PDF(Pubmed)
Abstract：
OBJECTIVE: Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA).
METHODS: E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA.
RESULTS: Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins.
CONCLUSIONS: Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.
METHODS: E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA.
RESULTS: Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins.
CONCLUSIONS: Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.
摘要：
目标：肠道细菌，如大肠杆菌，可以在胺饥饿的条件下乙酰化它们的蛋白质组。据推测，（肠道）微生物组参与破坏对修饰的自身蛋白的免疫耐受，从而导致抗修饰蛋白抗体（AMPAs），标志血清阳性类风湿性关节炎（RA）。我们的目的是确定乙酰化的细菌蛋白是否可以诱导与修饰的自身蛋白交叉反应的AMPA反应并被人AMPA（hAMPA）识别。
方法：E.大肠杆菌在胺饥饿下生长，产生内源性乙酰化细菌蛋白。此外，大肠杆菌蛋白被化学乙酰化。通过蛋白质印迹和ELISA分析hAMPA对这些蛋白质的识别;通过pSyk(Syk磷酸化)活化测定分析携带修饰的蛋白质反应性B细胞受体(BCR)的B细胞的识别。C57BL/6小鼠用（修饰的）细菌蛋白部分免疫，通过ELISA分析和血清。
结果：化学修饰的细菌蛋白质级分含有高水平的乙酰化蛋白质，易于被hAMPA识别，并能够激活携带修饰的蛋白质反应性BCR的B细胞。可能是由于乙酰化水平大大降低，hAMPA或表达hAMPA的B细胞不识别内源性乙酰化的蛋白质组分。用化学修饰的蛋白质部分免疫小鼠诱导强烈的交叉反应性AMPA反应，靶向各种修饰的抗原，包括瓜氨酸化蛋白。
结论：乙酰化细菌蛋白可被hAMPA识别，并能够在小鼠中诱导交叉反应性AMPA。这些观察结果为涉及细菌蛋白质组（内源性）乙酰化的新机制提供了第一个概念性证据。允许破坏对修饰蛋白的耐受性并形成交叉反应性AMPA。
方法：E.大肠杆菌在胺饥饿下生长，产生内源性乙酰化细菌蛋白。此外，大肠杆菌蛋白被化学乙酰化。通过蛋白质印迹和ELISA分析hAMPA对这些蛋白质的识别;通过pSyk(Syk磷酸化)活化测定分析携带修饰的蛋白质反应性B细胞受体(BCR)的B细胞的识别。C57BL/6小鼠用（修饰的）细菌蛋白部分免疫，通过ELISA分析和血清。
结果：化学修饰的细菌蛋白质级分含有高水平的乙酰化蛋白质，易于被hAMPA识别，并能够激活携带修饰的蛋白质反应性BCR的B细胞。可能是由于乙酰化水平大大降低，hAMPA或表达hAMPA的B细胞不识别内源性乙酰化的蛋白质组分。用化学修饰的蛋白质部分免疫小鼠诱导强烈的交叉反应性AMPA反应，靶向各种修饰的抗原，包括瓜氨酸化蛋白。
结论：乙酰化细菌蛋白可被hAMPA识别，并能够在小鼠中诱导交叉反应性AMPA。这些观察结果为涉及细菌蛋白质组（内源性）乙酰化的新机制提供了第一个概念性证据。允许破坏对修饰蛋白的耐受性并形成交叉反应性AMPA。
