- Email: [email protected]
Cloning and Expression of Equine β-Nerve Growth Factor
Accepted Manuscript Cloning and expression of equine β-nerve growth factor Yosuke Amagai, DVM, PhD, Hiroaki Sato, DVM, Saori Ishizaka, DVM, Kenshiro Matsuda, BS, Christine Aurich, DVM, PhD, Akane Tanaka, DVM, PhD, Hiroshi Matsuda, DVM, PhD PII:
S0737-0806(15)30125-8
DOI:
10.1016/j.jevs.2016.06.001
Reference:
YJEVS 2118
To appear in:
Journal of Equine Veterinary Science
Received Date: 19 December 2015 Revised Date:
1 June 2016
Accepted Date: 1 June 2016
Please cite this article as: Amagai Y, Sato H, Ishizaka S, Matsuda K, Aurich C, Tanaka A, Matsuda H, Cloning and expression of equine β-nerve growth factor, Journal of Equine Veterinary Science (2016), doi: 10.1016/j.jevs.2016.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Cloning and expression of equine β-nerve growth factor
a,1
, Hiroaki Sato DVM
a,1
, Saori Ishizaka DVM
a,b,1
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Yosuke Amagai DVM, PhD
Kenshiro Matsuda BS a , Christine Aurich DVM, PhD b, Akane Tanaka DVM, PhD
,
Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications
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a
a,c
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Hiroshi Matsuda DVM, PhD a,d,*
,
and System Engineering, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan b
Centre for Artificial Insemination and Embryo Transfer, University for Veterinary
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Sciences Vienna, Vienna, Austria
Laboratories of c Comparative Animal Medicine and d Veterinary Molecular Pathology
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and Therapeutics, Division of Animal Life Science, Institute of Agriculture, Tokyo
1
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University of Agriculture and Technology, Fuchu, Tokyo, Japan
These authors equally contributed this work.
* Corresponding author at: Hiroshi Matsuda DVM, PhD, Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, 1
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Japan
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SC
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E-mail address: [email protected] (H. Matsuda).
2
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Abstract Nerve growth factor (NGF) is a neurotrophic factor that is essential for the
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maintenance of peripheral and central neurons. While importance of such physiological activities as well as the potential of it as a stress marker in Equus caballus has been implicated, the sequence of equine NGF remained unknown. In this study, we identified
SC
the sequence of equine NGF from the mRNA expressed in the peripheral blood in 7
M AN U
Thoroughbreds and 3 warmblood horses. There were no polymorphisms among samples analyzed and the homology is more than 90% in comparison to human, mouse, rat, dog, and cow. When the sequence corresponding to the biologically active β-NGF was expressed in CHO-K1 cells, they were stained with an anti-NGF antibody that
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recognizes human NGF. Thus it provides a rationale for using antibodies that react to
Key Words
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other species’ NGF to measure the equine NGF for further studies.
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Nerve growth factor, cloning, horse
3
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1. Introduction Nerve growth factor (NGF) is a polypeptide necessary for the growth and support
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of neuron cells, which is produced from various kinds of cells such as hematopoietic cells, keratinocytes, and fibroblasts [1–4]. Besides nerve cell maintenance, it is involved in a wide variety of physiological activities such as pain sensation and wound healing
SC
[2–7]. In Equus caballus, NGF has been implicated as a novel stress marker because
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serum NGF levels elevated after running exercise or truck transportation [8,9]. In those studies, we estimated the elevation of NGF in an in vitro assay using rat PC12 pheochromocytoma cell line based on the premise that equine NGF sequence is highly conserved and cross-reacts among species, leaving the sequence of equine NGF
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remained undetermined. Recently, Wade et al. [10] provided a predicted sequence of equine NGF from a shotgun sequence analysis of a Thoroughbred genome. It indicates
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that equine NGF shares high homology with other species, though more data are required to conclude the equine NGF sequences taking polymorphisms into account.
AC C
In this study, we identified a sequence of equine NGF using blood samples from 10
horses and analyzed whether a commercially available antibody against human NGF can be applicable for the detection of the protein.
