Bláha Jan, Pachl Petr, Novák Petr, Vaněk Ondřej
Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 12840, Czech Republic.
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, Prague 16610, Czech Republic.
Protein Expr Purif. 2015 May;109:7-13. doi: 10.1016/j.pep.2015.01.006. Epub 2015 Jan 24.
Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.
凝集素样转录本1（LLT1，基因clec2d）被鉴定为自然杀伤细胞（NK）和NK-T淋巴细胞表面存在的单个人NKR-P1受体的配体。正常情况下，LLT1在NK细胞表面表达，刺激γ干扰素的产生，并且在其他免疫细胞激活后会上调，例如经Toll样受体（TLR）刺激的树突状细胞和B细胞，或经T细胞受体激活的T细胞。在正常组织中，LLT1与NKR-P1的相互作用（代表另一种“缺失自我”识别系统）在调节NK细胞与抗原呈递细胞之间的串扰中发挥免疫调节作用，而在最致命的肿瘤之一胶质母细胞瘤细胞中LLT1上调，它在其中充当胶质瘤细胞免疫逃逸的介质。在此，我们报告了LLT1胞外域可溶性His176Cys突变体在人悬浮适应型HEK293S GnTI(-)细胞系的真核表达系统中的瞬时表达及特性分析，该细胞系具有均一的N-聚糖。如质谱分析所示，His176Cys突变对于C型凝集素样结构域的稳定性至关重要，可导致LLT1中第三个典型二硫键的重建。纯化后的可溶性LLT1具有均一性、可去糖基化，并且形成非共价同源二聚体，其二聚化不依赖于其N-聚糖的存在。作为可溶性LLT1生产的一部分，我们使HEK293S GnTI(-)细胞系适应于在便于瞬时转染的培养基中悬浮生长，并优化了新的高细胞密度转染方案，极大提高了蛋白质产量。该转染方案通常适用于此细胞系内的蛋白质生产，尤其适用于蛋白质晶体学研究。
