Human Epididymis Protein 4 Quantification and Interaction Network Analysis in Seminal Plasma

Author(s): Krishna Kant, Anil K. Tomar, Pankaj Sharma, Bishwajit Kundu, Sarman Singh, Savita Yadav*.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 6 , 2019

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Abstract:

Background: A well-known tissue marker of ovarian cancer, Human Epididymis protein 4 (HE4) is the member of whey acidic four-disulfide core proteins family. Purified from human seminal plasma and characterized as a cross-class protease inhibitor, HE4 was proposed to shield spermatozoa against proteolytic factors. However, its exact biological function is unknown. Proteins usually function in conjunction with other proteins in the system and thus, identification and analysis of protein networks become essential to decode protein functions.

Objective: This study was performed to explore possible role(s) of HE4 in reproductive physiology via identification of its interactome in human seminal plasma.

Methods: HE4 binding proteins were identified through co-immunoprecipitation and MALDITOF/ MS analysis. Also, HE4 was quantified by ELISA in fertile and infertile human seminal plasma samples.

Results: Ten HE4 binding proteins were identified, viz. protein phosphatase 1 regulatory subunit 21, protein kinase CLK3, Ankyrin repeat domain-containing protein36A, prostatic acid phosphatase, KIF5C, Spectrin repeat containing, nuclear envelope 1, isoform CRAf, tropomyosin 4, vezatin, utrophin and fibronectin1. This interaction network suggests that HE4 plays multiple roles, specifically in capacitation, sperm motility and maturation. Further, HE4 concentration in human seminal plasma samples was determined by Elisa. Higher HE4 expression in normozoospermia compared to azoospermia and asthenozoospermia affirms its importance in fertilization.

Conclusion: Based on identified interactome, it is plausible that HE4 plays a crucial role in fertilization, specifically in sperm maturation, motility and capacitation.

Keywords: Human Epididymis protein 4 (HE4), co-immunoprecipitation, ELISA, fertilization, protein-protein interactions, protease inhibitor.