4
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2. Materials and Methods 2.1. Animals
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All experiments with horses had ethical permission along with the standards specified in the guidelines of the University Animal Care and Use Committee of the Tokyo University of Agriculture and Technology, as well as with the guidelines for the
SC
use of laboratory animals provided by the Science Council of Japan. A total of 10
M AN U
healthy male adults horses (7 Thoroughbreds and 3 warmblood horses) were included. Approximately 30 ml of blood were obtained from one Vena jugularis externa of each horse into conical tubes containing EDTA (Maxim Biotech Inc., San Francisco, CA). After hemolysis with ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM
TE D
Na2EDTA, pH 7.2), total RNA from white blood cells was extracted with ISOGEN (Nippongene, Tokyo, Japan) and reverse-transcribed into cDNA by using random
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oligo(dT) primers and PrimeScript (Takara Bio, Shiga, Japan) according to the
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manufacturer’s instructions.
2.2. Cloning of equine NGF Polymerase chain reactions (PCRs) with PrimeStar Max DNA polymerase (Takara
Bio) were carried out using a pair of primers 5’-ATGTCCATGTTGTTCTACACT-3’ (sense primer) and 5’-TCAGGCTTTTCTCCCGGTTTT-3’ (antisense primer), and the equine blood cDNA as templates. PCR products were cloned into a pGEM-T TA 5
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cloning vector (Promega, Madison, WI), followed by PCR reactions using BigDye Terminator v3.1 Cycle Sequencing kits (Life Technologies, Gaithersburg, MD) with
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M13-forward and M13-reverse primers. The sequence was analyzed by ABI3100 sequencer (Life Technologies) according to the manufacturer’s instruction.
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2.3. Cell culture
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CHO-K1 cells were obtained from Riken Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humid
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atmosphere of 5% CO2 in air at 37°C.
2.4. Expression of equine NGF and immunocytochemistry
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The β-NGF sequence was cloned into pIRES2-AcGFP vector (Clontech, Palo Alto, CA), which is designated pIRES2-βNGF-AcGFP, and transfected into CHO-K1 cells by
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using a Cell Line Nucleofector kit T (Lonza, Walkersville, MD) with a Nucleofector I electroporation device (Lonza) according to the manufacturer's instructions. Cells were cultured for 48 hours in the culture medium, and then fixed with 4% paraformaldehyde (Wako, Osaka, Japan). Subsequently, the cells were permeabilized with Triton-X and stained with an anti-NGF rabbit polyclonal antibody (Santa Cruz, Santa Cruz, CA; H-20), followed by anti-rabbit IgG-Alexa647 antibody (Life Technologies). Images 6
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were captured using a fluorescence microscope (model BZ-9000; Keyence, Osaka, Japan)
after
treatment
with
the
Prolong
Gold
antifading
reagent
with
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4'6-diamidino-2-phenylindole (DAPI; Life Technologies).
2.5. Homology search
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Sequences of NGF of each species were searched through GenBank database and
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compared using the multiple sequence alignment program CLUSTAL W [11]. The following Genbank databases were used as references; human (accession number NP_002497), mouse (NP_038637), rat (NP_001263984), dog (NP_00181879.1), cow (NP_001092832), cat (XP_004001166), Przewalski's horse (XP_008513352.1), and
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donkey (XP_014703734).
