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Computational and biological approach for studying structure-function of inhibin chimeric peptide antibodies in Clarias batrachus
Accepted Manuscript Computational and biological approach for studying structurefunction of inhibin chimeric peptide antibodies in Clarias batrachus
Irshad Ahmad, Irfan Ahmad Bhat, Dhanashree D. Jagtap, Nafisa H. Balasinor, C. Selvaa Kumar, T.I. Chanu, Babitha Rani, Showkat Ahmad Dar, Tasok Leya, Adnan Hussain Gora, Neelam Saharan PII: DOI: Reference:
S0044-8486(18)31925-2 https://doi.org/10.1016/j.aquaculture.2018.11.016 AQUA 633678
To appear in:
aquaculture
Received date: Revised date: Accepted date:
6 September 2018 5 November 2018 8 November 2018
Please cite this article as: Irshad Ahmad, Irfan Ahmad Bhat, Dhanashree D. Jagtap, Nafisa H. Balasinor, C. Selvaa Kumar, T.I. Chanu, Babitha Rani, Showkat Ahmad Dar, Tasok Leya, Adnan Hussain Gora, Neelam Saharan , Computational and biological approach for studying structure-function of inhibin chimeric peptide antibodies in Clarias batrachus. Aqua (2018), https://doi.org/10.1016/j.aquaculture.2018.11.016
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ACCEPTED MANUSCRIPT Computational and biological approach for studying structurefunction of inhibin chimeric peptide antibodies in Clarias batrachus
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Irshad Ahmad1 , Irfan Ahmad Bhat1 , Dhanashree D. Jagtap2 , Nafisa H Balasinor2 , Selvaa Kumar C3 , Chanu T.I4 , Babitha Rani4 , Showkat Ahmad Dar4 , Tasok Leya4 , Adnan Hussain Gora5 , Neelam Saharan4
1
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Affiliations:
College of Fisheries Science, Gumla, Birsa Agricultural University, Ranchi-834006,
2
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India
Indian Council of Medical Research-National Institute for Research in Reproductive
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Bioinformatics Department, School of Biotechnology and Bioinformatics, D.Y. Patil
University, Navi Mumbai-400614, India
Indian Council of Agricultural Research-Central Institute of Fisheries Education,
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Mumbai- 400061, India
ICAR-CMFRI, Biotechnology Center, Madras Chennai, India
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5
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Health, Mumbai-400012, India
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Corresponding Author:
Prof. Neelam Saharan ICAR-Central Institute of Fisheries Education Department of Aquatic Environment management India, Mumbai-400061
Fax: +91 2226361573 Email: [email protected]/[email protected]
ACCEPTED MANUSCRIPT Abstract Inhibins are heterodimeric glycoproteins that belong to transforming growth factor-β superfamily. The present study was aimed at structural elucidation of immunogenic inhibin chimeric peptide (ICP) using immunoinformatics tools, to develop anti-ICP
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antibodies and to understand the mechanism of interaction between HPG-axis and
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anti-ICP antibodies in adult female Clarias batrachus. Homology modelling predicted
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that the ICP adopts a turn and random coil, which was further confirmed by circular dichroism (CD). Competitive binding ELISA with ICP and different unrelated peptides
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confirmed that the developed anti-ICP antiserum is highly specific and critical for the
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direct interaction to the inhibin-α subunit. The anti- ICP antibodies increased the mRNA transcript levels of follicle stimulating hormone receptor (FSHr), luteinizing hormone
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receptor (LHr), activin receptor type I (Act RI) and activin receptor type II (Act RII).
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Further, the serum concentrations of follicle stimulating hormone (FSH), 17β-estradiol (E2) and progesterone increased significantly in anti-ICP antisera treated groups
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compared to control. Modelled inhibin- and ICP exhibits better interaction with each
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other through two salt bridge formations. Also, ICP showed better interaction with betaglycan through positive and polar-based interactions. The results of the present study
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suggest that immunoneutralization of inhibin bioactivity, confirms the physiological role of inhibin as a component of ovarian feedback mechanism regulating FSH secretion through augmentation of gonadotropin and activin receptor signalling pathways that could affect the reproductive success in fishes.
Keywords: Inhibin chimeric peptide, circular dichroism, protein-peptide docking, antipeptide antibody, activin receptor signalling
ACCEPTED MANUSCRIPT 1. Introduction Inhibin proteins are structurally related to the Transforming Growth Factor-β (TGF-β) superfamily. Biologically active forms; inhibin A and inhibin B are heterodimeric glycoproteins composed of an α-subunit and one of the forms of β-subunit namely βA or
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βB (Makanji et al., 2014). Inhibin binds to betaglycan with high affinity via its α-subunit
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and this binding markedly triggers the binding of the inhibin-β subunit to the activin
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receptor type II A (Act RIIA) (Lewis et al., 2000; Ahmad et al., 2018a). Inhibin action is
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regulated when inhibin binds to activin receptor type II preventing activin- induced heteromerization of type I and type II receptors, which play a crucial role in activating the
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activin-dependent intracellular signalling cascade (Lebrun and Vale, 1997). Precise mechanisms of hormonal regulation of the hypothalamic-pituitary-
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gonadal (HPG) axis are essential for normal reproductive functioning in all animal
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species. The pulsatile secretion of hypothalamic gonadotropin-releasing hormone stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone
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(LH) from the anterior pituitary (Stamatiades and Kaiser, 2017). Despite the central role
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of gonadal inhibin in the regulation of the reproductive axis, little is known regarding its structure and molecular mechanism of action in lower vertebrates. Designing an
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immunogenic peptide of inhibin-α and elucidating the structure of the designed peptide using immunoinformatics tools and also to understand receptor expression and hormonal physiology is of importance. Immunogenic peptides designed against inhibitory proteins (inhibins) could be one the approaches towards the development of novel targets to improve breeding performances in fishes. Anti-peptide antibodies are important tools in clinical laboratory
ACCEPTED MANUSCRIPT diagnostics primarily because they recognize native proteins (Trier and Houen, 2017). Designing new immunogenic peptides and developing antibodies with desired properties could have a profound impact on fish reproduction. In captivity, the breeding performances of fishes are generally low due to the poor quality of female brooders
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(Zohar, 1989 and Mylonas and Zohar 2001). This is mainly because of the lack of
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required environmental parameters for natural reproductive performances (Mylonas et al.,
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2010). The purpose of the present study was to determine whether the presence of antibodies against endogenous inhibins could affect the secretion of some important
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reproductive genes, hormones and the timing of ovulation in female Clarias batrachus,
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an important air-breathing catfish found in Indian waters. We aimed to designed speciesspecific inhibin chimeric peptide (ICP) and to elucidate its structure using bioinformatics
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approach. Furthermore, the in-vivo effect of the anti-ICP antibody on receptor expression
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and hormonal levels were also studied in adult female C. batrachus.
2. Materials and methods
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2.1. Homology modelling
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Inhibin-α was cloned and sequenced in C. batrachus (Ahmad et al., 2018a) and the putative protein sequence was used for the prediction of continuous B- and T-cell
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epitopes using the IEDB (http://www.iedb.org). Based on this information, inhibin-α chimeric peptide (ICP) was designed from in silico perspective. To model the 3D structure of the ICP, 3D crystal structure was much needed. For the same, BLAST-PDB search (Altschul et al., 1990) was initiated to identify potential template with a default matrix of BLOSUM62. However, this step went futile as there was no reported structure available from Protein Data Bank (Berman et al., 2000) based on sequence homology. As
ACCEPTED MANUSCRIPT an alternative, we opted for a fold-based method using pDOMTHREADER (Lobley et al., 2009) that is an accurate and sensitive superfamily discrimination method. Highly accurate domain alignments were generated through the collective information from both sequence and structure. The template with better score listed by pDOMTHREADER was
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considered for homology modelling using Modeller 9.18 standalone version (Sali and
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Blundell, 1993). ‘align2d.py’ and ‘model-single.py’ were used for the template-query
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alignment and model building respectively. Next, to identify a potential template for inhibin- BLAST-PDB search was initiated with a default matrix of BLOSUM62. of all
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the listed templates, the crystal structure with better query coverage and identity was
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considered for sequence-based homology modelling using Modeller9.18. Ten models were generated for ICP and inhibin- α that was later validated through DOPE (Discrete
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Optimized Protein Energy) and RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/
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rampage.php) (Richardson, 1981). This is the process of evaluating reliability for 3Datomic models of large biological molecules and also identify the model with the best
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geometry.
