In silico characterization of interferon-stimulated gene (ISG15) as a biomarker for early pregnancy diagnosis in Bubalus bubalis
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Keywords:
Biomarker, Bubalus bubalis, Characterization, In silico, ISG15, Pregnancy detection Present address, 1Research Associate (drkanishtbatra @gmail.Abstract
Reproductive management of livestock is the foremost requirement for increasing production from any dairy animal. High reproductive efficiency can be achieved by implementation of one of the crucial management practices which involves early differentiation of pregnant and non-pregnant animals. The early and precise pregnancy diagnosis is the major problem in Bubalus bubalis (buffalo) due to absence of estrous signs instead of normal cyclicity of reproductive organs. Therefore, in the present study, one of the conceptus derived protein interferon stimulated protein (15 kDa) released during 18 to 21 days after insemination in response to implantation of embryo was in silico characterized. The protein expressed by interferon-stimulated gene-15 (ISG15) was analyzed by isolation and sequencing of coding region of mRNA. The primary and secondary structures were predicted from the protein sequence to decipher the interaction of ISG15 protein with other molecules. The functional characterization identifies various motifs present in ISG15 protein which are responsible for its interaction with other proteins. Physiochemical properties predicted the ISG15 protein nature during in vitro conditions which are required for any of the assays development. In addition, immunogenic studies revealed ISG15 protein is strongly antigenic in nature and can be used for antibody production. In conclusion, ISG15 protein expression from buffalo is a good indicator of conceptus implantation and has suitable properties for being used as target to develop early pregnancy diagnostic kits.Downloads
References
Arnold K L, Bordoli J K and Schwede T. 2006. The SWISS- MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201. DOI: https://doi.org/10.1093/bioinformatics/bti770
Austin K J, Ward S K, Teixeira M G, Dean V C, Moore D W and Hansen T R. 1996. Ubiquitin cross-reactive protein is released by the bovine uterus in response to interferon during early pregnancy. Biology of Reproduction 54: 600–606. DOI: https://doi.org/10.1095/biolreprod54.3.600
Austin K J, Bany B M, Belden E L, Rempel L A, Cross J C and Hansen T R. 2003. Interferon stimulated gene-15 (Isg15) expression is up-regulated in the mouse uterus in response to the implanting conceptus. Endocrinology 144: 3107–13. DOI: https://doi.org/10.1210/en.2002-0031
Balhara A K, Gupta M, Mohanty A K, Phulia S K, Sharma R K, Singh S and Singh I. 2014. Changes in sera proteome in relation to day of pregnancy in early pregnant buffaloes. Indian Journal of Animal Sciences 84(4): 400–409.
Batra K. 2017. ‘In vitro expression, molecular and immunological characterization of early pregnancy related interferon tau stimulated gene of Bubalus bubalis.’ PhD Thesis, LUVAS, Hisar, Haryana, India.
Batra K, Nanda T, Kumar A, Kumar V, Gopal G J and Maan S. 2018a. Molecular cloning and expression kinetics of serum Interferon stimulated gene for early pregnancy detection. Indian Journal of Animal Research. DOI: 10.18805/ijar. B-3636. DOI: https://doi.org/10.18805/ijar.B-3636
Batra K, Nanda T, Kumar A, Gupta A K, Kumari R, Kumar V, Sheoran N and Maan S. 2018b. Molecular cloning and characterization of Mx2 for early pregnancy diagnosis in Bubalus bubalis. Proceedings of the National Academy of Sciences India Section B Biological Sciences. https://doi.org/10.1007/s40011-018-0993-x DOI: https://doi.org/10.1007/s40011-018-0993-x
Batra K, Kumar A, Maan S, Kumar V, Kumari R and Nanda T. 2018c. Recombinant interferon stimulated protein 15 (rISG15) as a molecular marker for detection of early pregnancy in Bubalus bubalis. Animal Reproduction Science 197: 106–16 DOI: https://doi.org/10.1016/j.anireprosci.2018.08.018
Bazer F W, Spencer T E, Johnson G A, Burghardt R C and Wu G. 2009. Comparative aspects of implantation. Reproduction 138: 195–209. DOI: https://doi.org/10.1530/REP-09-0158
Bebington C, Doherty F J and Fleming S D. 1999. Ubiquitin cross- reactive protein gene expression is increased in decidualized endometrial stromal cells at the initiation of pregnancy. Molecular Human Reproduction 5: 966–72. DOI: https://doi.org/10.1093/molehr/5.10.966
