Rigo Mauricio M, Borges Thiago J, Lang Benjamin J, Murshid Ayesha, Wolfgeher Donald, Calderwood Stuart K, Truman Andrew W, Bonorino Cristina
School of Medicine, Pontificia Universidade Catolica do Rio Grande do Sul, Av. Ipiranga, 6681, Porto Alegre Rio Grande do Sul, Zip Code: 90619-900, Brazil.
Schuster Family Transplantation Research Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave, Boston, MA, 02115, USA.
FEBS J. 2020 Mar 6. doi: 10.1111/febs.15279.
The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.
使用模式生物进行重组蛋白生产会导致添加模式特异性的翻译后修饰（PTM），这些修饰会影响蛋白质的结构、电荷和功能。70 kDa热休克蛋白（Hsp70）最初被描述为具有ATP酶和折叠酶活性的细胞内伴侣蛋白。最近，已鉴定出Hsp70蛋白的新的细胞外活性（例如作为免疫调节剂）。虽然一些研究表明细胞外Hsp70蛋白具有促炎潜力，但其他研究则表明其具有免疫抑制活性。我们推测，在不同表达系统中生产重组Hsp70会导致添加不同的PTM，这或许至少可以解释其中一些相反的免疫学结果。我们从两种不同的系统，即大肠杆菌和毕赤酵母中生产并纯化了结核分枝杆菌DnaK，并通过质谱分析了蛋白质制剂，从计算机模拟和体外角度研究了PTM 的影响。计算机模拟中DnaK结构的比较突出表明，存在取决于表达系统的静电和拓扑差异。在真核系统中生产DnaK会显著影响其ATP酶活性，并显著改变其下调小鼠树突状细胞（DC）上MHC II和CD86表达的能力。对DnaK进行磷酸酶处理表明，其中一些差异与磷酸化特别相关。总之，我们的数据表明PTM是表达系统的一个重要特征，其差异会影响热休克蛋白与其配体的相互作用以及随后的功能活性。
