Windsor W T, Syto R, Tsarbopoulos A, Zhang R, Durkin J, Baldwin S, Paliwal S, Mui P W, Pramanik B, Trotta P P
Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
Biochemistry. 1993 Aug 31;32(34):8807-15. doi: 10.1021/bi00085a011.
Interleukin 10 (IL-10), which was first discovered by its ability to inhibit the synthesis of various cytokines, most notably gamma interferon, from Th1 helper cells, displays pleiotropic immunoregulatory properties. Human and murine IL-10 have a high amino acid sequence identity (ca. 73%) which includes the conservation of all four cysteine residues in human IL-10 and the first four out of five cysteine residues for murine IL-10. Chemical analysis was used to determine that both recombinant human and recombinant murine IL-10 contain two disulfide bonds. The disulfide pairs for each were determined by mass spectrometric and reversed-phase HPLC analysis of trypsin-derived polypeptide fragments. The disulfide bond assignments for both species were similar in that the first cysteine residue in the sequence paired with the third and the second paired with the fourth. The fifth cysteine in murine IL-10 was determined by chemical modification to be unpaired. Far-UV circular dichroism analysis indicated that the secondary structure of recombinant human and murine IL-10 are composed of ca. 60% alpha-helix. Reduction of the disulfide bonds structurally destabilized the protein and led to a structure containing only 53% alpha-helix. The reduced protein displayed no in vitro biological activity in a mast cell proliferation assay. These studies indicate that IL-10 is a highly alpha-helical protein containing two disulfide bonds, either one or both of which are critical for its structure and function. In addition, these properties suggest that this interesting cytokine may belong to the alpha helical cytokine class of hematopoietic ligands.
白细胞介素10（IL-10）最初因其能够抑制多种细胞因子（最显著的是γ干扰素）从Th1辅助细胞的合成而被发现，具有多效性免疫调节特性。人和小鼠的IL-10具有高度的氨基酸序列同一性（约73%），其中包括人IL-10中所有四个半胱氨酸残基的保守性以及小鼠IL-10五个半胱氨酸残基中的前四个。化学分析用于确定重组人IL-10和重组小鼠IL-10均含有两个二硫键。通过对胰蛋白酶衍生的多肽片段进行质谱分析和反相高效液相色谱分析，确定了每种物质的二硫键对。两种物种的二硫键分配相似，即序列中的第一个半胱氨酸残基与第三个配对，第二个与第四个配对。通过化学修饰确定小鼠IL-10中的第五个半胱氨酸未配对。远紫外圆二色性分析表明，重组人IL-10和小鼠IL-10的二级结构约由60%的α-螺旋组成。二硫键的还原在结构上使蛋白质不稳定，并导致一种仅含有53%α-螺旋的结构。在肥大细胞增殖试验中，还原后的蛋白质没有体外生物学活性。这些研究表明，IL-10是一种高度α-螺旋的蛋白质，含有两个二硫键，其中一个或两个对其结构和功能至关重要。此外，这些特性表明这种有趣的细胞因子可能属于造血配体的α螺旋细胞因子类别。
