Studies toward Chemical Synthesis of Homogeneously Glycosylated Interleukin-10

doi:10.6023/cjoc202410001

Abstract

Human cytokine interleukin-10 (IL-10) sequence contains a conserved N-glycosylation sequon, Asn-Lys-Ser, but the presence and functional significance of this glycosylation have not been previously studied. Here, the chemical synthesis and characterization of IL-10 and its glycosylated form were explored. Using solid-phase peptide synthesis, native chemical ligation, and metal-free desulfurization, the full-length IL-10 was assembled and the non-glycosylated protein was successfully obtained through optimizing the refolding conditions. Building on this synthetic route, an N-acetylglucosamine (N-GlcNAc) modification was introduced into the peptide segment via cassette strategy, and further elaborated into Asn116 N-mono- saccharide-modified IL-10. Mass spectrometry (MS) and circular dichroism analysis of the synthesized IL-10 variants have suggested that they have similar structures to that of the biologically expressed counterpart.

Key words: chemical protein synthesis, glycoprotein, native chemical ligation, protein folding, human interleukin-10

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Weidong Dong, Haiyun Liu, Xinyu Li, Biao Wang, Bo Cheng, Suwei Dong. Studies toward Chemical Synthesis of Homogeneously Glycosylated Interleukin-10[J]. Chinese Journal of Organic Chemistry, 2025, 45(3): 951-958.

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Fig. & Tab. 5

Figure 1. Structure and retrosynthetic analysis of IL-10 Plausible ligation sites depicted in purple; Met mutated to Nle, depicted in green; Intrachain disulfides depicted in blue dashed lines

Scheme 1. Preparation of peptide segments Reaction conditions: (i) Fmoc-based SPPS; (ii) TFA/TIS/H2O (V∶V∶V=95∶2.5∶2.5), r.t., 2.5 h; (iii) N2H4•H2O/DMF (V∶V=5∶95), r.t., 16 h.

Scheme 2. Segment assembling of IL-10 Reaction conditions: (I) 1 (4.5 mmol/L) and 150 mmol/L acetylacetone (1.0 equiv.) in buffer 1 (6 mol/L Gnd•HCl, 200 mmol/L Na2HPO4, 200 mmol/L MPAA, pH=3), r.t., 5 h, then 2 (1.0 equiv., 4.5 mmol/L) in buffer 2 (6 mol/L Gnd•HCl, 200 mmol/L Na2HPO4, 150 mmol/L TCEP, pH=7.0) was added, pH=6.9, 2 h; (II) 3 (1.0 equiv., 4.5 mmol/L) in buffer 2 was added to (I), pH=6.9, overnight, 32% yield over three steps; (III) 4a/4b (4.9 mmol/L) and 150 mmol/L acetylacetone (4.0 equiv.) in buffer 1, r.t., 5 h, then 5 (0.5 equiv., 2.0 mmol/L) in buffer 2 was added, pH=6.9, 2 h, 70%/68% yield; (IV) buffer 3 (6 mol/L Gnd•HCl, 200 mmol/L Na2HPO4, pH=7.0), 0.2 mol/L Togni-II (in methanol), t-BuSH, 0.5 mol/L TCEP (0.5 mol/L in water), 50 ℃, 5 h, 65%/61% yield; (V) buffer 3, PdCl2 (56 mmol/L in buffer 3), 37 ℃, 30 min, then DTT buffer (0.5 mol/L DTT, 6 mol/L Gnd•HCl, 200 mmol/L Na2HPO4), 64%/63% yield; (VI) 7 (1.0 mmol/L) and 150 mmol/L acetylacetone (4.0 equiv.) in buffer 1, r.t., 5 h, then 10a/10b (1.0 equiv., 1.0 mmol/L) in buffer 2 was added, pH=6.9, 6 h, 46%/45% yield.

Entry	Folding redox (conc.)	Buffer	Process	Results^b
1^[11]	GSH/GSSG (2/0.2 mmol/L)	6 mol/L Gnd•HCl, 5 mmol/L dithiothreitol (DTT); then 50 mmol/L Tris-HCl, 50 mmol/ L NaCl, 5 mmol/L EDTA, pH 8.0	2~8 ℃, 16 h	Complete aggregation
2^[32]	Cysteine/Cystine (4/0.5 mmol/L)	100 mmol/L Tris-HCl, pH 8.5, Gnd•HCl from 6 mol/L to 3 mol/L to 1 mol/L	Stepwise dialysis, 48 h in total	Complete aggregation
3^[19]	Cysteine/Cystamine (5/1 mmol/L)	10 mmol/L DTT, 6 mol/L Gnd•HCl, 100 mmol/L NaCl, 5 mmol/L ethylenediamine- tetraacetic acid (EDTA), 20 mmol/L Tris•HCl; then 500 mmol/L Gnd•HCI, 20 mmol/ L Tris, 15% Glycerol, 1 mol/L Arginine, pH 9.0	2~8 ℃, 72 h	Products with 4 less of molecular weight than that of the reduced forms

