Rational Design of Hyper-glycosylated Human Chorionic Gonadotropin Analogs (A Bioinformatics Approach)

Author(s): Zahra Nabizadeh, Zarrin Minuchehr*, Ali Akbar Shabani*

Journal Name: Letters in Drug Design & Discovery

Volume 17 , Issue 8 , 2020


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Abstract:

Background: Protein pharmaceuticals routinely display a series of intrinsic physicochemical instabilities during their production and administration that can unfavorably affect their therapeutic effectiveness. Glycoengineering is one of the most desirable techniques to improve the attributes of therapeutic proteins. One aspect of glycoengineering is the rational manipulation of the peptide backbone to introduce new N-glycosylation consensus sequences (Asn-X-Ser/Thr, where X is any amino acid except proline).

Methods: In this work, the amino acid sequence of human chorionic gonadotropin (hCG) was analyzed to identify suitable positions in order to create new N-glycosylation sites. This survey led to the detection of 46 potential N-glycosylation sites. The N-glycosylation probability of all the potential positions was measured with the NetNGlyc 1.0 server. After theoretical reviews and the removal of unsuitable positions, the five acceptable ones were selected for more analyses. Then, threedimensional (3D) structures of the selected analogs were generated and evaluated by SPDBV software. The molecular stability and flexibility profile of five designed analogs were examined using Molecular Dynamics (MD) simulations.

Results: Finally, three analogs with one additional N-glycosylation site (V68T, V79N and R67N) were proposed as the qualified analogs that could be glycosylated at the new sites.

Conclusion: According to the results of this study, further experimental investigations could be guided on the three analogs. Therefore, our computational strategy can be a valuable method due to the reduction in the number of the expensive, tiresome and time-consuming experimental studies of hCG analogs.

Keywords: Rational design, hCG analogs, glycoengineering, hyper-glycosylated hCG, bioinformatic approach, hyperglycosylation.

