Dudich Elena, Dudich Igor, Semenkova Lidia, Benevolensky Sergey, Morozkina Elena, Marchenko Aleksey, Zatcepin Sergey, Dudich Dmitry, Soboleva Galina, Khromikh Luidmila, Roslovtceva Olga, Tatulov Eduard
Institute of Immunological Engineering, Lyubuchany, Moscow Region, Chekhov District 142380, Russia.
Protein Expr Purif. 2012 Jul;84(1):94-107. doi: 10.1016/j.pep.2012.04.008. Epub 2012 Apr 26.
Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.
甲胎蛋白（AFP）是一种在自身免疫性疾病、癌症及再生医学治疗方面具有高度药用潜力的生物药物候选物。大规模生产重组人甲胎蛋白（rhAFP）对于结构与功能研究以及应用研究而言是很有必要的。在本研究中，我们在具有多个染色体整合型合成人AFP基因的酿酒酵母菌株中，克隆并以分泌形式表达了野生型糖基化人rhAFP和非糖基化突变体rhAFP(0)（N233S）。rhAFP和rhAFP(0)已成功从培养液中生产并纯化出来，成为具有天然折叠活性的蛋白质。与野生型蛋白相比，通过突变消除糖基化使rhAFP(0)的分泌减少了约三倍，这表明N-连接聚糖对异源蛋白折叠和分泌起着关键作用。通过圆二色性技术证实了rhAFP和rhAFP(0)与天然胚胎eAFP的结构相似性。功能测试表明，与天然eAFP相比，重组体rhAFP和rhAFP(0)具有相似类型的肿瘤抑制和免疫抑制活性。据记载，rhAFP和rhAFP(0)的这两种生物活性均归因于肿瘤细胞和丝裂原活化淋巴细胞中凋亡的快速诱导。尽管rhAFP和rhAFP(0)的肿瘤抑制活性与eAFP相比略显低效，但与糖基化rhAFP和eAFP相比，rhAFP(0)在诱导体外淋巴细胞增殖抑制能力方面有统计学上显著的提高。我们得出结论，rhAFP的N-连接糖基化对于有效折叠和分泌是必需的。然而，N-连接糖部分的存在对于肿瘤抑制活性并不重要，但对其免疫调节活性至关重要，这表明AFP的这两种生物功能活性涉及不同的分子机制。