Table 1. Screening of refolding conditionsa

Figure 2. Characterization of the synthetic IL-10 (A) Ribbon diagram of IL-10; (B) SDS-PAGE analysis of refolded proteins 12a/12b and recombinant IL-10 (rIL-10); (C) Circular dichroism (CD) analysis of refolded proteins 12a/b and expressed IL-10; (D) Deconvoluted Hi-res Mass data from UPLC/Q-TOF-MS analysis of refolded protein 12a (left) and 12a treated with 50 mmol/L DTT(right); (E) Deconvoluted Hi-res Mass data from UPLC/Q-TOF-MS analysis of refolded protein 12b (left) and 12b treated with 50 mmol/L DTT(right).

References 33

[1]	(a) Fiorentino, D. F.; Bond, M. W.; Mosmann, T. R. J. Exp. Med. 1989, 170, 2081.
	(b) Chaudhry, A.; Samstein, R. M.; Treuting, P.; Liang, Y.; Pils, M. C.; Heinrich, J.-M.; Jack, R. S.; Wunderlich, F. T.; Brüning, J. C.; Müller, W.; Rudensky, A. Y. Immunity 2011, 34, 566.
	(c) Murai, M.; Turovskaya, O.; Kim, G.; Madan, R.; Karp, C. L.; Cheroutre, H.; Kronenberg, M. Nat. Immunol. 2009, 10, 1178.
[2]	(a) Yoon, S. I.; Logsdon, N. J.; Sheikh, F.; Donnelly, R. P.; Walter, M. R. Biol. Chem. 2006, 281, 35088.
	(b) Murray, P. J. Curr. Opin. Pharmacol. 2006, 6, 379.
[3]	Kühn, R.; Löhler, J.; Rennick, D.; Rajewsky, K.; Müller, W. Cell 1993, 75, 263.
[4]	Couper, K. N.; Blount, D. G.; Riley, E. M. J. Immunol. 2008, 180, 5771.
[5]	Burmeister, A. R.; Marriott, I. Front. Cell. Neurosci. 2018, 12, 458.
[6]	Rajbhandari, P.; Thomas, B. J.; Feng, A.-C.; Hong, C.; Wang, J.; Vergnes, L.; Sallam, T.; Wang, B.; Sandhu, J.; Seldin, M. M.; Lusis, A. J.; Fong, L. G.; Katz, M.; Lee, R.; Young, S. G.; Reue, K.; Smale, S. T.; Tontonoz, P. Cell 2018, 172, 218.
[7]	Quiros, M.; Nishio, H.; Neumann, P. A.; Siuda, D.; Brazil, J. C.; Azcutia, V.; Hilgarth, R.; O'Leary, M. N.; Garcia-Hernandez, V.; Leoni, G. J. Clin. Invest. 2017, 127, 3510.
[8]	King, A.; Balaji, S.; Le, L. D.; Crombleholme, T. M.; Keswani, S. G. Adv. Wound Care 2014, 3, 315.
[9]	Wang, X.; Wong, K.; Ouyang, W.; Rutz, S. Cold Spring Harbor Perspect. Biol. 2019, 11, a028548.
[10]	Vieira, P.; de Waal-Malefyt, R.; Dang, M. N.; Johnson, K. E.; Kastelein, R.; Fiorentino, D. F.; de Vries, J. E.; Roncarolo, M. G.; Mosmann, T. R.; Moore, K. W. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 1172.
[11]	Zdanov, A.; Schalk-Hihi, C.; Gustchina, A.; Tsang, M.; Weatherbee, J.; Wlodawer, A. Structure 1995, 3, 591.
[12]	Logsdon, N. J.; Jones, B. C.; Allman, J. C.; Izotova, L.; Schwartz, B.; Pestka, S.; Walter, M. R. J. Mol. Biol. 2004, 342, 503.
[13]	Josephson, K.; DiGiacomo, R.; Indelicato, S. R.; Iyo, A. H.; Nagabhushan, T. L.; Parker, M. H.; Walter, M. R. J. Biol. Chem. 2000, 275, 13552.
[14]	(a) Windsor, W. T.; Syto, R.; Tsarbopoulos, A.; Zhang, R.; Durkin, J.; Baldwin, S.; Paliwal, S.; Mui, P. W.; Pramanik, B.; Trotta, P. P. Biochemistry 1993, 32, 8807.
	(b) Moore, K. W.; de Waal Malefyt, R.; Coffman, R. L.; O'Garra, A. Annu. Rev. Immunol. 2001, 19, 683.