[1]
Oparin, A.I.; Gladilin, K.L. Evolution of self-assembly of probionts. Bio Systems, 1980, 12(3-4), 133-145.
[2]
Segre, D.; Lancet, D. Composing life. EMBO Reports., 2000, 1(3), 217-222.
[3]
O’Brien, D.A.; Welch, J.E.; Fulcher, K.D.; Eddy, E.M. Expression of mannose 6-phosphate receptor messenger ribonucleic acids in mouse spermatogenic and Sertoli cells. Biol. Reprod., 1994, 50(2), 429-435.
[4]
Yanagimachi, R. Fertility of mammalian spermatozoa: Its development and relativity. Zygote, 1994, 2(4), 371-372.
[5]
Wassarman, P.M.; Jovine, L.; Litscher, E.S. A profile of fertilization in mammals. Nat. Cell Biol., 2001, 3(2), E59-E64.
[6]
Bonetta, L. Protein-protein interactions: Interactome under construction. Nature, 2010, 468(7325), 851-854.
[7]
De Las Rivas, J.; Fontanillo, C. Protein-protein interactions essentials: Key concepts to building and analyzing interactome networks. PLoS Comput. Biol., 2010, 6(6)e1000807
[8]
Huang, B.X.; Kim, H.Y. Effective identification of Akt interacting proteins by two-step chemical crosslinking, co-immunoprecipitation and mass spectrometry. PloS One,2013, 8(4), e61430; (b) Lin, J.S.; Lai, E.M. Protein-Protein Interactions: Co-Immunoprecipitation. Methods Mol. Biol., 2017, 1615, 211-219.
[9]
Schummer, M.; Ng, W.V.; Bumgarner, R.E.; Nelson, P.S.; Schummer, B.; Bednarski, D.W.; Hassell, L.; Baldwin, R.L.; Karlan, B.Y.; Hood, L. Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas. Gene, 1999, 238(2), 375-385.
[10]
Karlsen, N.S.; Karlsen, M.A.; Høgdall, C.K.; Høgdall, E.V. HE4 tissue expression and serum HE4 levels in healthy individuals and patients with benign or malignant tumors: A systematic review. Cancer Epidemiol. Biomarkers Prev., 2014, 23(11), 2285-2295.
[11]
Chhikara, N.; Saraswat, M.; Tomar, A.K.; Dey, S.; Singh, S.; Yadav, S. Human epididymis protein-4(HE-4): A novel cross-class protease inhibitor. PLoS One, 2012, 7(11)e47672
[12]
Szklarczyk, D.; Franceschini, A.; Wyder, S.; Forslund, K.; Heller, D.; Huerta-Cepas, J.; Simonovic, M.; Roth, A.; Santos, A.; Tsafou, K.P.; Kuhn, M.; Bork, P.; Jensen, L.J.; von Mering, C. STRING v10: Protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res., 2015, 43, D447-D452.
[13]
Tomar, A.K.; Sooch, B.S.; Raj, I.; Singh, S.; Yadav, S. Interaction analysis identifies semenogelin I fragments as new binding partners of PIP in human seminal plasma. Int. J. Biol. Macromol., 2013, 52, 296-299.
[14]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685.
[15]
Alam, M.; Selladurai, M.; Nagpal, S.; Tomar, A.K.; Saraswat, M.; Raziuddin, M.; Mittal, S.; Singh, T.P.; Yadav, S. Sample complexity reduction aids efficient detection of low-abundant proteins from human amniotic fluid. J. Sep. Sci., 2010, 33(12), 1723-1739.
[16]
Breitbart, H.; Etkovitz, N. Role and regulation of EGFR in actin remodeling in sperm capacitation and the acrosome reaction. Asian J. Androl., 2010, 13(1), 106-110.
[17]
Ota, Y.; Shimoya, K.; Zhang, Q.; Moriyama, A.; Chin, R.; Tenma, K.; Kimura, T.; Koyama, M.; Azuma, C.; Murata, Y. The expression of secretory leukocyte protease inhibitor(SLPI) in the Fallopian tube: SLPI protects the acrosome reaction of sperm from inhibitory effects of elastase. Hum. Reprod., 2002, 17(10), 2517-2522.
[18]
Wang, J.; Qi, L.; Huang, S.; Zhou, T.; Guo, Y.; Wang, G.; Guo, X.; Zhou, Z.; Sha, J. Quantitative phosphoproteomics analysis reveals a key role of insulin growth factor 1 receptor(IGF1R) tyrosine kinase in human sperm capacitation. Mol. Cell. Proteomics, 2015, 14(4), 104-112.
[19]