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2.2. Secondary structure estimation of ICP by Circular dichroism (CD) CD was recorded on a Jasco J-815 spectrometer (Jasco, Easton, MD, USA) from 195 to
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260nm, in a 1cm path length quartz cell. Fifty micromolar synthetic ICP (United BioSystems Inc. USA) in 25mM sodium phosphate buffer, pH 7.5 at 25 0 C was used. All solutions were filtered with a 0.22mm pore nylon filter prior to performing the experiments. Three consecutive scans were accumulated and the spectra thus obtained were averaged. The percentages of different secondary structures were estimated using the data provided by (Yang et al., 1986) for the reference spectra (Sharma et al., 2000; Kene et al., 2005; Chitnis et al., 2009).
ACCEPTED MANUSCRIPT 2.3. Production of anti-ICP antibody Antibodies against ICP were developed in rabbits (Belgian white strain) of approximately 3 kg body weight following our previous reports (Ahmad et al., 2018b). Use of the animal was approved by the Institutional Animal Ethics Committee of ICMR-NIRRH
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(IAEC no. 15/16) and all the experiments were conducted as per the norms of the
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Committee for the Purpose of Control and Supervision of Experiments on Animals
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(CPCSEA), India. 2.4. Competitive ELISA
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For competition experiments, serial dilutions of the ICP and unrelated peptide were pre-
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incubated with the antisera before addition to the microtiter wells following (Jagtap et al., 2002).
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2.5. Western blotting
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Binding of anti-ICP antibodies to the whole brain lysate of female C. batrachus was checked by Western blotting following Navalakhe et al. (2013). Frozen whole brain
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tissue was homogenized in ice-cold lysis buffer containing protease inhibitor cocktail and
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was centrifuged (Ahmad et al., 2018b). The protein concentration of the supernatant was determined by micro-BCA protein assay kit (Thermo Fisher, Waltham, MA, USA) and
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the supernatant was stored at -80°C till further use. 30 μg of the stored brain supernatant was resolved on a 12% SDS-PAGE and the proteins were transferred onto a nitrocellulose membrane (0.45 μm Immobilon; GE Health-care) using the Trans-Blot wet transfer apparatus (Bio-Rad, Richmond, CA, USA) at 4°C for 1h. After transfer, the membrane was cut into two and blocked with 5 % non-fat dry milk (NFDM; Anik Industries Ltd, Mumbai, India) in TBS-T (Tris-buffered saline containing 0.1% Tween 20). Next, one membrane blot was incubated with anti-ICP antibodies (test) and the other
ACCEPTED MANUSCRIPT blot was incubated with pre- immune rabbit serum (control) in 1 % NFDM in TBS-T for 1h at room temperature. The membranes were then washed with TBS-T followed by treatment with the secondary antibody (Goat anti-rabbit IgG HRP, 1:2000 dilution). After washing the membranes, the immunoreactive bands were visualized with ECL plus
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reagent according to the manufacturer’s instructions (GE Healthcare).
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2.6. Animals
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The adult mature female C. batrachus (> 1-year-old) in the late pre-spawning stage with average weight; 78.66 ± 0.89g and length; 21 ± 1.09cm were stocked in cemented tanks
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(4.5×2×1m). Females were fed ad libitum with commercially available pelleted feed
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(Charoen Pokphand (CP) feeds, India) having crude protein-(36%), crude lipid-(5%), fibre- (4%), moisture-(11%) and ash-(16%) respectively. The experiment was conducted
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at the freshwater fish farm Balabhadrapuram, Kakinada centre of ICAR-Central Institute
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of Fisheries Education, Andhra Pradesh, India. The animals used for the study were reared and handled under the approved protocol that followed the policies and guidelines
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of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on
India.
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Animals), Ministry of Environment and Forests (Animal Welfare Division), Govt. of
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To check the effect of anti-ICP antibodies on the receptor expression and hormonal profiles, 80 females fishes were randomly assigned to four treatment groups of 20 fishes each. The T1 group was injected with 200µl pre- immune rabbit serum (control), T2 group with 100µl anti-ICP (low dose), T3 group with 200µl anti-ICP (medium dose) and T4 group with 400µl anti-ICP (high dose) antisera respectively. The antisera were administered intramuscularly to each female fish on day 1, day 7 and day 14 under light
ACCEPTED MANUSCRIPT anesthesia by immersion method using clove oil (Merck, Germany) @ 40 μl/l of water (Ahmad et al., 2018b). 2.7. mRNA receptor expression Three fishes from each treatment group were randomly selected and sacrificed on day 7,
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day 14 and day 21 for the collection of whole brain and ovarian tissues. Total RNA was
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extracted from ovary and brain tissues as per our previous reports (Ahmad et al., 2018).
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To remove any genomic DNA contamination, the extracted RNA was treated with DNaseI using DNaseI RNase-free kit (Thermo Scientific, USA). DNaseI treated RNA
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(1µg) was then reverse transcribed using AffinityScript QPCR cDNA Synthesis Kit
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according to the manufacturer's protocol (Agilent technologies, USA). To study the modulatory effect of anti- ICP antibodies, mRNA expression of
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different receptors involved in reproduction was performed by real-time quantitative
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polymerase chain reaction (qPCR). The receptors selected were follicle stimulating hormone receptor (FSHr), luteinizing hormone receptor (LHr), activin receptor type I and
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II (Act RI and II). The quantitative RT-PCR experiments for these genes were conducted
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in duplicates (Table 1).
β-actin was used as an internal control in the present study as it exhibited lesser
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variations compared to other housekeeping genes. The softwares GeNorm, best keeper, delta Ct method and NormFinder were used for the validation of housekeeping genes. Further, the serial dilution of primers was also done to determine the efficiency of the primers. PCR was performed using Light Cycler1 Real-time PCR detection system (Roche, USA) using Brilliant III Ultra-Fast SYBR® Green qPCR master mix (Agilent Technologies, USA). Cycle threshold (Ct) values were obtained from the exponential phase of PCR amplification, and the expression of genes was normalized to the
ACCEPTED MANUSCRIPT expression of β-actin generating a ΔCt value (Ct of target gene -ΔCt of β-actin). The relative expression level of the target gene to the β-actin gene was determined using the 2- ΔΔCt method proposed by (Livak and Schmittgen, 2001) which gives the value that corresponds to the n-fold difference relative to the control.
