×26,000).1 and 2. Halo of secretory granules.
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2.2. Transmission electron microscopy For ultra structural study, sections of CL tissues (1 mm) were fixed in 2.5% glutaraldehyde for 24 h. After fixation they were washed in 0.1 M phosphate buffer (PBS) and postfixed with OSO4 in 0.1 M PBS for 1 h. Slices were finally washed, dehydrated and flat-embedded in Epon812. Sections were stained with uranyl acetate and lead citrate. The sections were examined and photographed with JEM-100SX (Japan electron) transmission electron microscopy (TEM). The experiment has been repeated for three times.
information of primary structure of the NSE protein, including relative molecular mass and theoretical isoelectric point (PI), was predicted by ProtParam. The potential transmembrane domains were analyzed by Online Programs TMPRED. Protein domain in goat NSE was identified by using the SMART (Simple Modular Architecture Research Tool) server at http://smart.embl-heidelberg.de/. S.I.F.T. tools (sort intolerant from tolerant) were applied to classify as tolerated or deleterious the substitutions occurring in NSE. The program conducts numerous comparisons of loaded amino-acid sequences and subsequently calculates the probability of all possible substitutions for each amino-acid residue (Ng and Henikoff, 2001).
2.3. Immunofluorescence staining 3. Results Tissue sections (7 μm) were deparaffinized in xylene, and rehydrated in a graded series of ethanol 100%I–100%II–95%–80%–70%. Immunofluorescence staining was operated according to the manufacturer's instructions (Immunol Fluorence Staining Kit with FITC, Beyotime Biotechnology, China) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime Biotech, Jiangsu, China) solution was added to stain cell nucleus for 5 min after immunofluorescence staining. The sections were blocked with 2% normal goat serum, the primary antibody was antisera vs. NSE (1:400) (rabbit monoclonal antibody ERP3377, Epitomics Biotechnology, USA), and the secondary antibody was fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H+L) (1:75) (CW114A, CoWin Bioscience Co., Ltd., Beijing, China). NSE and cell nucleus were monitored with fluorescence microscope inspired by green and blue lights, respectively. Negative controls were produced by substituting the primary antibody with 0.1 M PBS. The experiment has been repeated for three times.
3.1. Ultra-structure of luteal cells The structural characteristics about nucleus and some organelles of luteal cells were shown in Fig. 1A. Electron microscopy data showed that some luteal cells had conspicuous, intracy-toplasmic, dense-core neurosecretory granules, which ranged from round to pleomorphic. Osmiophilic granule existed in luteal cells (Fig. 1B). The granules scattered in the cytoplasm, about 100–500 μm in diameter, are round or oval. There was a peri-theca around the granules and a nucleoid with different electron emission densities and shapes located centrally or leaning toward one side of the granules. Some secretory granules were hollow (Fig. 1C). The nuclei were round or slightly irregular, with small nucleoli. Halo, which has clearance in various widths existed between the nucleoid and the peri-theca (Figs. 1C, D). 3.2. Localization of NSE immunoreactive cells
2.4. RNA extraction and RT-PCR Total RNA was isolated using RNAiso Plus (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions. The first strand of cDNA was obtained using Reverted First Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). PCR primer pairs which cover the whole CDS of the NSE gene were designed using Primer Premier 5.0 based on the conserved sequence of NSE in Rattus norvegicus and Bos taurus. The forward and reverse primers, 5′ATGTCCATAGAG AAGATTTGGGCC-3′ and 5′-TCACAGCACACTGGGGT TGCG-3′, respectively, were synthesized and stored at −20 °C. A 1305 bp nucleotide of the goat NSE