Passadaki, T.; Asimakopoulos, B.; Zeginiadou, T.; Nikolettos, N. Soluble FAS and FAS ligand levels in seminal plasma: Association with basic parameters of semen analysis. In Vivo, 2013, 27(2), 285-287.
[20]
Luborsky, J.L.; Yu, Y.; Edassery, S.L.; Jaffar, J.; Yip, Y.Y.; Liu, P.; Hellstrom, K.E.; Hellstrom, I. Autoantibodies to mesothelin in infertility. Cancer Epidemiol. Biomarkers Prev., 2011, 20(9), 1970-1978.
[21]
Salvolini, E.; Buldreghini, E.; Lucarini, G.; Vignini, A.; Lenzi, A.; Di Primio, R.; Balercia, G. Involvement of sperm plasma membrane and cytoskeletal proteins in human male infertility. Fertil. Steril., 2013, 99(3), 697-704.
[22]
Chakrabarti, R.; Cheng, L.; Puri, P.; Soler, D.; Vijayaraghavan, S. Protein phosphatase PP1 gamma 2 in sperm morphogenesis and epididymal initiation of sperm motility. Asian J. Androl., 2007, 9(4), 445-452.
[23]
Cohen, P.T. Protein phosphatase 1--targeted in many directions. J. Cell Sci., 2002, 115(Pt 2), 241-256.
[24]
Fardilha, M.; Esteves, S.L.; Korrodi-Gregorio, L.; Pelech, S.; da Cruz, E.S.O.A.; da Cruz, E.S.E. Protein phosphatase 1 complexes modulate sperm motility and present novel targets for male infertility. Mol. Human. Reprod., 2011, 17(8), 466-477.
[25]
Nayler, O.; Stamm, S.; Ullrich, A. Characterization and comparison of four serine- and arginine-rich(SR) protein kinases. Biochemical. J., 1997, 326(Pt 3), 693-700.
[26]
Menegay, H.; Moeslein, F.; Landreth, G. The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa. Exp. Cell Res., 1999, 253(2), 463-473.
[27]
Barbonetti, A.; Vassallo, M.R.; Cordeschi, G.; Venetis, D.; Carboni, A.; Sperandio, A.; Felzani, G.; Francavilla, S.; Francavilla, F. Protein tyrosine phosphorylation of the human sperm head during capacitation: Immunolocalization and relationship with acquisition of sperm-fertilizing ability. Asian J. Androl., 2010, 12(6), 853-861.
[28]
Goldfarb, D.A.; Stein, B.S.; Shamszadeh, M.; Petersen, R.O. Age-related changes in tissue levels of prostatic acid phosphatase and prostate specific antigen. J. Urol., 1986, 136(6), 1266-1269.
[29]
Lee, C.L.; Li, S.S.; Chu, T.M. Immunologically reactive tryptic fragments of human prostatic acid phosphatase. Biochem. J., 1984, 223(3), 871-877.
[30]
Luchter-Wasyl, E.; Ostrowski, W. Subunit structure of human prostatic acid phosphatase. Biochim. Biophys. Acta, 1974, 365(2), 349-359.
[31]
Ronnberg, L.; Vihko, P.; Sajanti, E.; Vihko, R. Clomiphene citrate administration to normogonadotropic subfertile men: Blood hormone changes and activation of acid phosphatase in seminal fluid. Int. J. AndrolM., 981 4(3), 372-378.
[32]
Yam, L.T. Clinical significance of the human acid phosphatases: A review. Am. J. Med., 1974, 56(5), 604-616.
[33]
Ahmann, F.R.; Schifman, R.B. Prospective comparison between serum monoclonal prostate specific antigen and acid phosphatase measurements in metastatic prostatic cancer. J. Urol., 1987, 137(3), 431-434.
[34]
Reif, A.E.; Schlesinger, R.M.; Fish, C.A.; Robinson, C.M. Acid phosphatase isozymes in cancer of the prostate. Cancer, 1973, 31(3), 689-699.
[35]
Simsek, U.; Kutlu, S.; Yavascaouglu, I.; Oktay, B.; Ozyurt, M. Seasonal variation of prostatic acid phosphate and prostate-specific antigen in patients without prostatic malignancy. Eur. Urol., 1992, 21(Suppl. 1), 111-114.
[36]
Coffey, D.S.; Pienta, K.J. New concepts in studying the control of normal and cancer growth of the prostate. Prog. Clin. Biol. Res., 1987, 239, 1-73.
[37]