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2.8. Hormonal assays
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After every 7 days interval, fishes from each treatment (n=5) were randomly collected
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and anaesthetized using clove oil (Merck, Germany) @ 40 μl/l (Ahmad et al., 2018) and the blood was collected without anticoagulant. The blood was allowed to clot for 4h and
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then centrifuged at 4000 rpm for 10 min, followed by the collection of serum. Serum was
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stored at -80°C for further analysis. The different reproductive hormones namely FSH, LH, 17β-estradiol (E2) and progesterone were measured in triplicate in the serum using
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commercial EIA kits following manufacturer's protocol (Cayman Chemical, USA). FSH
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and LH immunometric assay was based on the sandwich technique and uses the Horse Radish Peroxidase (HRP)- labeled detection antibody. For steroid hormones (E2 and
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progesterone) the assay was based on the competitive immunoreactions between the free
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hormone and steroid- linked acetylcholinesterase conjugate. The sensitivity, intra-assay and inter-assay coefficients of variations were determined for all the hormo nes and are
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presented in Supplementary Table 1. Enzyme immunoassay was performed in 96-well plate format and the absorbance was recorded on an ELISA reader for different hormones. 2.9. Protein-peptide docking 3D-modelled ICP and inhibin- were considered for protein-peptide docking to understand their overall binding affinity using Cluspro server (Kozakov et al., 2017). This study was mainly intended to understand the effect of ICP binding with inhibin-
ACCEPTED MANUSCRIPT and their interference to activin receptor type II. In total Cluspro generates 30 poses, of which based on the literature survey and antigenicity report from SVMTrip software (Yao et al., 2012) best-docked pose was selected. Cluspro only generates poses and not the binding energy. To calculate the same, here we opted for DCOMPLEX software (Liu
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et al., 2004) that gives energy in kcal/mol. Finally, the docked pose was visualized using
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CHIMERA software (Pettersen et al., 2004).
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2.10. Betaglycan-ICP docking
Betaglycan was downloaded from the PubChem database (Kim et al., 2016) Structure ID:
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CID_439262) in sdf file format. Furthermore, a stable conformer was generated after
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structure minimization using FROG2 (Miteva et al., 2010). In the option box ‘minimize' was selected as ‘Yes' and ‘Produce' was ‘single'. Which means the given betaglycan will
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be energy minimized and a single conformer with least energy will be considered.
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Gasteiger charges were added and the optimized structure was considered for docking with modelled ICP using Hex8.0.0 (Ritchie, 2003). For correlation type, and Post-
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processing we opted for "Shape+Electro+DARS" and for "DARS Minimization"
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respectively. Standard docking with a grid dimension of 0.6Å and solution of 2000 was preferred. After docking, top ten listed docked poses were considered for amino acid
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interaction analysis using CHIMERA software (Pettersen et al., 2004). Finally, we selected the pose with the better binding affinity between the betaglycan and ICP. Our main intention behind this study was to understand the overall interaction between the ligand and the ICP. 2.11. Statistical analysis The differences in mRNA transcript and hormonal levels were tested for statistical significance using the statistical package SPSS 17.0 (USA). One-way analysis of
ACCEPTED MANUSCRIPT variance (ANOVA), followed by Tukey's multiple comparison test and P < 0.05 was considered statistically significant. The results were expressed as the mean ± standard error.
3. Results
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3.1. Homology Modelling
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The predicted B-cell and T-cell epitopes were used to design the inhibin chimeric peptide
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(IEKLQRPSPEKDDTYGGGDTVSSEDP) for C. batrachus (Ahmad et al., 2018). With
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no reported template available based on amino acid identity, the short chimeric peptide was designed using the fold-based method using pDOMTHREADER. The listed TGF-β2
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with PDB ID: 2TGI (Daopin et al., 1992) has a confidence of HIGH with a net score of 4.65. This template was considered for modelling (26 amino acid ICP) using Modeller
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standalone software and ten models were generated (Fig.1). The 3D model with the least
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DOPE score of -13503.70 was selected. Furthermore, the modelled structure was validated using RAMPAGE tool. In ICP, 91.7% is in the favoured region and 8.3% in
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the allowed region with no outliers (Supplementary Figure 1A). This modelled structure
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is a random coil with no reported secondary structure. Inhibin-α was modelled based on 5HLY Chain A with the query coverage of 24-343 residues. Of the ten- modelled
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structure, the 3D structure with the least DOPE score of -31191.13 was selected. Inhibinα, 90.6% residues are in the favoured region; 6.2% in the allowed region; 3.2% residues in the outlier region (Supplementary Figure 1B).
ACCEPTED MANUSCRIPT 3.2. CD profile of ICP ICP (5mg) with 90% purity was commercially synthesized from United BioSystems Inc. USA. Secondary structure estimated by CD analysis revealed that the ICP comprises 21.9% α-helix, 15.7% β-sheets, 33.3% turn and 29.1% random-coil structure.
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3.3. Competitive ELISA
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In competitive inhibition studies, homologous peptide competitively inhibited the binding
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of the ICP anti-peptide antibodies to the immobilized peptide while the unrelated peptides did not (Fig. 2).
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3.4. Inhibin-α in whole brain lysate
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Whole brain lysate of C. batrachus was probed with anti-ICP antibodies that detected a dark band at a molecular weight of ∼18 kDa. No band was detected when the lysate was
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3.4. mRNA expression studies
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probed with pre-immune rabbit sera (negative control) (Supplementary Figure 2).
Treatment with anti-ICP antisera on the expression of different mRNA transcripts in the
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brain and ovary of C. batrachus were studied on day 7, 14 and 21. In the present study,
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the coefficient of determination (R2 ) of the primers above 0.98 was accepted. mRNA expression levels of the 4 genes assayed, exhibited an up-regulation following the anti-
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ICP antibody treatment (Fig. 3). For Act RI, significantly higher levels were observed in the brain on day 21 in T4 treatment while in ovary higher levels were seen in T3 on day 7 and on day 21 in T4 treatment groups respectively. Act RII were lowest in the brain while it followed more or less similar trend as that of Act RI in the ovary. FSHr mRNA levels were significantly highest in the ovaries in the treatment T3 followed by T2 on day 7. Similarly significantly highest mRNA expression levels of LHr were observed in the ovary on day 7 in the T3 treatment group.
ACCEPTED MANUSCRIPT 3.5. Changes in reproductive hormonal levels Hormone assays (FSH, LH, estradiol and progesterone) were carried out to check the effect of the administered anti-ICP antibody on reproductive hormonal levels in the serum on days 7, 14 and 21 post- injection. Anti-ICP antibody injected treatment T2 and
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T3 showed significantly higher FSH levels till day 14. LH surge was seen in treatment T2
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on day 7 and in T3 on day 21 after anti-ICP antibody treatment. The E2 surge was
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significantly higher in T2 on day 7 and T3 on day 14 and day 21. Progesterone levels were higher in treatment T2 on day 7 and T3 on 21 days (Fig. 4).
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3.6. Protein-peptide docking
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Modelled inhibin-α and the 26 residues ICP was docked using the Cluspro software. The best docked pose based on DCOMPLEX software reports a binding score of -17.80
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kcal/mol, which has a pose matching with the available literature and the gene rated
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antigenicity report (Figure 5). Two salt bridges were observed between ICP and inhibinα. These interactions were observed between Asp25-Arg246 and Glu2- Lys278 of
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chimeric peptide and inhibin-α respectively.
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3.7. Ligand-peptide docking
Betaglycan and the ICP were docked using Hex.8.0.0 software. Hydrogen bond
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interaction was investigated between the Betaglycan and the chimeric peptide using CHIMERA software. It was observed that ICP interacts with betaglycan through Lys3 and Ser8 with a binding energy of -255.60 kcal/mol
(Fig. 6). Thus positive and polar
amino acid interactions were observed between the ICP and betaglycan.
4. Discussion
ACCEPTED MANUSCRIPT Immunization against inhibin promotes the secretion of gonadotropins, that promotes the gonadal growth and gametogenesis (Yan et al., 2015). Despite the immense importance of inhibin hormone in reproduction little is known about its molecular mechanism of action in lower vertebrates. Given the increasing exploitation of antibodies in different
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contexts such as molecular diagnostics and therapeutics, it would be beneficial to raise
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antibodies against the in silico designed peptides and to validate their effect in animal
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models like fish. In the present study, a computational approach was used to design ICP and the antibodies were developed against it and subsequently, the antibodies were
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administered in adult female C. batrachus to check their effect on gametogenesis and on
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the mRNA levels of some important reproductive receptors and the hormones. Modelled 3D structure of inhibin- based on homology modelling when docked
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with the fold based modelled ICP showed strong interactions through two salt bridges
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formation. Furthermore, the protein- ligand interaction between ICP and betaglycan shows interaction with positive residues like Lys3 and polar residues like Ser8. This
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interaction could certainly interfere with activin binding to its receptor type II.