Blom N, Gammeltoft S and Brunak S. 1999. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of Molecular Biology 294(5): 1351–62. DOI: https://doi.org/10.1006/jmbi.1999.3310
Chauhan J S, Rao A and Raghava G P S. 2013. In silico Platform for prediction of N-, O-and C-glycosites in eukaryotic protein sequences. PloS one 8(6):e67008. DOI: https://doi.org/10.1371/journal.pone.0067008
Dinkel H, Van K R, Michael S, Kumar M, Uyar B, Altenberg B, Milchevskaya V, Schneider M, Kuhn H, Damerell V, Diebel S, Kalman S, Klein S, Knudsen A C, Mader C, Merrill S, Staudt A, Thiel V, Welti L, Davey NE, Diella F and Gibson T J. 2016. ELM 2016 –data update and new functionality of the eukaryotic linear motif resource. Nucleic Acids Research 4(44): 294–300. DOI: https://doi.org/10.1093/nar/gkv1291
Forde N, Carter F, Spencer T E, Bazer F W, Sandra O, Mansouri- Attia N, Okumu L A, McGettigan P A, Mehta J P, McBride R, O’Gaora P, Roche J F and Lonergan P. 2011. Conceptus- induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?. Biology of Reproduction 85: 144–56. DOI: https://doi.org/10.1095/biolreprod.110.090019
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M R and Appel R D. 2005. Protein Identification and Analysis Tools on the ExPASy Server, pp 571–607. The proteomics protocols Handbook. (Eds) John M. Walker. Humana Press. DOI: https://doi.org/10.1385/1-59259-890-0:571
Gill S C and Von Hippel P H. 1989. Extinction coefficient. Analytical Biochemistry 182: 319–28. DOI: https://doi.org/10.1016/0003-2697(89)90602-7
Green J A, Parks T E, Avalle M P, Telugu B P, McLain A L, Peterson A J, McMillan W, Mathialagan N, Hook R R, Xie S and Roberts R M. 2005. The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers. Theriogenology 63(5): 1481–1503. DOI: https://doi.org/10.1016/j.theriogenology.2004.07.011
Green J C, Okamura C S, Poock S E and Lucy M C. 2010. Measurement of interferon-tau (IFN-tau) stimulated gene expression in blood leukocytes for pregnancy diagnosis within 18–20 d after insemination in dairy cattle. Animal Reproduction Science 121: 24–33. DOI: https://doi.org/10.1016/j.anireprosci.2010.05.010
Guex N and Peitsch M C. 1997. SWISS-MODEL and the Swiss- PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714–23. DOI: https://doi.org/10.1002/elps.1150181505
Guruprasad K, Reddy B and Pandit M W. 1990. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Engineering 4:155–164. DOI: https://doi.org/10.1093/protein/4.2.155
Hamerman J A, Hayashi F, Schroeder L A, Gygi S P, Haas A L, Hampson L, Coughlin P, Aebersold R and Aderem A. 2002. Serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog. Journal of Immunology 168: 2415– 23. DOI: https://doi.org/10.4049/jimmunol.168.5.2415
Han H, Austin K J, Rempel L A and Hansen T R. 2006. Low blood ISG15 mRNA and progesterone levels are predictive of non-pregnant dairy cows. Journal of Endocrinology 191: 505– 12. DOI: https://doi.org/10.1677/joe.1.07015
Hansen T R, Austin K J and Johnson G A. 1997. Transient ubiquitin cross-reactive protein gene expression in the bovine endometrium. Endocrinology 138: 5079–82. DOI: https://doi.org/10.1210/endo.138.11.5655
Hansen T R, Austin K J, Perry D J, Pru J K, Teixeira M G and Johnson G A. 1999. Mechanism of action of interferon-tau in the uterus during early pregnancy. Journal of Reproduction Fertility 54: 329–39.
Ikai A J. 1980. Thermo stability and aliphatic index of globular proteins. Journal of Biochemistry 88: 1895–98.
Jain A, Baviskar P S, Kandasamy S, Kumar R, Singh R, Kumar S, Agarwal S K, Joshi P and Mitra A. 2012. Interferon stimulated gene 15 (ISG15): molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis). Animal Reproduction Science 133(3–4): 159–68. DOI: https://doi.org/10.1016/j.anireprosci.2012.06.023
Johnson G A, Austin K J, Van Kirk E A and Hansen T R. 1998. Pregnancy and interferon-tau induce conjugation of bovine ubiquitin cross-reactive protein to cytosolic uterine proteins. Biology of Reproduction 58: 898–904. DOI: https://doi.org/10.1095/biolreprod58.4.898
Johnson G A, Spencer T E, Hansen T R, Austin K J, Burghardt R C and Bazer F W. 1999. Expression of the interferon tau inducible ubiquitin crossreactive protein in the ovine uterus. Biology of Reproduction 61: 312–18. DOI: https://doi.org/10.1095/biolreprod61.1.312
Joyce M M, Hansen T R and Johnson G A. 2002. Interferon- stimulated gene 17 is expressed in the porcine uterus and may be critical to placental development across species. Biology of Reproduction 66: 185.