[1]
Berger, P.; Sturgeon, C. Pregnancy testing with hCG--future prospects. Trends Endocrinol. Metab., 2014, 25(12), 637-648.
[http://dx.doi.org/10.1016/j.tem.2014.08.004] [PMID: 25246381]
[2]
Fares, F. The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists. Biochim. Biophys. Acta, 2006, 1760(4), 560-567.
[http://dx.doi.org/10.1016/j.bbagen.2005.12.022] [PMID: 16527410]
[3]
Stenman, U.H.; Tiitinen, A.; Alfthan, H.; Valmu, L. The classification, functions and clinical use of different isoforms of HCG. Hum. Reprod. Update, 2006, 12(6), 769-784.
[http://dx.doi.org/10.1093/humupd/dml029] [PMID: 16877746]
[4]
Ludwig, M.; Doody, K.J.; Doody, K.M. Use of recombinant human chorionic gonadotropin in ovulation induction. Fertil. Steril., 2003, 79(5), 1051-1059.
[http://dx.doi.org/10.1016/S0015-0282(03)00173-0] [PMID: 12738494]
[5]
Kim, S.O.; Ryu, K.H.; Hwang, I.S.; Jung, S.I.; Oh, K.J.; Park, K. Penile growth in response to human chorionic gonadotropin (HCG) treatment in patients with idiopathic hypogonadotrophic hypogonadism. Chonnam Med. J., 2011, 47(1), 39-42.
[http://dx.doi.org/10.4068/cmj.2011.47.1.39] [PMID: 22111055]
[6]
Makedonsky, I.A. The use of human chorionic gonadotropin (HCG) for penile reconstruction in bladder exstrophy and total epispadias patients. Eur. J. Pediatr. Surg., 2006, 16(6), 428-431.
[http://dx.doi.org/10.1055/s-2006-924746] [PMID: 17211793]
[7]
Solá, R.J.; Griebenow, K. Effects of glycosylation on the stability of protein pharmaceuticals. J. Pharm. Sci., 2009, 98(4), 1223-1245.
[http://dx.doi.org/10.1002/jps.21504] [PMID: 18661536]
[8]
Solá, R.J.; Griebenow, K. Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs, 2010, 24(1), 9-21.
[http://dx.doi.org/10.2165/11530550-000000000-00000] [PMID: 20055529]
[9]
Han, M.; Wang, W.; Jiang, G.; Wang, X.; Liu, X.; Cao, H.; Tao, Y.; Yu, X. Enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide. Biotechnol. Lett., 2014, 36(12), 2467-2471.
[http://dx.doi.org/10.1007/s10529-014-1620-4] [PMID: 25048243]
[10]
Han, M.; Yu, X. Enhanced expression of heterologous proteins in yeast cells via the modification of N-glycosylation sites. Bioengineered, 2015, 6(2), 115-118.
[http://dx.doi.org/10.1080/21655979.2015.1011031] [PMID: 25671496]
[11]
Sagt, C.M.J.; Kleizen, B.; Verwaal, R.; de Jong, M.D.M.; Müller, W.H.; Smits, A.; Visser, C.; Boonstra, J.; Verkleij, A.J.; Verrips, C.T. Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts. Appl. Environ. Microbiol., 2000, 66(11), 4940-4944.
[http://dx.doi.org/10.1128/AEM.66.11.4940-4944.2000] [PMID: 11055947]
[12]
Samoudi, M.; Minuchehr, Z.; Harcum, S.W.; Tabandeh, F.; Omid Yeganeh, N.; Khodabandeh, M. Rational design of glycoengineered interferon-β analogs with improved aggregation state: experimental validation. Protein Eng. Des. Sel., 2017, 30(1), 23-30.
[PMID: 27881683]
[13]
Liu, Y.; Yi, X.; Zhuang, Y.; Zhang, S. Limitations in the process of transcription and translation inhibit recombinant human chorionic gonadotropin expression in CHO cells. J. Biotechnol., 2015, 204, 63-69.
[http://dx.doi.org/10.1016/j.jbiotec.2014.12.005] [PMID: 25529346]
[14]
Sinclair, A.M.; Elliott, S. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J. Pharm. Sci., 2005, 94(8), 1626-1635.
[http://dx.doi.org/10.1002/jps.20319] [PMID: 15959882]
[15]