was amplified in a 50 μL reaction mixture which was made up according to the manufacturer's instructions (TaKaRa Biotechnology, Dalian, China). PCR reaction was performed in the Alpha Unit Block Assembly for DNA Engine Systems (Bio-Rad, USA) as follows: Touchdown PCR, denaturation at 94 °C for 3 min, 30 amplification cycles including denaturation at 94 °C for 30 s, annealing for 30 s (the annealing temperature of the reaction was decreased 0.5 °C every second cycle from 65 °C to a ‘touchdown’ at 50°C, at which temperature 30 cycles were carried out) and extension at 72 °C for 90 s, followed by a final 10 min extension at 72 °C. The amplified DNAs were confirmed by electrophoresis on a 1% agarose gel and then purified using a DNA fragment gel purification and extraction kit (BioDev-Tech, China). 2.5. Sequencing and genotyping The purified DNAs were ligated into the pMD18-T vector (TransGen, China) according to the manufacturer's instructions. The ligated vector was transformed into DH5α competent cells (Transgen Biotech, Beijing, China) and the plasmid DNA was confirmed by enzyme digestion with EcoR I and Hind III. The successfully constructed plasmid was purified and sequenced at both directions by GenScript (Nanjing, China). The NSE gene sequences and deduced amino acid alignment were analyzed using DNASTAR (Version 5.01), and were compared with NSE sequences of other mammals available in the GenBank database. The biological
The immunofluorescence study revealed that NSE immunopositive cells exhibited widespread cytoplasmic staining throughout the entire CL (Fig. 2A). The background was black under the Laser scanning confocal microscope. Immunoreactive cells were clearly visible in green with dark green stained granules in the cytoplasm and a blue nucleus that is located centrally or laterally in the cell. Cells were in various shapes including round, oval, and triangular. The large luteal cells were about 22–25 μm in diameter and had a low nuclear to cytoplasmic ratio; the small luteal cells were about 12–20 μm in diameter and had a high nuclear to cytoplasmic ratio. The positive reaction occurred mainly in the large luteal cells; however, it was also found in the small luteal cells. No cytoplasmic stained cells were evident when the primary antibody was replaced by 0.1 M PBS (Fig. 2B). 3.3. Results of RT-PCR and restriction enzyme digestion The NSE CDS of dairy goats was amplified, and the target PCR product had an approximately 1305 bp nucleotide fragment as shown by electrophoresis on 1% agarose gel (Fig. 3A). Enzyme digestion of the constructed plasmid by ligating the amplified NSE fragment to the pMD18-T vector yielded the 1305 bp fragment (Fig. 3B). 3.4. Sequence analysis of the CDS of dairy goat NSE gene The NSE CDS sequence was analyzed by the DNASTAR software and aligned with the consensus sequence of other known mammals available in the GenBank database. The newly determined dairy goat NSE CDS sequence has been deposited in the GenBank database with accession number JN887466. When compared with other mammalian NSE, the nucleotide sequence alignments of caprine NSE (JN887466) showed high identity with that of B. taurus (NM_001101125, 97.9%), R. norvegicus (NM_139325.2, 89.3%), M. musculus (NM_013509.2, 90%), and H. sapiens (NM_001975.2, 92.6%) (Fig. 4A). When compared with other mammalian NSE, the amino acid sequence alignments of caprine NSE showed high identity with that
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Fig. 2. Localization of NSE protein in corpus luteum. (A) NSE immunoactive cell in corpus luteum (×100). Green fluorescent NSE in the cytoplasm of luteal cells was observed with FITC filter; nuclei were counterstained blue with DAPI. LLC: large luteal cells; SLC: small luteal cells. (B) Control for (A) without primary antibody (×100). No detectable cytoplasmic staining was observed; nuclei were counterstained blue with DAPI.
