Identification of KRT16 as a target of an autoantibody response in complex regional pain syndrome.
Identification of KRT16 as a target of an autoantibody response in complex regional pain syndrome.
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DOI：
10.1016/j.expneurol.2016.10.011
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发表时间：
2017-01
影响因子：
5.3
通讯作者：
Clark JD
中科院分区：
文献类型：
--
作者：
Tajerian M;Hung V;Khan H;Lahey LJ;Sun Y;Birklein F;Krämer HH;Robinson WH;Kingery WS;Clark JD
使用复杂区域疼痛综合征（CRPS）的小鼠模型，我们的目标是识别患肢皮肤中的自身抗原。使用胫骨骨折/石膏固定模型诱导CRPS样状态。骨折后三周，将后爪皮肤匀浆，在2-d凝胶上电泳，并用骨折小鼠和对照小鼠的血清进行探测。通过液相色谱-质谱（LC-MS）分析感兴趣的斑点，并通过检查其丰度和亚细胞定位验证靶标列表。为了测量所选蛋白质靶标的自身抗原性，我们定量了对照和骨折小鼠血清以及对照和CRPS人血清中IgM与重组蛋白的结合。我们显示骨折皮肤提取物和骨折血清之间的独特结合，表明存在自身抗原。LC-MS分析为我们提供了一系列潜在的靶点，其中一些在骨折后上调（KRT 16，eEF 1a 1和PRPH），而另一些则显示出亚细胞再分布和膜定位增加（ANXA 2和ENO 3）。未观察到蛋白质瓜氨酸或氨甲酰化的变化。除了增加的丰度，KRT 16表现出自身抗原性，因为来自骨折小鼠和CRPS患者的血清显示与重组kRT 16蛋白结合的自身抗体增加。寻求自身免疫对CRPS的贡献提供了一种新的方法来了解病情，并可能允许开发基于机制的治疗方法。在小鼠和人类中鉴定抗KRT 16的自身抗体作为生物标志物是实现这些目标的关键一步，也是将CRPS重新定义为具有自身免疫病因的关键一步。
Using a mouse model of complex regional pain syndrome (CRPS), our goal was to identify autoantigens in the skin of the affected limb. A CRPS-like state was induced using the tibia fracture/cast immobilization model. Three weeks after fracture, hindpaw skin was homogenized, run on 2-d gels, and probed by sera from fracture and control mice. Spots of interest were analyzed by liquid chromatography-mass spectroscopy (LC-MS) and the list of targets validated by examining their abundance and subcellular localization. In order to measure the autoantigenicity of selected protein targets, we quantified the binding of IgM in control and fracture mice sera, as well as in control and CRPS human sera, to the recombinant protein. We show unique binding between fracture skin extracts and fracture sera, suggesting the presence of auto-antigens. LC-MS analysis provided us a list of potential targets, some of which were upregulated after fracture (KRT16, eEF1a1, and PRPH), while others showed subcellular-redistribution and increased membrane localization (ANXA2 and ENO3). No changes in protein citrullination or carbamylation were observed. In addition to increased abundance, KRT16 demonstrated autoantigenicity, since sera from both fracture mice and CRPS patients showed increased autoantibody binding to recombinant kRT16 protein. Pursuing autoimmune contributions to CRPS provides a novel approach to understanding the condition and may allow the development of mechanism-based therapies. The identification of autoantibodies against KRT16 as a biomarker in mice and in humans is a critical step towards these goals, and towards redefining CRPS as having an autoimmune etiology.