Weenen, C.; Peña, J.E.; Pollak, S.V.; Klein, J.; Lobel, L.; Trousdale, R.K.; Palmer, S.; Lustbader, E.G.; Ogden, R.T.; Lustbader, J.W. Long-acting follicle-stimulating hormone analogs containing N-linked glycosylation exhibited increased bioactivity compared with o-linked analogs in female rats. J. Clin. Endocrinol. Metab., 2004, 89(10), 5204-5212.
[http://dx.doi.org/10.1210/jc.2004-0425] [PMID: 15472227]
[16]
Elliott, S.; Lorenzini, T.; Asher, S.; Aoki, K.; Brankow, D.; Buck, L.; Busse, L.; Chang, D.; Fuller, J.; Grant, J.; Hernday, N.; Hokum, M.; Hu, S.; Knudten, A.; Levin, N.; Komorowski, R.; Martin, F.; Navarro, R.; Osslund, T.; Rogers, G.; Rogers, N.; Trail, G.; Egrie, J. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat. Biotechnol., 2003, 21(4), 414-421.
[http://dx.doi.org/10.1038/nbt799] [PMID: 12612588]
[17]
Ceaglio, N.; Etcheverrigaray, M.; Kratje, R.; Oggero, M. Novel long-lasting interferon alpha derivatives designed by glycoengineering. Biochimie, 2008, 90(3), 437-449.
[http://dx.doi.org/10.1016/j.biochi.2007.10.013] [PMID: 18039474]
[18]
Samoudi, M.; Tabandeh, F.; Minuchehr, Z.; Ahangari Cohan, R.; Nouri Inanlou, D.; Khodabandeh, M.; Sabery Anvar, M. Rational design of hyper-glycosylated interferon beta analogs: a computational strategy for glycoengineering. J. Mol. Graph. Model., 2015, 56, 31-42.
[http://dx.doi.org/10.1016/j.jmgm.2014.12.001] [PMID: 25544388]
[19]
Wu, H.; Lustbader, J.W.; Liu, Y.; Canfield, R.E.; Hendrickson, W.A. Structure of human chorionic gonadotropin at 2.6 A resolution from MAD analysis of the selenomethionyl protein. Structure, 1994, 2(6), 545-558.
[http://dx.doi.org/10.1016/S0969-2126(00)00054-X] [PMID: 7922031]
[20]
Lapthorn, A. J.; Harris, D. C.; Littlejohn, A.; Lustbader, J. W.; Canfield, R. E.; Machin, K. J.; Morgan, F.; Isaacs, N. W. crystal structure of human chorionic gonadotropin. nature, 1994, 369(6480), 455-461.
[21]
Banerjee, A.; Venkatesh, N.; Murthy, G.S. Structure function analysis: Lessons from human chorionic gonadotropin. Indian J. Exp. Biol., 2002, 40(4), 434-447.
[22]
Gupta, R.; Brunak, S. Prediction of glycosylation across the human proteome and the correlation to protein function. Pac. Symp. Biocomput., 2002, 7, 310-322.
[PMID: 11928486]
[23]
Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T.J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Söding, J.; Thompson, J.D.; Higgins, D.G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol., 2011, 7(539), 539.
[http://dx.doi.org/10.1038/msb.2011.75] [PMID: 21988835]
[24]
Guex, N.; Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis, 1997, 18(15), 2714-2723.
[http://dx.doi.org/10.1002/elps.1150181505] [PMID: 9504803]
[25]
Momany, F.A.; Rone, R. Validation of the general purpose QUANTA® 3.2/CHARMm® force field. J. Comput. Chem., 1992, 13(7), 888-900.
[http://dx.doi.org/10.1002/jcc.540130714]
[26]
Ahmad, S.; Gromiha, M.; Fawareh, H.; Sarai, A. ASAView: database and tool for solvent accessibility representation in proteins. BMC Bioinformatics, 2004, 5(51), 51.
[http://dx.doi.org/10.1186/1471-2105-5-51] [PMID: 15119964]
[27]
Laskowski, R.A. PDBsum: summaries and analyses of PDB structures. Nucleic Acids Res., 2001, 29(1), 221-222.
[http://dx.doi.org/10.1093/nar/29.1.221] [PMID: 11125097]
[28]
Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput., 2008, 4(3), 435-447.
[http://dx.doi.org/10.1021/ct700301q] [PMID: 26620784]
[29]
Schmid, N.; Eichenberger, A.P.; Choutko, A.; Riniker, S.; Winger, M.; Mark, A.E.; van Gunsteren, W.F. Definition and testing of the GROMOS force-field versions 54A7 and 54B7. Eur. Biophys. J., 2011, 40(7), 843-856.