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In the present study, a homologous competitive ELISA determined the specific binding of the anti-ICP antibody with ICP and not with other unrelated peptides. Further,
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the anti-ICP antiserum was when treated with C. batrachus whole brain lysate showed a specific band of 18 kDa, thus confirming the specificity of the anti- inhibin antibody. Transcript expression levels of different receptors were upregulated after treatment with anti-ICP antibody in the whole brain and ovarian tissues. To functionally antagonize activin molecules, inhibin- α subunit interacts with the activin receptors type II and type I (Lebrun and Vale, 1997). Activin molecules in the goldfish and zebrafish stimulate mRNA expression of FSHβ but suppress LHβ expression in the pituitary (Yam et al.,
ACCEPTED MANUSCRIPT 1999; Poon et al., 2009). The effect of inhibin immunoneutralization is realized by a reduction in the inhibin/activin ratio, which in turn reduces the antagonizing effect of inhibins (Cai et al., 2015). The present study expands our understanding of how inhibin achieves potent antagonism of activin action through a combined mechanism of
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competitive binding of both Act RII by each subunit of the inhibin heterodimer, in
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conjunction with the co-receptor betaglycan, to block activin receptor- ligand binding and
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downstream signalling. Inhibin precursors have been found to compete with FSH binding at its receptor (FSHr), which reduces FSHr signalling and subsequent FSH stimulation of
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granulosa cell function (Schneyer et al.,1991). In the present study, FSHr transcript levels
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were up-regulated in the ovaries, which is in accordance with the reports of (Cai et al., 2015; Mao et al., 2016). The increase in FSH is accompanied by the enhancement in the
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E2 synthesis that in turn upregulates the expression of LHr (Cai et al., 2015; Ge, 2005).
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The E2 hormonal levels and mRNA expression of LHr in present work exhibited an upsurge that confirmed the role of anti-ICP antisera in ovulation of fish. The results of the
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present study were in agreement with the find ings, where the immunization against
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inhibin increased LHr expression (Cai et al., 2015). We anticipate a dose of 200 μl would yield a better spawning performance and ovulation in fishes for further trials.
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The results the present study indicate that endogenous inhibin is a primary factor that regulates gametogenesis, mainly by the controlling the synthesis and secretion of FSH from the anterior pituitary as described previously (Ahmad et al., 2018b). FSH concentration increased in a dose-dependent manner in the present study, which is similar to the earlier studies, wherein passive immunization against inhibin improved serum FSH concentrations in immature rats (Ishigame et al., 2005). It is well accepted that LH surge is essential for ovulation in fishes (Swanson et al., 1991). In addition, some recent studies
ACCEPTED MANUSCRIPT have indicated that increased level of LH in the circulation is also involved in follicular development and differentiation with increased ability to produce progesterone in response to gonadotropins (Conneely, 2001; Luo, 2011; Hill, 2012). The results of the present study suggest that anti- ICP antibody stimulated the sustained rise in plasma FSH
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concentration that may, in turn, increases the E2 levels. Similarly, the anti-ICP antibody
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may increase the LH levels that could help in the improving the spawning success and
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final ovulation in fishes. The increase in the hormonal level resulted in the advancement
gonadal maturation by acting on the HPG-axis.
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Conclusion
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of the gonadal development in fish. This denotes that anti-ICP antibody improves the
A species-specific ICP was designed, synthesized and antibodies were developed against
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ICP to study its effects in adult female C. batrachus. The results from the present study
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suggest that immunoneutralization of inhibin bioactivity occurs through augmentation of gonadotropin and activin receptor signalling. Furthermore, the immunoneutralization of
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inhibin improved reproductive function in C. batrachus.
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Conflict of interest
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All authors declared that they have no conflict of interest.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector
Acknowledgement
ACCEPTED MANUSCRIPT The authors are thankful to the Director, ICAR-Central Institute of Fisheries Education, Mumbai, India. We are also thankful to the Director ICMR-NIRRH, Mumbai for allowing us to work in Structural Biology and Neuroendocrinology. We would also like to acknowledge Mr Bhalchandra Kulkarni and Mr Suryakant M for technical assistance
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in the laboratory aspects of this study.
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Effect of FSH Receptor‐ Binding Inhibitor‐ 8 on FSH‐ Mediated Granulosa Cell
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ACCEPTED MANUSCRIPT Sali, A., Blundell, T.L., 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815. Schneyer, A.L., Sluss, P.M., Whitcomb, R.W., Martin, K.A., Sprengel, R., Crowley, Jr, W.F., 1991. Precursors of alpha- inhibin modulate follicle-stimulating hormone
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Stamatiades, G.A., Kaiser, U.B., 2017. Gonadotropin regulation by pulsatile GnRH:
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Signaling and gene expression. Molecular and cellular endocrinology 463, 131-141. Swanson, K., Suzuki, H., Kawauchi, W., Dickhoff., 1991. Isolation and characterization
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of two coho salmon gonadotropins, GTH I and GTH II. Biology of Reproduction 44,
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ACCEPTED MANUSCRIPT Yao, B., Zhang, L., Liang, S., Zhang, C., 2012. SVMTriP: A method to predict antigenic epitopes using support vector machine to integrate tri-peptide similarity and propensity. PLoS ONE 7(9), 451-52. Zohar, Y., 1989. Endocrinology and fish farming: aspects in reproduction, growth, and
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smoltification. Fish Physiology and Biochemistry, 7(1-6), pp.395-405.