of B. taurus (NP_001094595, 99.1%), R. norvegicus (NP_647541, 97%), M. musculus (NP_038537, 97.2%), and H. sapiens (NP_001966, 98.2%) (Fig. 4B). The deduced amino acid alignment of 5 mammalian NSE proteins was shown in Fig. 5A. The amino acid sequence of caprine NSE started with the consensus sequence MSIEK, which is the characteristic of the animals analyzed in this study. The starting sequence was different from that of R. norvegicus. In both the C-terminal domain and the N-terminal domain, there were some highly stable regions, showing high conservation. In the N-terminal domain, a fragment with the sequence Asp-Ser-ArgGly-Asn-Pro-Thr-Val-Glu (approximately residues 13–21) was one such region. Another highly stable region in the N-terminal domain was ProSer-Gly-Ala-Ser-Thr-Gly (residues 36–42). The N-terminal domain ended with highly nonvariable elements Gly-Ala-Asn-Ala-Ile-Leu-GlyVal-Ser-Leu-Ala-Val (residues 107–118). The C-terminal region started with highly nonvariable fragment Leu-Pro-Val-Pro (residues 145–148). The C-terminal domain (~290 amino-acid residues), was relatively rich in conservative elements, such as Gly-Asp-Glu-Gly-Gly-Phe-Ala-Pro (residues 208–215), Val/Leu-Ser-His-Arg (residues 369–372), and Gly-GlnIle-Lys-Thr (residues 391–395), divided by weakly variable fragments. In the 5 mammalian NSE molecules 18 sites of tolerated substitutions were present, eleven of which lay in the N-terminal domain. Meanwhile, caprine NSE had 6 sites of tolerated substitutions, five of which lay in the N-terminal domain. The phylogenetic tree of 5 mammalian NSE proteins is presented in Fig. 5B. Caprine and B. taurus reside in the same group with small
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variations between them, and are phylogenetically distant from other mammals including H. sapiens, R. norvegicus and M. musculus. 4. Discussion As NSE is a unique, generalized molecular marker for DNES cells, NSE was used as the key molecular marker to identify the DNES cells. Previous classification of DNES cells has had to rely on staining and physiological properties which occasionally proved difficult to document for a given cell class, the presence of NSE provides a simple and useful method for identifying members of this group (Schmechel et al., 1978). The present study provided the distribution of NSE in the caprine CL. As NSE antiserum reacts specifically and uniformly with DNES cells, NSE positive cells were DNES cells. Additionally, we found that the luteal cells bore physical signs of DNES cells according to their morphological and ultra-structural characteristics (Barakat et al., 2004). Considering the above results, it is certain that DNES cells exist in CL of goats. We identified the DNES cells in CL by using molecular marker associated with morphological and ultra-structural characteristics of luteal cells. NSE is known as a glycolytic enzyme that catalyzes the interconversion between 2-phospho-D-glycerate (2PG) and phosphoenolpyruvate (PEP) (Tracy and Hedges, 2000). NSE is highly expressed in the neuronal neuroendocrine tissues and in lung cancer tissues (Ferrigno et al., 2003). Many reports are available demonstrating the direct correlation between increased expression of enolase and progression of neuroendocrine tumors, neuroblastoma and lung cancer (Eriksson et
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Fig. 3. Results of RT-PCR and restriction enzyme digestion. (A) Analysis of goat NSE PCR product by agarose gel electrophoresis.M. DNA marker DL1000; 1. dairy goat NSE CDS. (B) Identification of recombinant plasmids by double-enzyme cleavage method. M. DNA marker DL5000; 1. Restriction enzyme cutting products.
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(B) Fig. 4. Sequence identity of 5 mammalian NSE protein genes. (A) Nucleotide sequence identity of 5 mammalian NSE protein genes.1. Caprine (JN_887466). 2. Bos taurus (NM_001101125). 3. Rattus norvegicus (NM_139325.2). 4. Mus musculus (NM_013509.2). 5. Homo sapiens (NM_001975.2). (B) Amino acid sequence identity of 5 mammalian NSE protein genes. 1. Caprine (AEV_53911). 2. B. taurus (NP_001094595). 3. R. norvegicus (NP_647541). 4. M. musculus (NP_038537). 5. H. sapiens (NP_001966).