7
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3. Results Because NGF production in monocytes/macrophages has been reported [12,13],
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we speculated that NGF mRNA can be amplified from blood samples. RT-PCR was conducted using the cDNA obtained from the equine blood and a primer set specific to NGF precursor sequence, which is highly conserved from rodents to humans [14,15]. In
SC
consequence, approximately 750 bp PCR product was obtained. Then TA cloning was
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conducted and the sequence of the product was analyzed. As shown in Fig. 1A, they were completely identical to the sequence registered in GenBank database (accession number XM_001496187), the predicted sequence of NGF from a Thoroughbred [10]. We carried out the same analyses using cDNAs from 10 horses (7 thoroughbreds and 3
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warmbloods respectively; Table 1) and did not find any polymorphisims at all. When compared the amino acid sequence of full length NGF (preproNGF form), the
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homology was > 90% among the human, mouse, rat, dog, cow, cat, and horse (Fig. 1B). We also compared the equine NGF sequence with predicted ones from a Przewalski's
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horse (Equus przewalskii; Genbank accession number XP_008513352.1) and a donkey (Equus asinus; Genbank accession number XP_014703734). As a result, NGF sequence was completely the same among the horse and donkey, and only one amino acid was different in the Przewalski's horse (Thr22Arg). β-NGF is the biologically active form resulting from cleavage and modification of preproNGF [16]. Amino acid sequence of cleavage site (118RSKR/SS in Fig. 1B), which is the target of furin [16], was also 8
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conserved in horses. Homology of C-terminal amino acids corresponding to predicted β-NGF was
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highly conserved in the horses. Especially, β-NGF sequence in the horse, human, dog, and cow were identical except a few amino acids in the C-terminal (Fig. 1B), suggesting that most antibodies which recognize β-NGF from other species will crossreact with
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equine β-NGF. To ascertain this, we carried out immunohistochemistry on CHO-K1
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cells expressing equine β-NGF. Judging from AcGFP expression, transfection efficacy of pIRES2-β-NGF-AcGFP with the cells was approximately 40%. As shown in Fig. 2, only the AcGFP-positive cells were stained with anti-human NGF polyclonal antibody (H-20). In contrast, AcGFP-positive cells resulting from pIRES2-AcGFP transfection
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were not stained with the anti-human NGF antibody (data not shown), indicating that
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the antibody specifically recognized equine β-NGF expressed in CHO-K1 cells.
9
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4. Discussion In this study, we cloned equine NGF and confirmed the cross-reactivity with
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commercially available antibodies. The sequence of NGF including the equine was highly conserved, and residues that interact with the receptor TrkA in human NGF (mainly AA124–143, 150–154, 173–180, 229–231 in Fig. 1B) [16] were almost
SC
identical between the human and horse. This probably suggests an involvement of NGF
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in fundamental biological processes such as supportive effects on neuronal cells and pain sensation independent of species-specific effects [3–7]. Recently, monoclonal antibodies against NGF were used in clinical trials, aiming at controlling pain associated with several diseases such as osteoarthritis [17,18]. Because NGF is a useful biomarker
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that reflects physical and mental stress in horses [8,9], such anti-NGF therapeutics may be of interest for the treatment of stress-mediated equine diseases like shipping fever
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and pneumonia. In addition, our results provide a rationale for using commercially
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available antibodies to directly determine equine NGF in future studies.
5. Conclusions
The present study demonstrates that the sequence of NGF in Equus caballus is
highly conserved among species.
10
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Acknowledgements This study was supported by the Japan Society for the Promotion of Science, the
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Grants-in-Aid for Scientific Program, Challenging Exploratory Research grant number 15K14868. The authors thank all members of the horse-riding club at the Tokyo University of Agriculture and Technology for their assistance with our experiment. No
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financial or personal relationships inappropriately influence or bias the content of this
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research.
References
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[1] Levi-Montalcini R, Angeletti PU. Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Dev Biol 1963;7:653–9.
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[2] Tanaka A, Wakita U, Kambe N, Iwasaki T, Matsuda H. An autocrine function of
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nerve growth factor for cell cycle regulation of vascular endothelial cells. Biochem Biophys Res Commun 2004;313:1009–14. [3] Scully JL, Otten U. NGF: Not just for neurons. Cell Biol Int 1995;19:459–69. [4] Matsuda H, Koyama H, Sato H, Sawada J, Itakura A, Tanaka A, et al. Role of nerve growth factor in cutaneous wound healing: Accelerating effects in normal and healing-impaired diabetic mice. J Exp Med 1998;187:297–306.
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[5] Mendell LM, Albers KM, Davis BM. Neurotrophins, nociceptors, and pain. Microsc Res Tech 1999;45:252–61.
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[6] McMahon SB. NGF as a mediator of inflammatory pain. Philos Trans R Soc Lond B Biol Sci 1996;351:431–40.
[7] Scholz J, Woolf CJ. The neuropathic pain triad: Neurons, immune cells and glia. Nat
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Neurosci 2007;10:1361–8.