ACCEPTED MANUSCRIPT Table 1 Primers used for the real-time quantitative PCR assay of genes Primer sequences (5′-3′)
Annealing temperature
FSHr
Upstream: CCAGGAGCCATACTTCTTCG Downstream: ATGAGGACACGCAGGAAAGT
63 0 C
LHr
Upstream: CCCTCCGCTGGAGAACCT Downstream: ACAGCGGCCTCCTTGTTGTC
65 0 C
Act RI
Upstream: CACACAGGGAAAGCCTGGCA Downstream: GACGGACTCGTGGCGTACAG
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62 0 C
Upstream: GCACCCCGTCCTGCTTACTGAG Downstream: CCAGACGGAGGATGGCATGG
570 C
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β-actin
67 0 C
Upstream: CCTGCGTAGGAGACAAGGAC Downstream: CACACTCCGTGCTGTCGTAG
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Act RII
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Gene
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Fig. 1. Labelled short inhibin chimeric peptide (ICP) structure wireframe. Fig. 2. Competitive inhibition antibodies binding studies of anti-ICP antibodies to the immobilized inhibin chimeric peptide (ICP), porcine inhibin- peptide and unrelated peptide. Various concentrations (3-100µg) of the ICP or the unrelated peptides were preincubated with antibody at 1:16000 dilution before addition to the peptide immobilized in microtitre wells for ELISA. Fig. 3. Change in the messenger RNA expression of four types of reproductive genes in female C. batrachus whole brain and ovarian tissue at day 7, day 14 and day 21 after treatments were injected with different doses: 200μl pre-immune rabbit serum (T1), 100μl anti-ICP antisera (T2), 200μl anti-ICP antisera (T3) and 400μl anti-ICP antisera (T4). The data are expressed as the mean ± SEM. Significant difference (P < 0.05) between the treated and control group is indicated with asterisk. Fig. 4. In-vivo effect of different anti- ICP antisera doses: T1 (200μl pre-immune rabbit serum), T2 (100μl), T3 (200μl) and T4 (400μl) on hormonal profile (FSH, LH, E2 and progesterone) in adult female C. batrachus. Values are expressed as mean ± SEM. Significant difference (P < 0.05) between the treated groups is indicated with different asterisk on different sampling days. Fig.5. Protein-peptide interaction analysis of inhibin- with ICP (A). Ribbon shaped structure of inhibin- docked with inhibin chimeric peptide (ICP). Two salt bridges were observed between (Asp25-Asp246; Glu2-Lys278) (B)
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Fig. 6. Ribbon shaped structure of inhibin chimeric peptide (ICP) interacting with betaglycan (A). Positive and polar residues Lys3 and Ser8 are involved in the protein ligand interaction. Surface view of inhibin chimeric peptide (ICP) docked with betaglycan (B)
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Highlights Novel species-specific inhibin chimeric peptide was designed for C. batrachus Structural characterization of ICP was determined using bioinformatics approach Anti-inhibin antibody significantly up regulated the receptor expression and hormonal levels
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Irshad Ahmad, Irfan Ahmad Bhat, Dhanashree D. Jagtap, Nafisa H. Balasinor, C. Selvaa Kumar, T.I. Chanu, Babitha Rani, Showkat Ahmad Dar, Tasok Leya, Adnan Hussain Gora, Neelam Saharan PII: DOI: Reference:
S0044-8486(18)31925-2 https://doi.org/10.1016/j.aquaculture.2018.11.016 AQUA 633678
To appear in:
aquaculture
Received date: Revised date: Accepted date:
6 September 2018 5 November 2018 8 November 2018
Please cite this article as: Irshad Ahmad, Irfan Ahmad Bhat, Dhanashree D. Jagtap, Nafisa H. Balasinor, C. Selvaa Kumar, T.I. Chanu, Babitha Rani, Showkat Ahmad Dar, Tasok Leya, Adnan Hussain Gora, Neelam Saharan , Computational and biological approach for studying structure-function of inhibin chimeric peptide antibodies in Clarias batrachus. Aqua (2018), https://doi.org/10.1016/j.aquaculture.2018.11.016
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ACCEPTED MANUSCRIPT Computational and biological approach for studying structurefunction of inhibin chimeric peptide antibodies in Clarias batrachus
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Irshad Ahmad1 , Irfan Ahmad Bhat1 , Dhanashree D. Jagtap2 , Nafisa H Balasinor2 , Selvaa Kumar C3 , Chanu T.I4 , Babitha Rani4 , Showkat Ahmad Dar4 , Tasok Leya4 , Adnan Hussain Gora5 , Neelam Saharan4
1
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Affiliations:
College of Fisheries Science, Gumla, Birsa Agricultural University, Ranchi-834006,
2
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India
Indian Council of Medical Research-National Institute for Research in Reproductive
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Bioinformatics Department, School of Biotechnology and Bioinformatics, D.Y. Patil
University, Navi Mumbai-400614, India
Indian Council of Agricultural Research-Central Institute of Fisheries Education,
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4
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Mumbai- 400061, India
ICAR-CMFRI, Biotechnology Center, Madras Chennai, India
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5
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Health, Mumbai-400012, India
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Corresponding Author:
Prof. Neelam Saharan ICAR-Central Institute of Fisheries Education Department of Aquatic Environment management India, Mumbai-400061
Fax: +91 2226361573 Email: [email protected]/[email protected]
ACCEPTED MANUSCRIPT Abstract Inhibins are heterodimeric glycoproteins that belong to transforming growth factor-β superfamily. The present study was aimed at structural elucidation of immunogenic inhibin chimeric peptide (ICP) using immunoinformatics tools, to develop anti-ICP
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antibodies and to understand the mechanism of interaction between HPG-axis and
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anti-ICP antibodies in adult female Clarias batrachus. Homology modelling predicted
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that the ICP adopts a turn and random coil, which was further confirmed by circular dichroism (CD). Competitive binding ELISA with ICP and different unrelated peptides
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confirmed that the developed anti-ICP antiserum is highly specific and critical for the
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direct interaction to the inhibin-α subunit. The anti- ICP antibodies increased the mRNA transcript levels of follicle stimulating hormone receptor (FSHr), luteinizing hormone
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receptor (LHr), activin receptor type I (Act RI) and activin receptor type II (Act RII).
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Further, the serum concentrations of follicle stimulating hormone (FSH), 17β-estradiol (E2) and progesterone increased significantly in anti-ICP antisera treated groups
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compared to control. Modelled inhibin- and ICP exhibits better interaction with each
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other through two salt bridge formations. Also, ICP showed better interaction with betaglycan through positive and polar-based interactions. The results of the present study
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suggest that immunoneutralization of inhibin bioactivity, confirms the physiological role of inhibin as a component of ovarian feedback mechanism regulating FSH secretion through augmentation of gonadotropin and activin receptor signalling pathways that could affect the reproductive success in fishes.
Keywords: Inhibin chimeric peptide, circular dichroism, protein-peptide docking, antipeptide antibody, activin receptor signalling
ACCEPTED MANUSCRIPT 1. Introduction Inhibin proteins are structurally related to the Transforming Growth Factor-β (TGF-β) superfamily. Biologically active forms; inhibin A and inhibin B are heterodimeric glycoproteins composed of an α-subunit and one of the forms of β-subunit namely βA or
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βB (Makanji et al., 2014). Inhibin binds to betaglycan with high affinity via its α-subunit
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and this binding markedly triggers the binding of the inhibin-β subunit to the activin
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receptor type II A (Act RIIA) (Lewis et al., 2000; Ahmad et al., 2018a). Inhibin action is
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regulated when inhibin binds to activin receptor type II preventing activin- induced heteromerization of type I and type II receptors, which play a crucial role in activating the
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activin-dependent intracellular signalling cascade (Lebrun and Vale, 1997). Precise mechanisms of hormonal regulation of the hypothalamic-pituitary-
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gonadal (HPG) axis are essential for normal reproductive functioning in all animal
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species. The pulsatile secretion of hypothalamic gonadotropin-releasing hormone stimulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone
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(LH) from the anterior pituitary (Stamatiades and Kaiser, 2017). Despite the central role
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of gonadal inhibin in the regulation of the reproductive axis, little is known regarding its structure and molecular mechanism of action in lower vertebrates. Designing an
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immunogenic peptide of inhibin-α and elucidating the structure of the designed peptide using immunoinformatics tools and also to understand receptor expression and hormonal physiology is of importance. Immunogenic peptides designed against inhibitory proteins (inhibins) could be one the approaches towards the development of novel targets to improve breeding performances in fishes. Anti-peptide antibodies are important tools in clinical laboratory
ACCEPTED MANUSCRIPT diagnostics primarily because they recognize native proteins (Trier and Houen, 2017). Designing new immunogenic peptides and developing antibodies with desired properties could have a profound impact on fish reproduction. In captivity, the breeding performances of fishes are generally low due to the poor quality of female brooders
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(Zohar, 1989 and Mylonas and Zohar 2001). This is mainly because of the lack of
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required environmental parameters for natural reproductive performances (Mylonas et al.,
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2010). The purpose of the present study was to determine whether the presence of antibodies against endogenous inhibins could affect the secretion of some important
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reproductive genes, hormones and the timing of ovulation in female Clarias batrachus,
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an important air-breathing catfish found in Indian waters. We aimed to designed speciesspecific inhibin chimeric peptide (ICP) and to elucidate its structure using bioinformatics
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approach. Furthermore, the in-vivo effect of the anti-ICP antibody on receptor expression
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and hormonal levels were also studied in adult female C. batrachus.