al., 2000). The expression level of enolase is too high for its contribution to glycolysis alone (Chai et al., 2004). Recent studies have demonstrated that several glycolytic enzymes are capable of functioning in different biological and physiological processes (Pancholi, 2001). Enolase is a multifunctional protein (Kim and Dang, 2005). The p19ras inhibits lung cancer cell proliferation by blocking of energy supply through the repression of NSE-enzyme activity (Jang et al., 2010), NSE gene silencing suppresses proliferation and promotes apoptosis of lung cancer cells in vitro (Zhou et al., 2011), all these results suggest that NSE gene expression promotes the cell proliferation and inhibits cell apoptosis. As NSE was located in the caprine CL, we speculate that NSE may play some certain role in the formation, function and apoptosis of CL, but its specific function in CL is still unclear and further study is needed to elucidate the NSE function in the CL. RNA interference (RNAi) is a valuable tool for the gene function analysis. We plan to use siRNA to silence the NSE gene in order to study the function of NSE in the CL. However, little is known about the sequence and molecular characteristics of the caprine NSE gene. In the present study, the CDS of caprine NSE gene was firstly isolated. The nucleotide sequence homology of NSE CDS of dairy goats was found to be 97.9%, 89.3%, 90% and 92.6% compared with that of B. taurus, R. norvegicus, M. musculus and H. sapiens, respectively, while the amino acid sequence homology was 99.1%, 97%, 97.2% and 98.2% respectively. These comparisons, similar to those of earlier work (Hannaert et al., 2000; Piast et al., 2005) demonstrated a high degree of nucleotide sequence and amino acid sequence conservation in NSE. The amino-acids critical for enzyme function are situated in especially well conserved fragments of the molecule. The active sites His-158, His190, Arg-372, Lys-394, Asp-318 and Lys-343, as well as the magnesium ion ligand binding sites Asp-245, Glu-293 and Asp-318 are conserved in
caprine NSE protein as they are in other mammalian NSE proteins (Lebioda and Stec, 1988; Mcaleese et al., 1988; Piast et al., 2005). Thiol groups may play a critical role in glycolytic enzyme activity and function (Banaś et al., 1988). It is worth mentioning that α and β enolase isoforms contain six cysteine residues (119, 337, 339, 357, 389 and 399). Besides, the NSE of B. taurus, R. norvegicus, M. musculus and H. sapiens contains all those mentioned above plus one additional Cys-270 (Piast et al., 2005). The present findings indicated that the goat NSE also contains all those six cysteine residues mentioned above. It might have important implications on structure and function of NSE. The above results proved that many DNES cells exist in the CL of dairy goats and the functional amino acids coded by the NSE gene were highly conserved in this species, as NSE gene expression promotes the cell proliferation and inhibits cell apoptosis in lung cancer cells of human with neuroendocrine differentiation (Zhou et al., 2011), we speculate that the gene NSE in caprine CL showed the same function as in human, that is, NSE gene expression may promote cell proliferation and inhibit cell apoptosis in caprine CL. 5. Conclusions In summary, the present study provided the distribution of NSE in the pregnant CL of dairy goats and proved that many DNES cells exist in the pregnant CL of dairy goats. It provided a morphological basis for further investigation on the role of pregnant CL in neuroendocrine and reproductive systems in dairy goats. In addition, the dairy goat NSE gene CDS was firstly identified in the present study. We found that the functional amino acids coded by the NSE gene were highly conserved in Caprine, B. taurus, R. norvegicus, M. musculus and H. sapiens, it was implied that the gene NSE in dairy
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Fig. 5. Protein sequence comparison of 5 mammalian NSE proteins. (A) Amino acid alignment of 5 mammalian NSE proteins, including Caprine (AEV_53911), Bos taurus (NP_001094595), Rattus norvegicus (NP_647541), Mus musculus (NP_038537) and Homo sapiens (NP_001966). The exclamation marks indicate the sites of tolerated substitutions. (B) The phylogenetic tree of 5 mammalian NSE proteins.
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Fig. 5 (continued).
goats had close homology to that of NSE of other species. These results may provide some new clue to reveal the function of NSE in the formation, function and apoptosis of pregnant CL of dairy goats. The revelation of the CDS of the dairy goats NSE gene will help construct siRNA to discover the relationship between NSE and the physiology of the pregnant CL during its formation and regression.
Acknowledgment This work was supported by the Natural Science Foundation of China (Project no. 30972151). We thank professor Walter H. Hsu for the critical proof reading of the manuscript and helpful suggestion.
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