Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2
Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2
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DOI：
10.1111/j.1462-5822.2006.00702.x
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发表时间：
2006-07-01
影响因子：
3.4
通讯作者：
Shibata, Ken-ichiro
中科院分区：
文献类型：
--
作者：
Kataoka, Hideo;Yasuda, Motoaki;Shibata, Ken-ichiro
在识别病原体相关的分子模式和形成功能性受体复合物中，与Toll样受体（TLR）连接的碳水化合物的作用的细节仍然未知。本研究旨在确定TLR 2胞外结构域的Asn 114、Asn 199、Asn 414和Asn 442残基连接的聚糖是否参与识别二酰化脂肽和脂蛋白。将单个和多个突变体与NF-κ B荧光素酶报告质粒一起转染到人胚肾（HEK）293细胞中。所有这些突变体都在表面上表达。转染子的SDS-PAGE表明，这些突变体迁移低于野生型TLR 2和它们的分子量随着突变的Asn残基的数目增加而降低。当用这些配体刺激时，TLIZ 12（N114 A）、TLR 2（N199 A）和TLR 2（N414 A）以及野生型TLR 2诱导NF-κ B活化，而TLR 2（N442 A）不能诱导NF-κ B活化。所有的三重和四重突变体均不能诱导NF-κ B活化，但在转染子中与野生型TLR 2和TLR 6相关。TLR 2（N114 A，N199 A）TLR 2（N114 A，N414 A）和在较小程度上的TLR 2（N114 A，N442 A），其中推测两个N-连接的聚糖暴露于TLR 2螺线管的凹面，不仅诱导NF-κ B活化，而且与野生型TLR 2和TLR 6相关。这些结果表明，Asn 442处的聚糖和推测暴露于TLR 2螺线管凹面的至少两个N-连接聚糖参与TLR 2对配体的识别和/或参与功能性TLR 2受体复合物的形成或成熟。
Details of roles of carbohydrates attached to Toll-like receptors (TLRs) in the recognition of pathogen-associated molecular patterns and in the formation of the functional receptor complex still remain unknown. This study was designed to determine whether the glycans linked at Asn114, Asn199, Asn414 and Asn442 residues of TLR2 ectodomain were involved in the recognition of diacylated lipopeptide and lipoprotein. Single and multiple mutants were transfected into human embryonic kidney (HEK) 293 cells together with a NF-kappa B luciferase reporter plasmid. All of these mutants were expressed on the surface. SDS-PAGE of the transfectants demonstrated that these mutants migrated lower than wild-type TLR2 and their molecular masses decreased as the number of mutated Asn residues increased. TLIZ12(N114A), TLR2(N199A) and TLR2(N414A) as well as wild-type TLR2 induced NF-kappa B activation when stimulated with these ligands, whereas TLR2(N442A) failed to induce NF-kappa B activation. All of triple and quadruple mutants failed to induce NF-kappa B activation, but were associated with both wildtype TLR2 and TLR6 in the transfectants. TLR2(N114A,N199A) TLR2(N114A,N414A) and, to a lesser extent, TLR2(N114A,N442A), in which two N-linked glycans are speculated to be exposed to the concave surface of TLR2 solenoid, not only induce NF-kappa B activation but also are associated with wild-type TLR2 and TLR6. These results suggest that the glycan at Asn442 and at least two N-linked glycans speculated to be exposed to the concave surface of TLR2 solenoid are involved in the recognition of ligands by TLR2 and/or in formation or maturation of a functional TLR2 receptor complex.