2. Materials and methods
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2.1. Homology modelling
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Inhibin-α was cloned and sequenced in C. batrachus (Ahmad et al., 2018a) and the putative protein sequence was used for the prediction of continuous B- and T-cell
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epitopes using the IEDB (http://www.iedb.org). Based on this information, inhibin-α chimeric peptide (ICP) was designed from in silico perspective. To model the 3D structure of the ICP, 3D crystal structure was much needed. For the same, BLAST-PDB search (Altschul et al., 1990) was initiated to identify potential template with a default matrix of BLOSUM62. However, this step went futile as there was no reported structure available from Protein Data Bank (Berman et al., 2000) based on sequence homology. As
ACCEPTED MANUSCRIPT an alternative, we opted for a fold-based method using pDOMTHREADER (Lobley et al., 2009) that is an accurate and sensitive superfamily discrimination method. Highly accurate domain alignments were generated through the collective information from both sequence and structure. The template with better score listed by pDOMTHREADER was
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considered for homology modelling using Modeller 9.18 standalone version (Sali and
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Blundell, 1993). ‘align2d.py’ and ‘model-single.py’ were used for the template-query
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alignment and model building respectively. Next, to identify a potential template for inhibin- BLAST-PDB search was initiated with a default matrix of BLOSUM62. of all
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the listed templates, the crystal structure with better query coverage and identity was
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considered for sequence-based homology modelling using Modeller9.18. Ten models were generated for ICP and inhibin- α that was later validated through DOPE (Discrete
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Optimized Protein Energy) and RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/
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rampage.php) (Richardson, 1981). This is the process of evaluating reliability for 3Datomic models of large biological molecules and also identify the model with the best
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geometry.
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2.2. Secondary structure estimation of ICP by Circular dichroism (CD) CD was recorded on a Jasco J-815 spectrometer (Jasco, Easton, MD, USA) from 195 to
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260nm, in a 1cm path length quartz cell. Fifty micromolar synthetic ICP (United BioSystems Inc. USA) in 25mM sodium phosphate buffer, pH 7.5 at 25 0 C was used. All solutions were filtered with a 0.22mm pore nylon filter prior to performing the experiments. Three consecutive scans were accumulated and the spectra thus obtained were averaged. The percentages of different secondary structures were estimated using the data provided by (Yang et al., 1986) for the reference spectra (Sharma et al., 2000; Kene et al., 2005; Chitnis et al., 2009).
ACCEPTED MANUSCRIPT 2.3. Production of anti-ICP antibody Antibodies against ICP were developed in rabbits (Belgian white strain) of approximately 3 kg body weight following our previous reports (Ahmad et al., 2018b). Use of the animal was approved by the Institutional Animal Ethics Committee of ICMR-NIRRH
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(IAEC no. 15/16) and all the experiments were conducted as per the norms of the
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Committee for the Purpose of Control and Supervision of Experiments on Animals
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(CPCSEA), India. 2.4. Competitive ELISA
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For competition experiments, serial dilutions of the ICP and unrelated peptide were pre-
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incubated with the antisera before addition to the microtiter wells following (Jagtap et al., 2002).
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2.5. Western blotting
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Binding of anti-ICP antibodies to the whole brain lysate of female C. batrachus was checked by Western blotting following Navalakhe et al. (2013). Frozen whole brain
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tissue was homogenized in ice-cold lysis buffer containing protease inhibitor cocktail and
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was centrifuged (Ahmad et al., 2018b). The protein concentration of the supernatant was determined by micro-BCA protein assay kit (Thermo Fisher, Waltham, MA, USA) and
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the supernatant was stored at -80°C till further use. 30 μg of the stored brain supernatant was resolved on a 12% SDS-PAGE and the proteins were transferred onto a nitrocellulose membrane (0.45 μm Immobilon; GE Health-care) using the Trans-Blot wet transfer apparatus (Bio-Rad, Richmond, CA, USA) at 4°C for 1h. After transfer, the membrane was cut into two and blocked with 5 % non-fat dry milk (NFDM; Anik Industries Ltd, Mumbai, India) in TBS-T (Tris-buffered saline containing 0.1% Tween 20). Next, one membrane blot was incubated with anti-ICP antibodies (test) and the other
ACCEPTED MANUSCRIPT blot was incubated with pre- immune rabbit serum (control) in 1 % NFDM in TBS-T for 1h at room temperature. The membranes were then washed with TBS-T followed by treatment with the secondary antibody (Goat anti-rabbit IgG HRP, 1:2000 dilution). After washing the membranes, the immunoreactive bands were visualized with ECL plus
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reagent according to the manufacturer’s instructions (GE Healthcare).
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2.6. Animals
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The adult mature female C. batrachus (> 1-year-old) in the late pre-spawning stage with average weight; 78.66 ± 0.89g and length; 21 ± 1.09cm were stocked in cemented tanks
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(4.5×2×1m). Females were fed ad libitum with commercially available pelleted feed
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(Charoen Pokphand (CP) feeds, India) having crude protein-(36%), crude lipid-(5%), fibre- (4%), moisture-(11%) and ash-(16%) respectively. The experiment was conducted
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at the freshwater fish farm Balabhadrapuram, Kakinada centre of ICAR-Central Institute
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of Fisheries Education, Andhra Pradesh, India. The animals used for the study were reared and handled under the approved protocol that followed the policies and guidelines
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of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on
India.
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Animals), Ministry of Environment and Forests (Animal Welfare Division), Govt. of
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To check the effect of anti-ICP antibodies on the receptor expression and hormonal profiles, 80 females fishes were randomly assigned to four treatment groups of 20 fishes each. The T1 group was injected with 200µl pre- immune rabbit serum (control), T2 group with 100µl anti-ICP (low dose), T3 group with 200µl anti-ICP (medium dose) and T4 group with 400µl anti-ICP (high dose) antisera respectively. The antisera were administered intramuscularly to each female fish on day 1, day 7 and day 14 under light
ACCEPTED MANUSCRIPT anesthesia by immersion method using clove oil (Merck, Germany) @ 40 μl/l of water (Ahmad et al., 2018b). 2.7. mRNA receptor expression Three fishes from each treatment group were randomly selected and sacrificed on day 7,
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day 14 and day 21 for the collection of whole brain and ovarian tissues. Total RNA was
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extracted from ovary and brain tissues as per our previous reports (Ahmad et al., 2018).
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To remove any genomic DNA contamination, the extracted RNA was treated with DNaseI using DNaseI RNase-free kit (Thermo Scientific, USA). DNaseI treated RNA
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(1µg) was then reverse transcribed using AffinityScript QPCR cDNA Synthesis Kit
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according to the manufacturer's protocol (Agilent technologies, USA). To study the modulatory effect of anti- ICP antibodies, mRNA expression of
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different receptors involved in reproduction was performed by real-time quantitative
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polymerase chain reaction (qPCR). The receptors selected were follicle stimulating hormone receptor (FSHr), luteinizing hormone receptor (LHr), activin receptor type I and
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II (Act RI and II). The quantitative RT-PCR experiments for these genes were conducted
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in duplicates (Table 1).
β-actin was used as an internal control in the present study as it exhibited lesser
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variations compared to other housekeeping genes. The softwares GeNorm, best keeper, delta Ct method and NormFinder were used for the validation of housekeeping genes. Further, the serial dilution of primers was also done to determine the efficiency of the primers. PCR was performed using Light Cycler1 Real-time PCR detection system (Roche, USA) using Brilliant III Ultra-Fast SYBR® Green qPCR master mix (Agilent Technologies, USA). Cycle threshold (Ct) values were obtained from the exponential phase of PCR amplification, and the expression of genes was normalized to the
ACCEPTED MANUSCRIPT expression of β-actin generating a ΔCt value (Ct of target gene -ΔCt of β-actin). The relative expression level of the target gene to the β-actin gene was determined using the 2- ΔΔCt method proposed by (Livak and Schmittgen, 2001) which gives the value that corresponds to the n-fold difference relative to the control.