Joyce M M, White F J, Burghardt R C, Muniz J J, Spencer T E, Bazer F W and Johnson G A. 2005. Interferon stimulated gene 15 conjugates to endometrial cytosolic proteins and is expressed at the uterineplacental interface throughout pregnancy in sheep. Endocrinology 146: 675–84. DOI: https://doi.org/10.1210/en.2004-1224
Karve T M and Cheema K A. 2011. Small changes huge impact: The role of protein posttranslational modifications in cellular homeostasis and disease. Journal of Amino Acids: 207691. DOI: https://doi.org/10.4061/2011/207691
Kelley L A, Mezulis S, Yates C M, Wass M N and Sternberg M J. 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10(6): 845–58. DOI: https://doi.org/10.1038/nprot.2015.053
Kizaki K, Shichijo-Kizaki A, Furusawa T, Takahashi T, Hosoe M and Hashizume K. 2013. Differential neutrophil gene expression in early bovine pregnancy. Reproductive Biology and Endocrinology 11: 6. DOI: https://doi.org/10.1186/1477-7827-11-6
Kwok S C, Mant C T and Hodges R S. 2002. Importance of secondary structural specificity determinants in protein folding: Insertion of a native b-sheet sequence into an á-helical coiled-coil. Protein Science 1(6): 1519–31. DOI: https://doi.org/10.1110/ps.4170102
Kyte J and Doolottle R F. 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157: 105–32. DOI: https://doi.org/10.1016/0022-2836(82)90515-0
Larsen J E, Lund O and Nielsen M. 2006. Bepipred Linear Epitope Prediction: Improved method for predicting linear B-cell epitopes. Immunome Research 2: 2. DOI: https://doi.org/10.1186/1745-7580-2-2
Loeb K R and Haas A L. 1992. The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. Journal of Biological Chemistry 267: 7806–13. DOI: https://doi.org/10.1016/S0021-9258(18)42585-9
Lovell S C, Davis I W, Arendall W B, de Bakker P I W, Word J M, Prisant M G, Richardson J S and Richardson D C. 2002. Structure validation by C alpha geometry: phi, psi and C beta deviation. Proteins: Structure Function and Genetics 50: 437– 50. DOI: https://doi.org/10.1002/prot.10286
Luthy R, Bowie J U and Eisenberg D. 1992. Assessment of protein models with three-dimensional profiles. Nature 6364: 83–85. Malakhov M P, Kim K I, Malakhova O A, Jacobs B S, Borden E C and Zhang D E. 2003. High-throughout immunoblotting. DOI: https://doi.org/10.1038/356083a0
Ubiquitin-like protein ISG15 modifies key regulators of signal transduction. Journal of Biological Chemistry 278: 16608–13.
