Warts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) syndrome is a primary immunodeficiency disease (PID) usually caused by autosomal dominant mutations in the chemokine receptor CXCR4 gene. To date, a total of nine different mutations including eight truncation mutations and one missense mutation (E343K, CXCR4E343K) distributed in the C-terminus of CXCR4 have been identified in humans. Studies have clarified that the loss of phosphorylation sites in the C-terminus of truncated CXCR4 impairs the desensitization process, enhances the activation of G-protein, prolongs downstream signaling pathways and introduces over immune responses, thereby causing WHIM syndrome. So far, there is only one reported case of WHIM syndrome with a missense mutation, CXCR4E343K, which has a full length of C-terminus with entire phosphorylation sites, no change in all potential phosphorylation sites. The mechanism of the missense mutation (CXCR4E343K) causing WHIM syndrome is unknown. This study aimed to characterize the effect of mutation at the 343 site of CXCR4 causing the replacement of arginine/E with glutamic acid/K on the receptor signal transduction, and elucidate the mechanism underling CXCR4E343K causing WHIM in the reported family. We completed a series of mutagenesis to generate different mutations at the 343 site of CXCR4 tail, and established a series of HeLa cell lines stably expressing CXCR4WT or CXCR4E343D (glutamic acid/E replaced with aspartic acid/D) or CXCR4E343K (glutamic acid/E replaced with lysine/K) or CXCR4E343R (glutamic acid/E replaced with arginine/R) or CXCR4E343A (glutamic acid/E replaced with alanine/A) and then systematically analyzed functions of the CXCR4 mutants above. Results showed that the cells overexpressing of CXCR4E343D had no functional changes with comparison that of wild type CXCR4. However, the cells overexpressing of CXCR4E343K or CXCR4E343R or CXCR4E343A had enhanced cell migration, prolonged the phosphorylation of ERK1/2, p38, JNK1/2/3, aggravated activation of PI3K/AKT/NF-κB signal pathway, introduced higher expression of TNFa and IL6, suggesting over immune response occurred in CXCR4 mutants with charge change at the 343 site of receptor tail, as a result, causing WHIM syndrome. Biochemical analysis of those mutations at the 343 site of CXCR4 above shows that CXCR4 mutants with no matter positive or neutral charge have aberrant signal pathways downstream of activated mutated CXCR4, only CXVR4 with negative charge residues at the site shows normal signal pathway post activation with stromal-derived factor (SDF1, also known as CXCL12). Taken together, our results demonstrated that the negative charge at the 343 site of CXCR4 plays an essential role in regulating the down-stream signal transduction of CXCR4 for physiological events, and residue charge changes, no matter positive or neutral introduce aberrant activities and functions of CXCR4, thus consequently lead to WHIM syndrome.

中文翻译：

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343位点的负电荷对于维持CXCR4的生理功能至关重要

疣、低丙种球蛋白血症、复发性细菌感染和骨髓增生异常综合征 (WHIM) 综合征是一种原发性免疫缺陷病 (PID)，通常由趋化因子受体 CXCR4 基因的常染色体显性突变引起。迄今为止，已在人类中鉴定出总共九种不同的突变，包括分布在 CXCR4 的 C 末端的八种截断突变和一种错义突变（E343K、CXCR4E343K）。研究已经阐明，截短的 CXCR4 的 C 端磷酸化位点的丢失会损害脱敏过程，增强 G 蛋白的活化，延长下游信号通路并引入过度免疫反应，从而导致 WHIM 综合征。到目前为止，只有一例 WHIM 综合征病例报告了错义突变，CXCR4E343K，它具有全长的 C 端和完整的磷酸化位点，所有潜在的磷酸化位点都没有变化。错义突变 (CXCR4E343K) 导致 WHIM 综合征的机制尚不清楚。本研究旨在表征 CXCR4 343 位点突变导致精氨酸/E 被谷氨酸/K 替代对受体信号转导的影响，并阐明 CXCR4E343K 引起报告家族中 WHIM 的机制。我们完成了一系列诱变，在CXCR4尾部的343位点产生不同的突变，并建立了一系列稳定表达CXCR4WT或CXCR4E343D（谷氨酸/E替换为天冬氨酸/D）或CXCR4E343K（谷氨酸/E替换为赖氨酸/K）或CXCR4E343R（谷氨酸/E替换为精氨酸/R）的HeLa细胞系) 或 CXCR4E343A（谷氨酸/E 被丙氨酸/A 取代），然后系统地分析了上述 CXCR4 突变体的功能。结果表明，与野生型CXCR4相比，过表达CXCR4E343D的细胞没有功能变化。然而，过表达CXCR4E343K或CXCR4E343R或CXCR4E343A的细胞增强了细胞迁移，延长了ERK1/2、p38、JNK1/2/3的磷酸化，加剧了PI3K/AKT/NF-κB信号通路的激活，引入了更高的TNFa表达和 IL6，提示CXCR4突变体发生过度免疫反应，受体尾部343位点电荷发生变化，导致WHIM综合征。上述CXCR4 343位点突变的生化分析表明，无论带正电荷或中性电荷的CXCR4突变体在激活突变CXCR4下游都有异常信号通路，只有该位点带负电荷残基的CXVR4在基质激活后显示正常信号通路。衍生因子（SDF1，也称为 CXCL12）。综上所述，我们的研究结果表明，CXCR4 343 位点的负电荷在调节 CXCR4 的下游信号转导中起着至关重要的作用，无论是正电荷还是中性的CXCR4，