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2.8. Hormonal assays
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After every 7 days interval, fishes from each treatment (n=5) were randomly collected
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and anaesthetized using clove oil (Merck, Germany) @ 40 μl/l (Ahmad et al., 2018) and the blood was collected without anticoagulant. The blood was allowed to clot for 4h and
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then centrifuged at 4000 rpm for 10 min, followed by the collection of serum. Serum was
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stored at -80°C for further analysis. The different reproductive hormones namely FSH, LH, 17β-estradiol (E2) and progesterone were measured in triplicate in the serum using
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commercial EIA kits following manufacturer's protocol (Cayman Chemical, USA). FSH
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and LH immunometric assay was based on the sandwich technique and uses the Horse Radish Peroxidase (HRP)- labeled detection antibody. For steroid hormones (E2 and
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progesterone) the assay was based on the competitive immunoreactions between the free
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hormone and steroid- linked acetylcholinesterase conjugate. The sensitivity, intra-assay and inter-assay coefficients of variations were determined for all the hormo nes and are
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presented in Supplementary Table 1. Enzyme immunoassay was performed in 96-well plate format and the absorbance was recorded on an ELISA reader for different hormones. 2.9. Protein-peptide docking 3D-modelled ICP and inhibin- were considered for protein-peptide docking to understand their overall binding affinity using Cluspro server (Kozakov et al., 2017). This study was mainly intended to understand the effect of ICP binding with inhibin-
ACCEPTED MANUSCRIPT and their interference to activin receptor type II. In total Cluspro generates 30 poses, of which based on the literature survey and antigenicity report from SVMTrip software (Yao et al., 2012) best-docked pose was selected. Cluspro only generates poses and not the binding energy. To calculate the same, here we opted for DCOMPLEX software (Liu
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et al., 2004) that gives energy in kcal/mol. Finally, the docked pose was visualized using
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CHIMERA software (Pettersen et al., 2004).
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2.10. Betaglycan-ICP docking
Betaglycan was downloaded from the PubChem database (Kim et al., 2016) Structure ID:
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CID_439262) in sdf file format. Furthermore, a stable conformer was generated after
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structure minimization using FROG2 (Miteva et al., 2010). In the option box ‘minimize' was selected as ‘Yes' and ‘Produce' was ‘single'. Which means the given betaglycan will
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be energy minimized and a single conformer with least energy will be considered.
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Gasteiger charges were added and the optimized structure was considered for docking with modelled ICP using Hex8.0.0 (Ritchie, 2003). For correlation type, and Post-
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processing we opted for "Shape+Electro+DARS" and for "DARS Minimization"
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respectively. Standard docking with a grid dimension of 0.6Å and solution of 2000 was preferred. After docking, top ten listed docked poses were considered for amino acid
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interaction analysis using CHIMERA software (Pettersen et al., 2004). Finally, we selected the pose with the better binding affinity between the betaglycan and ICP. Our main intention behind this study was to understand the overall interaction between the ligand and the ICP. 2.11. Statistical analysis The differences in mRNA transcript and hormonal levels were tested for statistical significance using the statistical package SPSS 17.0 (USA). One-way analysis of
ACCEPTED MANUSCRIPT variance (ANOVA), followed by Tukey's multiple comparison test and P < 0.05 was considered statistically significant. The results were expressed as the mean ± standard error.
3. Results
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3.1. Homology Modelling
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The predicted B-cell and T-cell epitopes were used to design the inhibin chimeric peptide
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(IEKLQRPSPEKDDTYGGGDTVSSEDP) for C. batrachus (Ahmad et al., 2018). With
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no reported template available based on amino acid identity, the short chimeric peptide was designed using the fold-based method using pDOMTHREADER. The listed TGF-β2
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with PDB ID: 2TGI (Daopin et al., 1992) has a confidence of HIGH with a net score of 4.65. This template was considered for modelling (26 amino acid ICP) using Modeller
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standalone software and ten models were generated (Fig.1). The 3D model with the least
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DOPE score of -13503.70 was selected. Furthermore, the modelled structure was validated using RAMPAGE tool. In ICP, 91.7% is in the favoured region and 8.3% in
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the allowed region with no outliers (Supplementary Figure 1A). This modelled structure
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is a random coil with no reported secondary structure. Inhibin-α was modelled based on 5HLY Chain A with the query coverage of 24-343 residues. Of the ten- modelled
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structure, the 3D structure with the least DOPE score of -31191.13 was selected. Inhibinα, 90.6% residues are in the favoured region; 6.2% in the allowed region; 3.2% residues in the outlier region (Supplementary Figure 1B).
ACCEPTED MANUSCRIPT 3.2. CD profile of ICP ICP (5mg) with 90% purity was commercially synthesized from United BioSystems Inc. USA. Secondary structure estimated by CD analysis revealed that the ICP comprises 21.9% α-helix, 15.7% β-sheets, 33.3% turn and 29.1% random-coil structure.
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3.3. Competitive ELISA
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In competitive inhibition studies, homologous peptide competitively inhibited the binding
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of the ICP anti-peptide antibodies to the immobilized peptide while the unrelated peptides did not (Fig. 2).
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3.4. Inhibin-α in whole brain lysate
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Whole brain lysate of C. batrachus was probed with anti-ICP antibodies that detected a dark band at a molecular weight of ∼18 kDa. No band was detected when the lysate was
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3.4. mRNA expression studies
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probed with pre-immune rabbit sera (negative control) (Supplementary Figure 2).
Treatment with anti-ICP antisera on the expression of different mRNA transcripts in the
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brain and ovary of C. batrachus were studied on day 7, 14 and 21. In the present study,
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the coefficient of determination (R2 ) of the primers above 0.98 was accepted. mRNA expression levels of the 4 genes assayed, exhibited an up-regulation following the anti-
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ICP antibody treatment (Fig. 3). For Act RI, significantly higher levels were observed in the brain on day 21 in T4 treatment while in ovary higher levels were seen in T3 on day 7 and on day 21 in T4 treatment groups respectively. Act RII were lowest in the brain while it followed more or less similar trend as that of Act RI in the ovary. FSHr mRNA levels were significantly highest in the ovaries in the treatment T3 followed by T2 on day 7. Similarly significantly highest mRNA expression levels of LHr were observed in the ovary on day 7 in the T3 treatment group.
ACCEPTED MANUSCRIPT 3.5. Changes in reproductive hormonal levels Hormone assays (FSH, LH, estradiol and progesterone) were carried out to check the effect of the administered anti-ICP antibody on reproductive hormonal levels in the serum on days 7, 14 and 21 post- injection. Anti-ICP antibody injected treatment T2 and
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T3 showed significantly higher FSH levels till day 14. LH surge was seen in treatment T2
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on day 7 and in T3 on day 21 after anti-ICP antibody treatment. The E2 surge was
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significantly higher in T2 on day 7 and T3 on day 14 and day 21. Progesterone levels were higher in treatment T2 on day 7 and T3 on 21 days (Fig. 4).
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3.6. Protein-peptide docking
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Modelled inhibin-α and the 26 residues ICP was docked using the Cluspro software. The best docked pose based on DCOMPLEX software reports a binding score of -17.80
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kcal/mol, which has a pose matching with the available literature and the gene rated
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antigenicity report (Figure 5). Two salt bridges were observed between ICP and inhibinα. These interactions were observed between Asp25-Arg246 and Glu2- Lys278 of
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chimeric peptide and inhibin-α respectively.
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3.7. Ligand-peptide docking
Betaglycan and the ICP were docked using Hex.8.0.0 software. Hydrogen bond
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interaction was investigated between the Betaglycan and the chimeric peptide using CHIMERA software. It was observed that ICP interacts with betaglycan through Lys3 and Ser8 with a binding energy of -255.60 kcal/mol
(Fig. 6). Thus positive and polar
amino acid interactions were observed between the ICP and betaglycan.