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[8] Matsuda H, Koyama H, Oikawa M, Yoshihara T, Kaneko M. Nerve growth Factor-like activity detected in equine peripheral blood after running exercise. Zentralbl Veterinarmed A 1991;38:557–9.
[9] Kawamoto K, Sato H, Oikawa M, Yoshihara T, Kaneko M, Matsuda H. Nerve
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growth factor activity detected in equine peripheral blood of horses with fever after truck transportation. J Equine Sci 1996;7:43–6.
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[10] Wade CM, Giulotto E, Sigurdsson S, Zoli M, Gnerre S, Imsland F, et al. Genome sequence, comparative analysis, and population genetics of the domestic horse. Science
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2009;326:865–7.
[11] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80. [12] Caroleo MC, Costa N, Bracci-Laudiero L, Aloe L. Human monocyte/macrophages activate by exposure to LPS overexpress NGF and NGF receptors. J Neuroimmunol 12
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2001;113:193–201. [13] Kanda N, Watanabe S. Regulatory roles of sex hormones in cutaneous biology and
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immunology. J Dermatol Sci 2005;38:1–7. [14] Varon S, Nomura J, Shooter E. The isolation of the mouse nerve growth factor protein in a high molecular weight form. Biochemistry 1967;6:2202–9.
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[15] Ullrich A, Gray A, Berman C, Dull TJ. Human β-nerve growth factor gene
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sequence highly homologous to that of mouse. Nature 1983;303:821–5. [16] Wiesmann C, De Vos A. Nerve growth factor: Structure and function. Cell Mol Life Sci 2001;58:748–59.
[17] Tiseo PJ, Kivitz AJ, Ervin JE, Ren H, Mellis SJ. Fasinumab (REGN475), an
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antibody against nerve growth factor for the treatment of pain: Results from a double-blind, placebo-controlled exploratory study in osteoarthritis of the knee. Pain
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2014;155:1245–52.
[18] Gow JM, Tsuji WH, Williams GJ, Mytych D, Sciberras D, Searle SL, et al. Safety,
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tolerability, pharmacokinetics, and efficacy of AMG 403, a human anti-nerve growth factor monoclonal antibody, in two phase I studies with healthy volunteers and knee osteoarthritis subjects. Arthritis Res Ther 2015;17:1–11.
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Horse
List of horses participated in this study Breed
Age
Sex
Castration
Warmblood
10
Male
Yes
2
Warmblood
14
Male
Yes
3
Warmblood
21
Male
Yes
4
Thoroughbred
8
Male
No
5
Thoroughbred
13
Male
Yes
6
Thoroughbred
15
Male
Yes
7
Thoroughbred
16
Male
Yes
8
Thoroughbred
17
9
Thoroughbred
18
10
Thoroughbred
22
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1
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Table 1.
Yes
Male
Yes
Male
Yes
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Male
14
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Figure legends Fig. 1. (A) Sequence of cloned NGF sequence. Square brackets indicate the beginning
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and the end of the predicted β-NGF sequence based on the amino acid comparison between species. (B) Comparison of amino acids of preproNGF in species. Framed
Fig.
2.
Immunohistochemical
analysis.
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sequences are corresponded to the β-NGF in each species.
CHO-K1
cells
transfected
with
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pIRES2-β-NGF-AcGFP were stained with anti-NGF polyclonal antibody, followed by the anti-rabbit IgG-Alexa647 antibody and DAPI. Original magnification, ×400. Bar; 50
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µm.
15
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1. We identified the sequence of equine β-NGF cDNA from the peripheral blood in 10 horses. 2. There were no polymorphisms among the samples. 3. The homology was more than 90% in comparison to other animals reported.
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4. Anti-human NGF antibody crossreacted with equine NGF expressed in CHO-K1 cells.