[http://dx.doi.org/10.1007/s00249-011-0700-9] [PMID: 21533652]
[30]
Zhong, X.; Wright, J.F. Biological Insights into therapeutic protein modifications throughout trafficking and their biopharmaceutical applications. Int. J. Cell Biol., 2013, 2013(273086)273086
[http://dx.doi.org/10.1155/2013/273086] [PMID: 23690780]
[31]
Mazola, Y.; Chinea, G.; Musacchio, A. Integrating bioinformatics tools to handle glycosylation. PLOS Comput. Biol., 2011, 7(12)e1002285
[http://dx.doi.org/10.1371/journal.pcbi.1002285] [PMID: 22219715]
[32]
Shakin-Eshleman, S.H.; Spitalnik, S.L.; Kasturi, L. The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. J. Biol. Chem., 1996, 271(11), 6363-6366.
[http://dx.doi.org/10.1074/jbc.271.11.6363] [PMID: 8626433]
[33]
Jones, J.; Krag, S.S.; Betenbaugh, M.J. Controlling N-linked glycan site occupancy. Biochim. Biophys. Acta, 2005, 1726(2), 121-137.
[http://dx.doi.org/10.1016/j.bbagen.2005.07.003] [PMID: 16126345]
[34]
Bause, E. Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem. J., 1983, 209(2), 331-336.
[http://dx.doi.org/10.1042/bj2090331] [PMID: 6847620]
[35]
Ryu, K.S.; Ji, I.; Chang, L.; Ji, T.H. Molecular mechanism of LH/CG receptor activation. Mol. Cell. Endocrinol., 1996, 125(1-2), 93-100.
[http://dx.doi.org/10.1016/S0303-7207(96)03951-2] [PMID: 9027347]
[36]
Pierce, J.G.; Parsons, T.F. Glycoprotein hormones: structure and function. Annu. Rev. Biochem., 1981, 50, 465-495.
[http://dx.doi.org/10.1146/annurev.bi.50.070181.002341] [PMID: 6267989]
[37]
Xia, H.; Chen, F.; Puett, D. A region in the human glycoprotein hormone alpha-subunit important in holoprotein formation and receptor binding. Endocrinology, 1994, 134(4), 1768-1770.
[http://dx.doi.org/10.1210/endo.134.4.7511092] [PMID: 7511092]
[38]
Xia, H.; Puett, D. Identification of conserved amino acid residues in the beta subunit of human choriogonadotropin important in holoprotein formation. J. Biol. Chem., 1994, 269(27), 17944-17953.
[PMID: 8027052]
[39]
Moyle, W.R.; Campbell, R.K.; Myers, R.V.; Bernard, M.P.; Han, Y.; Wang, X. Co-evolution of ligand-receptor pairs. Nature, 1994, 368(6468), 251-255.
[PMID: 8145825]
[40]
Keutmann, H.T.; Mason, K.A.; Kitzmann, K.; Ryan, R.J. Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin. Mol. Endocrinol., 1989, 3(3), 526-531.
[http://dx.doi.org/10.1210/mend-3-3-526] [PMID: 2747659]
[41]
Suganuma, N.; Matzuk, M.M.; Boime, I. Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit. J. Biol. Chem., 1989, 264(32), 19302-19307.
[PMID: 2478557]
[42]
Chen, F.; Puett, D. A single amino acid residue replacement in the beta subunit of human chorionic gonadotrophin results in the loss of biological activity. J. Mol. Endocrinol., 1992, 8(1), 87-89.
[http://dx.doi.org/10.1677/jme.0.0080087] [PMID: 1311931]
[43]
Chen, F.; Wang, Y.; Puett, D. The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin. Mol. Endocrinol., 1992, 6(6), 914-919.
[http://dx.doi.org/10.1210/mend.6.6.1379673] [PMID: 1379673]
[44]
Chen, F.; Puett, D. Contributions of arginines-43 and -94 of human choriogonadotropin β to receptor binding and activation as determined by oligonucleotide-based mutagenesis. Biochemistry, 1991, 30(42), 10171-10175.
[http://dx.doi.org/10.1021/bi00106a014] [PMID: 1931947]
[45]
Sen Gupta, C.; Dighe, R.R. Biological activity of single chain chorionic gonadotropin, hCGalphabeta, is decreased upon deletion of five carboxyl terminal amino acids of the alpha subunit without affecting its receptor binding. J. Mol. Endocrinol., 2000, 24(2), 157-164.