4. Discussion
ACCEPTED MANUSCRIPT Immunization against inhibin promotes the secretion of gonadotropins, that promotes the gonadal growth and gametogenesis (Yan et al., 2015). Despite the immense importance of inhibin hormone in reproduction little is known about its molecular mechanism of action in lower vertebrates. Given the increasing exploitation of antibodies in different
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contexts such as molecular diagnostics and therapeutics, it would be beneficial to raise
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antibodies against the in silico designed peptides and to validate their effect in animal
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models like fish. In the present study, a computational approach was used to design ICP and the antibodies were developed against it and subsequently, the antibodies were
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administered in adult female C. batrachus to check their effect on gametogenesis and on
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the mRNA levels of some important reproductive receptors and the hormones. Modelled 3D structure of inhibin- based on homology modelling when docked
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with the fold based modelled ICP showed strong interactions through two salt bridges
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formation. Furthermore, the protein- ligand interaction between ICP and betaglycan shows interaction with positive residues like Lys3 and polar residues like Ser8. This
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interaction could certainly interfere with activin binding to its receptor type II.
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In the present study, a homologous competitive ELISA determined the specific binding of the anti-ICP antibody with ICP and not with other unrelated peptides. Further,
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the anti-ICP antiserum was when treated with C. batrachus whole brain lysate showed a specific band of 18 kDa, thus confirming the specificity of the anti- inhibin antibody. Transcript expression levels of different receptors were upregulated after treatment with anti-ICP antibody in the whole brain and ovarian tissues. To functionally antagonize activin molecules, inhibin- α subunit interacts with the activin receptors type II and type I (Lebrun and Vale, 1997). Activin molecules in the goldfish and zebrafish stimulate mRNA expression of FSHβ but suppress LHβ expression in the pituitary (Yam et al.,
ACCEPTED MANUSCRIPT 1999; Poon et al., 2009). The effect of inhibin immunoneutralization is realized by a reduction in the inhibin/activin ratio, which in turn reduces the antagonizing effect of inhibins (Cai et al., 2015). The present study expands our understanding of how inhibin achieves potent antagonism of activin action through a combined mechanism of
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competitive binding of both Act RII by each subunit of the inhibin heterodimer, in
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conjunction with the co-receptor betaglycan, to block activin receptor- ligand binding and
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downstream signalling. Inhibin precursors have been found to compete with FSH binding at its receptor (FSHr), which reduces FSHr signalling and subsequent FSH stimulation of
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granulosa cell function (Schneyer et al.,1991). In the present study, FSHr transcript levels
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were up-regulated in the ovaries, which is in accordance with the reports of (Cai et al., 2015; Mao et al., 2016). The increase in FSH is accompanied by the enhancement in the
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E2 synthesis that in turn upregulates the expression of LHr (Cai et al., 2015; Ge, 2005).
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The E2 hormonal levels and mRNA expression of LHr in present work exhibited an upsurge that confirmed the role of anti-ICP antisera in ovulation of fish. The results of the
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present study were in agreement with the find ings, where the immunization against
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inhibin increased LHr expression (Cai et al., 2015). We anticipate a dose of 200 μl would yield a better spawning performance and ovulation in fishes for further trials.
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The results the present study indicate that endogenous inhibin is a primary factor that regulates gametogenesis, mainly by the controlling the synthesis and secretion of FSH from the anterior pituitary as described previously (Ahmad et al., 2018b). FSH concentration increased in a dose-dependent manner in the present study, which is similar to the earlier studies, wherein passive immunization against inhibin improved serum FSH concentrations in immature rats (Ishigame et al., 2005). It is well accepted that LH surge is essential for ovulation in fishes (Swanson et al., 1991). In addition, some recent studies
ACCEPTED MANUSCRIPT have indicated that increased level of LH in the circulation is also involved in follicular development and differentiation with increased ability to produce progesterone in response to gonadotropins (Conneely, 2001; Luo, 2011; Hill, 2012). The results of the present study suggest that anti- ICP antibody stimulated the sustained rise in plasma FSH
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concentration that may, in turn, increases the E2 levels. Similarly, the anti-ICP antibody
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may increase the LH levels that could help in the improving the spawning success and
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final ovulation in fishes. The increase in the hormonal level resulted in the advancement
gonadal maturation by acting on the HPG-axis.
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Conclusion
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of the gonadal development in fish. This denotes that anti-ICP antibody improves the
A species-specific ICP was designed, synthesized and antibodies were developed against
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ICP to study its effects in adult female C. batrachus. The results from the present study
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suggest that immunoneutralization of inhibin bioactivity occurs through augmentation of gonadotropin and activin receptor signalling. Furthermore, the immunoneutralization of
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inhibin improved reproductive function in C. batrachus.
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Conflict of interest
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All authors declared that they have no conflict of interest.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector
Acknowledgement
ACCEPTED MANUSCRIPT The authors are thankful to the Director, ICAR-Central Institute of Fisheries Education, Mumbai, India. We are also thankful to the Director ICMR-NIRRH, Mumbai for allowing us to work in Structural Biology and Neuroendocrinology. We would also like to acknowledge Mr Bhalchandra Kulkarni and Mr Suryakant M for technical assistance
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in the laboratory aspects of this study.
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smoltification. Fish Physiology and Biochemistry, 7(1-6), pp.395-405.
ACCEPTED MANUSCRIPT Table 1 Primers used for the real-time quantitative PCR assay of genes Primer sequences (5′-3′)
Annealing temperature
FSHr
Upstream: CCAGGAGCCATACTTCTTCG Downstream: ATGAGGACACGCAGGAAAGT
63 0 C
LHr
Upstream: CCCTCCGCTGGAGAACCT Downstream: ACAGCGGCCTCCTTGTTGTC
65 0 C
Act RI
Upstream: CACACAGGGAAAGCCTGGCA Downstream: GACGGACTCGTGGCGTACAG
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62 0 C
Upstream: GCACCCCGTCCTGCTTACTGAG Downstream: CCAGACGGAGGATGGCATGG
570 C
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β-actin
67 0 C
Upstream: CCTGCGTAGGAGACAAGGAC Downstream: CACACTCCGTGCTGTCGTAG
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Act RII
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Gene
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Fig. 1. Labelled short inhibin chimeric peptide (ICP) structure wireframe. Fig. 2. Competitive inhibition antibodies binding studies of anti-ICP antibodies to the immobilized inhibin chimeric peptide (ICP), porcine inhibin- peptide and unrelated peptide. Various concentrations (3-100µg) of the ICP or the unrelated peptides were preincubated with antibody at 1:16000 dilution before addition to the peptide immobilized in microtitre wells for ELISA. Fig. 3. Change in the messenger RNA expression of four types of reproductive genes in female C. batrachus whole brain and ovarian tissue at day 7, day 14 and day 21 after treatments were injected with different doses: 200μl pre-immune rabbit serum (T1), 100μl anti-ICP antisera (T2), 200μl anti-ICP antisera (T3) and 400μl anti-ICP antisera (T4). The data are expressed as the mean ± SEM. Significant difference (P < 0.05) between the treated and control group is indicated with asterisk. Fig. 4. In-vivo effect of different anti- ICP antisera doses: T1 (200μl pre-immune rabbit serum), T2 (100μl), T3 (200μl) and T4 (400μl) on hormonal profile (FSH, LH, E2 and progesterone) in adult female C. batrachus. Values are expressed as mean ± SEM. Significant difference (P < 0.05) between the treated groups is indicated with different asterisk on different sampling days. Fig.5. Protein-peptide interaction analysis of inhibin- with ICP (A). Ribbon shaped structure of inhibin- docked with inhibin chimeric peptide (ICP). Two salt bridges were observed between (Asp25-Asp246; Glu2-Lys278) (B)
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Fig. 6. Ribbon shaped structure of inhibin chimeric peptide (ICP) interacting with betaglycan (A). Positive and polar residues Lys3 and Ser8 are involved in the protein ligand interaction. Surface view of inhibin chimeric peptide (ICP) docked with betaglycan (B)
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Highlights Novel species-specific inhibin chimeric peptide was designed for C. batrachus Structural characterization of ICP was determined using bioinformatics approach Anti-inhibin antibody significantly up regulated the receptor expression and hormonal levels
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