S0737-0806(15)30125-8
DOI:
10.1016/j.jevs.2016.06.001
Reference:
YJEVS 2118
To appear in:
Journal of Equine Veterinary Science
Received Date: 19 December 2015 Revised Date:
1 June 2016
Accepted Date: 1 June 2016
Please cite this article as: Amagai Y, Sato H, Ishizaka S, Matsuda K, Aurich C, Tanaka A, Matsuda H, Cloning and expression of equine β-nerve growth factor, Journal of Equine Veterinary Science (2016), doi: 10.1016/j.jevs.2016.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Cloning and expression of equine β-nerve growth factor
a,1
, Hiroaki Sato DVM
a,1
, Saori Ishizaka DVM
a,b,1
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Yosuke Amagai DVM, PhD
Kenshiro Matsuda BS a , Christine Aurich DVM, PhD b, Akane Tanaka DVM, PhD
,
Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications
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a
a,c
SC
Hiroshi Matsuda DVM, PhD a,d,*
,
and System Engineering, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan b
Centre for Artificial Insemination and Embryo Transfer, University for Veterinary
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Sciences Vienna, Vienna, Austria
Laboratories of c Comparative Animal Medicine and d Veterinary Molecular Pathology
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and Therapeutics, Division of Animal Life Science, Institute of Agriculture, Tokyo
1
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University of Agriculture and Technology, Fuchu, Tokyo, Japan
These authors equally contributed this work.
* Corresponding author at: Hiroshi Matsuda DVM, PhD, Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, 1
ACCEPTED MANUSCRIPT
Japan
AC C
EP
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SC
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E-mail address: [email protected] (H. Matsuda).
2
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Abstract Nerve growth factor (NGF) is a neurotrophic factor that is essential for the
RI PT
maintenance of peripheral and central neurons. While importance of such physiological activities as well as the potential of it as a stress marker in Equus caballus has been implicated, the sequence of equine NGF remained unknown. In this study, we identified
SC
the sequence of equine NGF from the mRNA expressed in the peripheral blood in 7
M AN U
Thoroughbreds and 3 warmblood horses. There were no polymorphisms among samples analyzed and the homology is more than 90% in comparison to human, mouse, rat, dog, and cow. When the sequence corresponding to the biologically active β-NGF was expressed in CHO-K1 cells, they were stained with an anti-NGF antibody that
TE D
recognizes human NGF. Thus it provides a rationale for using antibodies that react to
Key Words
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other species’ NGF to measure the equine NGF for further studies.
AC C
Nerve growth factor, cloning, horse
3
ACCEPTED MANUSCRIPT
1. Introduction Nerve growth factor (NGF) is a polypeptide necessary for the growth and support
RI PT
of neuron cells, which is produced from various kinds of cells such as hematopoietic cells, keratinocytes, and fibroblasts [1–4]. Besides nerve cell maintenance, it is involved in a wide variety of physiological activities such as pain sensation and wound healing
SC
[2–7]. In Equus caballus, NGF has been implicated as a novel stress marker because
M AN U
serum NGF levels elevated after running exercise or truck transportation [8,9]. In those studies, we estimated the elevation of NGF in an in vitro assay using rat PC12 pheochromocytoma cell line based on the premise that equine NGF sequence is highly conserved and cross-reacts among species, leaving the sequence of equine NGF
TE D
remained undetermined. Recently, Wade et al. [10] provided a predicted sequence of equine NGF from a shotgun sequence analysis of a Thoroughbred genome. It indicates
EP
that equine NGF shares high homology with other species, though more data are required to conclude the equine NGF sequences taking polymorphisms into account.
AC C
In this study, we identified a sequence of equine NGF using blood samples from 10
horses and analyzed whether a commercially available antibody against human NGF can be applicable for the detection of the protein.
4
ACCEPTED MANUSCRIPT
2. Materials and Methods 2.1. Animals
RI PT
All experiments with horses had ethical permission along with the standards specified in the guidelines of the University Animal Care and Use Committee of the Tokyo University of Agriculture and Technology, as well as with the guidelines for the
SC
use of laboratory animals provided by the Science Council of Japan. A total of 10
M AN U
healthy male adults horses (7 Thoroughbreds and 3 warmblood horses) were included. Approximately 30 ml of blood were obtained from one Vena jugularis externa of each horse into conical tubes containing EDTA (Maxim Biotech Inc., San Francisco, CA). After hemolysis with ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM
TE D
Na2EDTA, pH 7.2), total RNA from white blood cells was extracted with ISOGEN (Nippongene, Tokyo, Japan) and reverse-transcribed into cDNA by using random
EP
oligo(dT) primers and PrimeScript (Takara Bio, Shiga, Japan) according to the
AC C
manufacturer’s instructions.
