The differential impact of disulfide bonds and N-linked glycosylation on the stability and function of CD14
The differential impact of disulfide bonds and N-linked glycosylation on the stability and function of CD14
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DOI：
10.1074/jbc.m707640200
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发表时间：
2008-02-08
影响因子：
4.8
通讯作者：
McKnight, C. James
中科院分区：
文献类型：
--
作者：
Meng, Jianmin;Parroche, Peggy;McKnight, C. James
先天免疫是抵抗病原体入侵的第一道防线。在革兰氏阴性细菌感染期间，Toll样受体4和MD-2复合物识别存在于细菌细胞壁中的脂多糖。这种识别可以通过CD14增强100 - 1000倍。然而，CD14提供的有益作用在脓毒症和脓毒性休克的情况下变得有害。因此，了解CD14的功能将有助于针对免疫抑制和免疫增强的治疗。在本研究中，我们使用定点突变来解决二硫键和N-连接的糖基化对CD14的作用。观察到五个二硫键对CD14折叠的不同影响，其中前两个（Cys（6）-Cys（17）和Cys（15）-Cys（32））是必不可少的，第三和第四个（Cys（168）-Cys（198）和Cys（222）-Cys（253））是重要的，最后一个（Cys（287）-Cys（333））是不重要的。观察到第一个二硫键的功能作用，因为C6A取代严重降低了CD14赋予脂多糖对U373细胞的反应性的能力。四个预测的糖基化位点中的两个，天冬酰胺132和263，实际上参与了N-连接的糖基化，导致CD14分子量的异质性。此外，Asn（132）处的糖基化在CD14运输和上游和/或下游配体相互作用中起作用。当映射到小鼠CD14的晶体结构上时，前两个二硫键和Asn132非常接近富含亮氨酸的重复结构域的初始β链。因此，二硫键和N-连接的糖基化在CD 14的内凹表面的初始β折叠的结构和功能至关重要。
Innate immunity is the first line defense against invading pathogens. During Gram-negative bacterial infection, the Toll-like receptor 4 and MD-2 complex recognize lipopolysaccharide present in the bacterial cell wall. This recognition can be enhanced 100-1000-fold by CD14. However, the beneficial role provided by CD14 becomes detrimental in the context of sepsis and septic shock. An understanding of how CD14 functions will therefore benefit treatments targeted at both immune suppression and immune enhancement. In the present study, we use site-directed mutagenesis to address the role of disulfide bonds and N-linked glycosylation on CD14. A differential impact is observed for the five disulfide bonds on CD14 folding, with the first two (Cys(6)-Cys(17) and Cys(15)-Cys(32)) being indispensable, the third and fourth (Cys(168)-Cys(198) and Cys(222)-Cys(253)) being important, and the last (Cys(287)-Cys(333)) being dispensable. A functional role is observed for the first disulfide bond because the C6A substitution severely reduces the ability of CD14 to confer lipopolysaccharide responsiveness to U373 cells. Two of the four predicted glycosylation sites, asparagines 132 and 263, are actually involved in N-linked glycosylation, resulting in heterogeneity in CD14 molecular weight. Furthermore, glycosylation at Asn(132) plays a role in CD14 trafficking and upstream and/or downstream ligand interactions. When mapped onto the crystal structure of mouse CD14, the first two disulfide bonds and Asn132 are in close proximity to the initial beta strands of the leucine rich repeat domain. Thus, disulfide bonds and N-linked glycosylation in the initial beta sheets of the inner concave surface of CD14 are crucial for structure and function.