[15]	Moore, K. W.; Vieira, P.; Fiorentino, D. F.; Trounstine, M. L.; Khan, T. A.; Mosmann, T. R. Science 1990, 248, 1230.
[16]	(a) Wang, P.; Dong, S.; Shieh, J.-H.; Peguero, E.; Hendrickson, R.; Moore, M. A. S.; Danishefsky, S. J. Science 2013, 342, 1357.
	(b) Murakami, M.; Kiuchi, T.; Nishihara, M.; Tezuka, K.; Okamoto, R.; Izumi, M.; Kajihara, Y. Sci. Adv. 2016, 2, e1500678.
[17]	Reif, A.; Lam, K.; Weidler, S.; Lott, M.; Boos, I.; Lokau, J.; Bretscher, C.; Mönnich, M.; Perkams, L.; Schmälzlein, M.; Graf, C.; Fischer, J.-P.; Lechner, C.; Hallstein, K.; Becker, S.; Weyand, M.; Steegborn, C.; Schultheiss, G.; Rose-John, S.; Garbers, C.; Unverzagt, C. Angew. Chem., Int. Ed. 2021, 60, 13380.
[18]	Sakamoto, I.; Tezuka, K.; Fukae, K.; Ishii, K.; Taduru, K.; Maeda, M.; Ouchi, M.; Yoshida, K.; Nambu, Y.; Igarashi, J.; Hayashi, N.; Tsuji, T.; Kajihara, Y. J. Am. Chem. Soc. 2012, 134, 5428.
[19]	Li, H.; Zhang, J.; An, C.; Dong, S. J. Am. Chem. Soc. 2021, 143, 2846.
[20]	Dong, W.; Yang, X.; Li, X.; Wei, S.; An, C.; Zhang, J.; Shi, X.; Dong, S. J. Am. Chem. Soc. 2024, 146, 18270.
[21]	Zhao, J.; Liu, X.; Liu, J.; Ye, F.; Wei, B.; Deng, M.; Li, T.; Huang, P.; Wang, P. J. Am. Chem. Soc. 2024, 146, 2615.
[22]	(a) Ye, F.; Li, C.; Liu, F.-L.; Liu, X.; Xu, P.; Luo, R.-H.; Song, W.; Zheng, Y.-T.; Ying, T.; Yu, B.; Wang, P. Natl. Sci. Rev. 2024, 11, nwae030.
	(b) Ye, F.; Zhao, J.; Xu, P.; Liu, X.; Yu, J.; Shangguan, W.; Liu, J.; Luo, X.; Li, C.; Ying, T.; Wang, J.; Yu, B.; Wang, P. Angew. Chem., Int. Ed. 2021, 60, 12904.
[23]	(a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
	(b) Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748.
[24]	Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776.
[25]	(a) Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed. 2007, 46, 9248.
	(b) Zhang, J.; Liu, H.; Teng, S.; Liao, Z.; Meng, L.; Wan, Q.; Dong, S. Chem. Commun. 2023, 59, 6513.
[26]	Maity, S. K.; Jbara, M.; Laps, S.; Brik, A. Angew. Chem., Int. Ed. 2016, 55, 8108.
[27]	(a) Fang, G.-M.; Li, Y.-M.; Shen, F.; Huang, Y.-C.; Li, J.-B.; Lin, Y.; Cui, H.-K.; Liu, L. Angew. Chem., Int. Ed. 2011, 50, 7645.
	(b) Huang, Y.-C.; Fang, G.-M.; Liu, L. Natl. Sci. Rev. 2016, 3, 107.
[28]	Li, H.; Dong, S. Sci. China: Chem. 2017, 60, 201.
[29]	Schwarz, J. B.; Kuduk, S. D.; Chen, X.-T.; Sames, D.; Glunz, P. W.; Danishefsky, S. J. J. Am. Chem. Soc. 1999, 121, 2662.
[30]	Flood, D. T.; Hintzen, J. C. J.; Bird, M. J.; Cistrone, P. A.; Chen, J. S.; Dawson, P. E. Angew. Chem., Int. Ed. 2018, 57, 11634.
[31]	Jbara M.; Laps S.; Morgan M.; Kamnesky, G.; Mann, G.; Wolberger, C.; Brik, A. Nat. Commun. 2018, 9, 3154.
[32]	Murakami, M.; Okamoto, R.; Izumi, M.; Kajihara, Y. Angew. Chem. Int., Ed. 2012, 51, 3567.
[33]	Thompson, R. E.; Chan, B.; Radom, L.; Jolliffe, K. A.; Payne, R. J. Angew. Chem., Int. Ed. 2013, 52, 9723.

均一糖基化修饰白细胞介素-10的化学合成研究