Guo Yugang, Li Rui, Song Xiaoping, Zhong Yongjun, Wang Chenguang, Jia Hao, Wu Lidan, Wang Dong, Fang Fang, Ma Jiajia, Kang Wenyao, Sun Jie, Tian Zhigang, Xiao Weihua
Hefei National Laboratory for Physical Sciences at Microscale, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China ; Center of Medical Biotechnology of Anhui Province, University of Science and Technology of China, Hefei, China.
Hefei National Laboratory for Physical Sciences at Microscale, Innovation Center for Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei, China.
PLoS One. 2014 Feb 20;9(2):e89264. doi: 10.1371/journal.pone.0089264. eCollection 2014.
CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications.
CD83是一种高度糖基化的I型跨膜糖蛋白，属于免疫球蛋白超家族。CD83在树突状细胞（DC）成熟过程中上调，这对适应性免疫反应的启动至关重要。CD83的可溶性异构体（sCD83）由全长CD83 mRNA的可变剪接编码，并抑制DC成熟，这表明sCD83作为一种潜在的免疫抑制剂发挥作用。在本研究中，我们开发了一种可靠的策略，用于在极高密度发酵中从毕赤酵母表达功能性sCD83。纯化的sCD83以单体形式表达，产量超过200 mg/L，并含有通过PNGase F消化鉴定的N-连接糖基化位点。体外试验表明，重组sCD83与单核细胞上其假定的对应物结合，并特异性阻断抗CD83抗体与DC细胞表面CD83的结合。此外，来自酵母的sCD83显著抑制ConA刺激的PBMC增殖。因此，从毕赤酵母表达的sCD83具有功能活性，可用于体内和体外研究以及未来的临床应用。