Matsuyama S, Kojima T, Kato S and Kimura K. 2012. Relationship between quantity of IFNT estimated by IFN- stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET. Reproductive Biology and Endocrinology 10: 21. DOI: https://doi.org/10.1186/1477-7827-10-21
Meyerholz M M, Mense K, Knaack H, Sandra O and Schmicke M.2016. Pregnancy-Induced ISG-15 and MX-1 Gene Expression is Detected in the Liver of Holstein-Friesian Heifers During Late Peri-Implantation Period. Reproduction in Domestic Animals 51(1): 175–77. DOI: https://doi.org/10.1111/rda.12638
Notredame C, Higgins D G and Heringa J. 2011. T-Coffee: A novel method for fast and accurate multiple sequence alignment. Journal of Molecular Biology 302(1): 205–17. DOI: https://doi.org/10.1006/jmbi.2000.4042
Perry D J, Austin K J and Hansen T R. 1999. Cloning of interferon- stimulated gene 17: the promoter and nuclear proteins that regulate transcription. Molecular Endocrinology 13: 1197–1206. DOI: https://doi.org/10.1210/mend.13.7.0294
Petersen B, Petersen T N, Andersen P, Nielsen M and Lundegaard C. 2009. A generic method for assignment of reliability scores applied to solvent accessibility predictions. BMC Structural Biology 9: 51. DOI: https://doi.org/10.1186/1472-6807-9-51
Ramachandran G N, Ramakrishnan C and Sasisekharan V. 1963. Stereochemistry of polypeptide chain configurations. Journal of Molecular Biology 7: 95–99. DOI: https://doi.org/10.1016/S0022-2836(63)80023-6
Rempel L A, Francis B R, Austin K J and Hansen T R. 2005. Isolation and sequence of an interferon-ô inducible, pregnancy and bovine interferon stimulated gene product 15 (ISG15)- specific, bovine ubiquitin activating E1-like (UBE1L) enzyme. Biology of Reproduction 72: 365–72. DOI: https://doi.org/10.1095/biolreprod.104.033027
Rodriguez M R, Monte K, Thackray L B and Lenschow D J. 2014. ISG15 Functions as an Interferon-Mediated Antiviral Effector Early in the Murine Norovirus Life Cycle. Journal of Virology 88(16): 9277–86. DOI: https://doi.org/10.1128/JVI.01422-14
Sahdev S, Khattar S K and Saini K S. 2008. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Molecular and Cellular Biochemistry 307: 249–264. DOI: https://doi.org/10.1007/s11010-007-9603-6
Sali A and Blundell T L. 1993. Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology 234: 779–815. DOI: https://doi.org/10.1006/jmbi.1993.1626
Sapay N, Guermeur Y and Deleage G. 2006. Prediction of amphipathic in-plane membranee anchors in monotopic proteins using a SVM classifier. BMC Bioinformatics 7(1):255. DOI: https://doi.org/10.1186/1471-2105-7-255
Sheikh A A, Hooda O K, Kalyan A, Kamboj A, Mohammed S, Alhussien M, Reddi S, Shimray P G, Rautela A, Pandita S, Kapila S, De S and Dang A K. 2018. Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows. Theriogenology 120: 61–67. DOI: https://doi.org/10.1016/j.theriogenology.2018.07.028
Shi S P, Qiu J D, Sun X Y, Suo S B, Huang S Y and Liang R P. 2012. PLMLA: prediction of lysine methylation and lysine acetylation by combining multiple feature. Molecular BioSystems 8(5): 1520–27. DOI: https://doi.org/10.1039/c2mb05502c
Singh S K, Agarwal S K, Shankar U and Gupta L K.2005. Purification and characterization of protein(s) from placental extracts in buffalo. Indian Journal of Animal Sciences 75 (7):769–800.
Smialowski P, Martin-Galiano A J, Mikolajka A, Girschick T, Holak T A and Frishman D.2007. Protein solubility: sequence based prediction and experimental verification. Bioinformatics 23(19): 2536–42. DOI: https://doi.org/10.1093/bioinformatics/btl623
Spencer T E, Sandra O and Wolf E. 2008. Genes involved in conceptus endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches. Reproduction 135: 165–79. DOI: https://doi.org/10.1530/REP-07-0327
Soumya N, Das D, Jeyakumar S, Mondal S, Mor A and Mundhe U. 2016. Differential expression of ISG 15 mRNA in peripheral blood mononuclear cells of nulliparous and multiparous pregnant versus non-pregnant Bos indicus cattle. Reproduction in Domestic Animals 52: 97–106. DOI: https://doi.org/10.1111/rda.12815
Thakur N, Singh G, Paul A, Bharati J, Rajesh G, Gm V, Chouhan V S, Bhure S K, Maurya V P, Singh G and Sarkar M. 2017. Expression and molecular cloning of interferon stimulated genes in buffalo (Bubalus bubalis). Theriogenology 100: 50– 58. DOI: https://doi.org/10.1016/j.theriogenology.2017.05.027
Thatcher W W, Guzeloglu A, Mattos R, Binelli M, Hansen T R and Pru J K. 2001. Uterine-conceptus interactions and reproductive failure in cattle. Theriogenology 56: 1435–50. DOI: https://doi.org/10.1016/S0093-691X(01)00645-8
Tokmakov A A, Kurotani A, Takagi T, Toyama M, Shirouzu M, Fukami Y and Yokoyama S. 2012. Multiple post-translational modifications affect heterologous protein synthesis. Journal of Biological Chemistry 287(32): 27106–16. DOI: https://doi.org/10.1074/jbc.M112.366351
Yin J, Li G, Ren X and Herrler G. 2007. Select what you need; a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. Journal of Biotechnology 127: 335–47. DOI: https://doi.org/10.1016/j.jbiotec.2006.07.012
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