Munch, J.; Rucker, E.; Standker, L.; Adermann, K.; Goffinet, C.; Schindler, M.; Wildum, S.; Chinnadurai, R.; Rajan, D.; Specht, A.; Gimenez-Gallego, G.; Sanchez, P.C.; Fowler, D.M.; Koulov, A.; Kelly, J.W.; Mothes, W.; Grivel, J.C.; Margolis, L.; Keppler, O.T.; Forssmann, W.G.; Kirchhoff, F. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell, 2007, 131(6), 1059-1071.
[38]
Olsen, J.S.; DiMaio, J.T.; Doran, T.M.; Brown, C.; Nilsson, B.L.; Dewhurst, S. Seminal plasma accelerates semen-derived enhancer of viral infection(SEVI) fibril formation by the prostatic acid phosphatase(PAP248-286) peptide. J. Biol. Chem., 2012, 287(15), 11842-11849.
[39]
Mannowetz, N.; Kartarius, S.; Wennemuth, G.; Montenarh, M. Protein kinase CK2 and new binding partners during spermatogenesis. Cell. Mol. Life Sci., 2010, 67(22), 3905-3913.
[40]
Hassoun, H.; Vassiliadis, J.N.; Murray, J.; Yi, S.J.; Hanspal, M.; Ware, R.E.; Winter, S.S.; Chiou, S.S.; Palek, J. Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis. J. Clin. Invest., 1995, 96(6), 2623-2629.
[41]
Bastian, Y.; Roa-Espitia, A.L.; Mujica, A.; Hernandez-Gonzalez, E.O. Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton. Reproduction, 2010, 140(5), 673-684.
[42]
Lehman, W.; Craig, R.; Vibert, P. Ca(2+)-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature, 1994, 368(6466), 65-67.
[43]
Gunning, P.; Weinberger, R.; Jeffrey, P. Actin and tropomyosin isoforms in morphogenesis. Anat. Embryol., 1997, 195(4), 311-315.
[44]
Yao, X.; Arst, H.N. Jr.; Wang, X.; Xiang, X. Discovery of a vezatin-like protein for dynein-mediated early endosome transport. Mol. Biol. Cell, 2015, 26(21), 3816-3827.
[45]
Kussel-Andermann, P.; El-Amraoui, A.; Safieddine, S.; Nouaille, S.; Perfettini, I.; Lecuit, M.; Cossart, P.; Wolfrum, U.; Petit, C. Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex. EMBO J., 2000, 19(22), 6020-6029.
[46]
Fusi, F.M.; Bronson, R.A. Sperm surface fibronectin. Expression following capacitation. J. Androl., 1992, 13(1), 28-35.
[47]
Wennemuth, G.; Meinhardt, A.; Mallidis, C.; Albrecht, M.; Krause, W.; Renneberg, H.; Aumuller, G. Assessment of fibronectin as a potential new clinical tool in andrology. Andrologia, 2001, 33(1), 43-46.
[48]
Akiyama, S.K.; Olden, K.; Yamada, K.M. Fibronectin and integrins in invasion and metastasis. Cancer Metastasis Rev., 1995, 14(3), 173-189.
[49]
Midwood, K.S.; Williams, L.V.; Schwarzbauer, J.E. Tissue repair and the dynamics of the extracellular matrix. Int. J. Biochem. Cell Biol., 2004, 36(6), 1031-1037.
[50]
Mosher, D.F. Physiology of fibronectin. Ann. Rev. Med., 1984, 35, 561-575.
[51]
Miranda, P.V.; Tezon, J.G. Characterization of fibronectin as a marker for human epididymal sperm maturation. Molecular. Reprod. Dev., 1992, 33(4), 443-450.
[52]
Lilja, H.; Oldbring, J.; Rannevik, G.; Laurell, C.B. Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen. J. Clin. Invest., 1987, 80(2), 281-285.
[53]
Malm, J.; Hellman, J.; Magnusson, H.; Laurell, C.B.; Lilja, H. Isolation and characterization of the major gel proteins in human semen, semenogelin I and semenogelin II. Eur. J. Biochem., 1996, 238(1), 48-53.
[54]
Robert, M.; Gibbs, B.F.; Jacobson, E.; Gagnon, C. Characterization of prostate-specific antigen proteolytic activity on its major physiological substrate, the sperm motility inhibitor precursor/semenogelin I. Biochemistry, 1997, 36(13), 3811-3819.
[55]
Bronson, R.A.; Fusi, F.M. Integrins and human reproduction. Mol. Hum. Reprod., 1996, 2(3), 153-168.


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Article Details

VOLUME: 26
ISSUE: 6
Year: 2019
Page: [458 - 465]
Pages: 8
DOI: 10.2174/0929866526666190327124919
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