Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
National Cell Bank, Pasteur Institute of Iran, Tehran, Iran.
J Biomol Struct Dyn. 2022 Nov;40(18):8169-8184. doi: 10.1080/07391102.2021.1908912. Epub 2021 Apr 10.
The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10-30, 40-60, 61-66, 88-120, and 130-140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency.Communicated by Ramaswamy H. Sarma.
重组人角质细胞生长因子（rhKGF）是一种高度聚集倾向的治疗蛋白。rhKGF 的高聚集倾向表现为单体状态的丧失，即使在中等温度下也会积累聚集物。在这里，我们使用几种基于序列的计算工具，包括 TANGO、ZipperDB、AGGRESCAN、Zyggregator、Camsol、PASTA、SALSA、WALTZ、SODA、Amylpred、AMYPDB 和基于结构的工具，包括 SolubiS、CamSol 结构校正、Aggrescan3D 和空间聚集倾向（SAP）算法，分析了 rhKGF 对聚集的脆弱性。rhKGF 中 APRs 的序列预测表明，它们主要位于位置 10-30、40-60、61-66、88-120 和 130-140。在 rhKGF 结构上的映射表明，这些残基中的大多数包括 F16-R25、I43、E45、R47-I56、F61、Y62、N66、L88-E91、E108-F110、A112、N114、T131 和 H133-T140 在天然状态下都是表面暴露的，这可以促进聚集而无需重大展开事件，或者可能在低聚物中发生构象变化。其他区域埋藏在天然状态下，它们对非天然聚集的贡献是通过之前的展开事件介导的。使用 SAP 工具对 APRs 进行基于结构的预测将鉴定的 APRs 的数量限制在动态暴露的疏水性残基上，包括 V12、A50、V51、L88、I89、L90、I118、L135 和 I139，这些残基介导天然状态下的聚集。我们对 rhKGF 中 APRs 的分析确定了决定 rhKGF 固有聚集倾向的区域，这些区域是工程改造 rhKGF 以降低其聚集倾向的候选位置。由 Ramaswamy H. Sarma 交流。
