Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.

我们此前曾报道，对人类外周血单个核细胞（human PBMCs）施以神经氨酸酶（neuraminidase, NA）预处理，可显著增强其对脂多糖（lipopolysaccharide, LPS）的细胞因子应答。为探究该现象的潜在机制，我们将编码TLR4、CD14与MD2（LPS受体复合物的三个组成部分）的质粒，以及NFκB荧光素酶报告系统，转染至HEK293T细胞中。上述质粒所编码的TLR4与MD2均发生了α-2,6唾液酸化修饰。转染了TLR4/MD2/CD14的HEK293T细胞可对LPS的添加产生强烈应答；但若省去MD2质粒，则该应答会被完全消除。向体系中加入MD2（sMD2）转染的HEK293T细胞培养上清，可恢复该应答，而重组非糖基化MD2则无此效果。对sMD2施以NA处理，可增强其对LPS的应答效果；向经TLR4/CD14转染的细胞中补充未处理的sMD2后，施以NA处理同样可增强该应答，但最优的LPS介导应答需同时使用经NA处理的TLR4/CD14转染细胞与经NA处理的sMD2方可实现。我们提出假说：TLR4复合物上聚糖的带负电荷唾液酸残基被移除后，可加速信号传导所需的TLR4单体二聚化过程。将分别携带YFP标签与FLAG标签的TLR4编码质粒共转染HEK293T细胞，随后施以NA处理并以LPS刺激，相较于未处理组细胞，经免疫共沉淀（co-immunoprecipitation）检测可见TLR4单体的时间依赖性二聚化出现更早且强度更高。该结果通过荧光共振能量转移（fluorescence resonance energy transfer, FRET）分析得到了验证。过表达人源Neu1可增强LPS介导的TLR4依赖型NFκB活化，而神经氨酸酶抑制剂则可抑制该活化过程。综上我们得出结论：(1) TLR4上的唾液酸残基可调控LPS应答，其潜在机制可能是促进TLR4同源二聚体的聚集；(2) 唾液酸及其他聚糖组分可调控LPS信号传导所需的MD2活性。我们推测，细胞活化过程中动员的内源性神经氨酸酶活性可能在该调控过程中发挥作用。