[http://dx.doi.org/10.1677/jme.0.0240157] [PMID: 10750017]
[46]
Mishra, A.K.; Mahale, S.D.; Iyer, K.S. Disulfide bonds Cys(9)-Cys(57), Cys(34)-Cys(88) and Cys(38)-Cys(90) of the β-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the alpha-subunit: experimental evidence for the conclusions from the crystal structure of hCG. Biochim. Biophys. Acta, 2003, 1645(1), 49-55.
[http://dx.doi.org/10.1016/S1570-9639(02)00501-0] [PMID: 12535610]
[47]
Furuhashi, M.; Ando, H.; Bielinska, M.; Pixley, M.R.; Shikone, T.; Hsueh, A.J.; Boime, I. Mutagenesis of cysteine residues in the human gonadotropin alpha subunit. Roles of individual disulfide bonds in secretion, assembly, and biologic activity. J. Biol. Chem., 1994, 269(41), 25543-25548.
[PMID: 7929256]
[48]
Chen, F.; Puett, D. Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity. J. Biol. Chem., 1991, 266(11), 6904-6908.
[PMID: 1707877]
[49]
Puett, D.; Angelova, K.; da Costa, M.R.; Warrenfeltz, S.W.; Fanelli, F. The luteinizing hormone receptor: insights into structure-function relationships and hormone-receptor-mediated changes in gene expression in ovarian cancer cells. Mol. Cell. Endocrinol., 2010, 329(1-2), 47-55.
[http://dx.doi.org/10.1016/j.mce.2010.04.025] [PMID: 20444430]
[50]
Jiang, X.; Dreano, M.; Buckler, D.R.; Cheng, S.; Ythier, A.; Wu, H.; Hendrickson, W.A.; el Tayar, N. Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure, 1995, 3(12), 1341-1353.
[http://dx.doi.org/10.1016/S0969-2126(01)00272-6] [PMID: 8747461]
[51]
Maiorov, V.N.; Crippen, G.M. Significance of root-mean-square deviation in comparing three-dimensional structures of globular proteins. J. Mol. Biol., 1994, 235(2), 625-634.
[http://dx.doi.org/10.1006/jmbi.1994.1017] [PMID: 8289285]
[52]
Ghasemi, F.; Zomorodipour, A.; Karkhane, A.A.; Khorramizadeh, M.R. In silico designing of hyper-glycosylated analogs for the human coagulation factor IX. J. Mol. Graph. Model., 2016, 68, 39-47.
[http://dx.doi.org/10.1016/j.jmgm.2016.05.011] [PMID: 27356208]
[53]
Karnik, S.; Mitra, J.; Singh, A.; Kulkarni, B.D.; Sundarajan, V.; Jayaraman, V.K. Third International Conference, PReMI 2009 New Delhi, 2009, pp. 146-151.
[54]
Shental-Bechor, D.; Levy, Y. Folding of glycoproteins: toward understanding the biophysics of the glycosylation code. Curr. Opin. Struct. Biol., 2009, 19(5), 524-533.
[http://dx.doi.org/10.1016/j.sbi.2009.07.002] [PMID: 19647993]
[55]
Petrescu, A.J.; Milac, A.L.; Petrescu, S.M.; Dwek, R.A.; Wormald, M.R. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology, 2004, 14(2), 103-114.
[http://dx.doi.org/10.1093/glycob/cwh008] [PMID: 14514716]
[56]
Elliott, S.; Chang, D.; Delorme, E.; Eris, T.; Lorenzini, T. Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin. J. Biol. Chem., 2004, 279(16), 16854-16862.
[http://dx.doi.org/10.1074/jbc.M311095200] [PMID: 14757769]
[57]
Flintegaard, T.V.; Thygesen, P.; Rahbek-Nielsen, H.; Levery, S.B.; Kristensen, C.; Clausen, H.; Bolt, G. N-glycosylation increases the circulatory half-life of human growth hormone. Endocrinology, 2010, 151(11), 5326-5336.
[http://dx.doi.org/10.1210/en.2010-0574] [PMID: 20826563]


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VOLUME: 17
ISSUE: 8
Year: 2020
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DOI: 10.2174/1570180817666200225101938
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