均一糖基化修饰白细胞介素-10的化学合成研究

doi:10.6023/cjoc202410001

摘要/Abstract

细胞因子人白细胞介素-10 (IL-10)序列中含有一个保守的天冬酰胺糖基化(N-糖基化)基序, 但尚无相关研究报道这一糖基化的存在与潜在功能. 探索了IL-10及其糖基化形式的化学合成与表征: 基于固相多肽合成、自然化学连接和无金属巯基脱除等策略, 实现IL-10全长序列的构建, 并通过优化折叠复性条件成功获得未经糖基修饰的IL-10. 在此合成路线基础上, 利用糖修饰氨基酸砌块插入策略获得N-乙酰葡糖胺(N-GlcNAc)修饰的糖多肽片断, 进一步获得了天冬酰胺116位点单糖修饰的IL-10. 最后, 经过对合成IL-10样品的质谱和圆二色谱评价, 证明其具有与生物表达样品相似的结构.

关键词: 蛋白质化学合成, 糖蛋白, 自然化学连接, 蛋白质折叠, 人白细胞介素-10

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

引用此文

董伟东, 刘海韵, 李信宇, 王标, 成波, 董甦伟. 均一糖基化修饰白细胞介素-10的化学合成研究[J]. 有机化学, 2025, 45(3): 951-958.

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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图/表 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).

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