2.2. Cloning of equine NGF Polymerase chain reactions (PCRs) with PrimeStar Max DNA polymerase (Takara
Bio) were carried out using a pair of primers 5’-ATGTCCATGTTGTTCTACACT-3’ (sense primer) and 5’-TCAGGCTTTTCTCCCGGTTTT-3’ (antisense primer), and the equine blood cDNA as templates. PCR products were cloned into a pGEM-T TA 5
ACCEPTED MANUSCRIPT
cloning vector (Promega, Madison, WI), followed by PCR reactions using BigDye Terminator v3.1 Cycle Sequencing kits (Life Technologies, Gaithersburg, MD) with
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M13-forward and M13-reverse primers. The sequence was analyzed by ABI3100 sequencer (Life Technologies) according to the manufacturer’s instruction.
SC
2.3. Cell culture
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CHO-K1 cells were obtained from Riken Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humid
TE D
atmosphere of 5% CO2 in air at 37°C.
2.4. Expression of equine NGF and immunocytochemistry
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The β-NGF sequence was cloned into pIRES2-AcGFP vector (Clontech, Palo Alto, CA), which is designated pIRES2-βNGF-AcGFP, and transfected into CHO-K1 cells by
AC C
using a Cell Line Nucleofector kit T (Lonza, Walkersville, MD) with a Nucleofector I electroporation device (Lonza) according to the manufacturer's instructions. Cells were cultured for 48 hours in the culture medium, and then fixed with 4% paraformaldehyde (Wako, Osaka, Japan). Subsequently, the cells were permeabilized with Triton-X and stained with an anti-NGF rabbit polyclonal antibody (Santa Cruz, Santa Cruz, CA; H-20), followed by anti-rabbit IgG-Alexa647 antibody (Life Technologies). Images 6
ACCEPTED MANUSCRIPT
were captured using a fluorescence microscope (model BZ-9000; Keyence, Osaka, Japan)
after
treatment
with
the
Prolong
Gold
antifading
reagent
with
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4'6-diamidino-2-phenylindole (DAPI; Life Technologies).
2.5. Homology search
SC
Sequences of NGF of each species were searched through GenBank database and
M AN U
compared using the multiple sequence alignment program CLUSTAL W [11]. The following Genbank databases were used as references; human (accession number NP_002497), mouse (NP_038637), rat (NP_001263984), dog (NP_00181879.1), cow (NP_001092832), cat (XP_004001166), Przewalski's horse (XP_008513352.1), and
AC C
EP
TE D
donkey (XP_014703734).
7
ACCEPTED MANUSCRIPT
3. Results Because NGF production in monocytes/macrophages has been reported [12,13],
RI PT
we speculated that NGF mRNA can be amplified from blood samples. RT-PCR was conducted using the cDNA obtained from the equine blood and a primer set specific to NGF precursor sequence, which is highly conserved from rodents to humans [14,15]. In
SC
consequence, approximately 750 bp PCR product was obtained. Then TA cloning was
M AN U
conducted and the sequence of the product was analyzed. As shown in Fig. 1A, they were completely identical to the sequence registered in GenBank database (accession number XM_001496187), the predicted sequence of NGF from a Thoroughbred [10]. We carried out the same analyses using cDNAs from 10 horses (7 thoroughbreds and 3
TE D
warmbloods respectively; Table 1) and did not find any polymorphisims at all. When compared the amino acid sequence of full length NGF (preproNGF form), the
EP
homology was > 90% among the human, mouse, rat, dog, cow, cat, and horse (Fig. 1B). We also compared the equine NGF sequence with predicted ones from a Przewalski's
AC C
horse (Equus przewalskii; Genbank accession number XP_008513352.1) and a donkey (Equus asinus; Genbank accession number XP_014703734). As a result, NGF sequence was completely the same among the horse and donkey, and only one amino acid was different in the Przewalski's horse (Thr22Arg). β-NGF is the biologically active form resulting from cleavage and modification of preproNGF [16]. Amino acid sequence of cleavage site (118RSKR/SS in Fig. 1B), which is the target of furin [16], was also 8
ACCEPTED MANUSCRIPT
conserved in horses. Homology of C-terminal amino acids corresponding to predicted β-NGF was
RI PT
highly conserved in the horses. Especially, β-NGF sequence in the horse, human, dog, and cow were identical except a few amino acids in the C-terminal (Fig. 1B), suggesting that most antibodies which recognize β-NGF from other species will crossreact with
SC
equine β-NGF. To ascertain this, we carried out immunohistochemistry on CHO-K1
M AN U
cells expressing equine β-NGF. Judging from AcGFP expression, transfection efficacy of pIRES2-β-NGF-AcGFP with the cells was approximately 40%. As shown in Fig. 2, only the AcGFP-positive cells were stained with anti-human NGF polyclonal antibody (H-20). In contrast, AcGFP-positive cells resulting from pIRES2-AcGFP transfection
TE D
were not stained with the anti-human NGF antibody (data not shown), indicating that
AC C
EP
the antibody specifically recognized equine β-NGF expressed in CHO-K1 cells.
9
ACCEPTED MANUSCRIPT
4. Discussion In this study, we cloned equine NGF and confirmed the cross-reactivity with
RI PT
commercially available antibodies. The sequence of NGF including the equine was highly conserved, and residues that interact with the receptor TrkA in human NGF (mainly AA124–143, 150–154, 173–180, 229–231 in Fig. 1B) [16] were almost
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identical between the human and horse. This probably suggests an involvement of NGF
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in fundamental biological processes such as supportive effects on neuronal cells and pain sensation independent of species-specific effects [3–7]. Recently, monoclonal antibodies against NGF were used in clinical trials, aiming at controlling pain associated with several diseases such as osteoarthritis [17,18]. Because NGF is a useful biomarker
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that reflects physical and mental stress in horses [8,9], such anti-NGF therapeutics may be of interest for the treatment of stress-mediated equine diseases like shipping fever
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and pneumonia. In addition, our results provide a rationale for using commercially
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5. Conclusions
The present study demonstrates that the sequence of NGF in Equus caballus is
highly conserved among species.
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Acknowledgements This study was supported by the Japan Society for the Promotion of Science, the
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Grants-in-Aid for Scientific Program, Challenging Exploratory Research grant number 15K14868. The authors thank all members of the horse-riding club at the Tokyo University of Agriculture and Technology for their assistance with our experiment. No
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financial or personal relationships inappropriately influence or bias the content of this
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[17] Tiseo PJ, Kivitz AJ, Ervin JE, Ren H, Mellis SJ. Fasinumab (REGN475), an
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Horse
List of horses participated in this study Breed
Age
Sex
Castration
Warmblood
10
Male
Yes
2
Warmblood
14
Male
Yes
3
Warmblood
21
Male
Yes
4
Thoroughbred
8
Male
No
5
Thoroughbred
13
Male
Yes
6
Thoroughbred
15
Male
Yes
7
Thoroughbred
16
Male
Yes
8
Thoroughbred
17
9
Thoroughbred
18
10
Thoroughbred
22
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Table 1.
Yes
Male
Yes
Male
Yes
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Male
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Figure legends Fig. 1. (A) Sequence of cloned NGF sequence. Square brackets indicate the beginning
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and the end of the predicted β-NGF sequence based on the amino acid comparison between species. (B) Comparison of amino acids of preproNGF in species. Framed
Fig.
2.
Immunohistochemical
analysis.
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sequences are corresponded to the β-NGF in each species.
CHO-K1
cells
transfected
with
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pIRES2-β-NGF-AcGFP were stained with anti-NGF polyclonal antibody, followed by the anti-rabbit IgG-Alexa647 antibody and DAPI. Original magnification, ×400. Bar; 50
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µm.
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1. We identified the sequence of equine β-NGF cDNA from the peripheral blood in 10 horses. 2. There were no polymorphisms among the samples. 3. The homology was more than 90% in comparison to other animals reported.
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4. Anti-human NGF antibody crossreacted with equine NGF expressed in CHO-K1 cells.