Calprotectin S100A9 Calcium-binding Loops I and II Are Essential for Keratinocyte Resistance to Bacterial Invasion
Calprotectin S100A9 Calcium-binding Loops I and II Are Essential for Keratinocyte Resistance to Bacterial Invasion
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DOI：
10.1074/jbc.m806605200
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发表时间：
2009-03-13
影响因子：
4.8
通讯作者：
Herzberg, Mark C.
中科院分区：
文献类型：
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作者：
Champaiboon, Chantrakorn;Sappington, Kaia J.;Herzberg, Mark C.
表达钙卫蛋白（S100 A8和S100 A9蛋白的异源二聚体）的上皮细胞对细菌入侵更具抵抗力。为了确定影响细菌入侵抗性的结构基序，在S100 A9中构建了靶向钙结合环I和II（E36 Q、E78 Q、E36 Q、E78 Q）和C末端（S100 A91 -99和S100 A91 -112）的突变，C末端含有推定的抗微生物锌结合和磷酸化位点。将S100 A8和突变的S100 A9编码质粒转染到钙卫蛋白阴性的KB癌细胞中。所有转染的细胞（除了KB-假）表达27 E10反应性异二聚体。在用单核细胞增生李斯特菌和鼠伤寒沙门氏菌（鼠伤寒沙门氏菌）进行的细菌侵入试验中，表达S100 A8与S100 A9 E36 Q、S100 A9 E78 Q、S100 A91 -99或S100 A91 -112突变体或S100 A91 -114（全长）钙卫蛋白复合物的细胞系比KB假手术更好地抵抗细菌侵入。当与KB-S100 A8/A91-114相比时，表达截短的S100 A91 -99或S100 A91 -112与S100 A8的细胞也显示出对细菌侵袭的抗性增加。相反，谷氨酸残基36和78在钙结合环I和II促进上皮细胞的抗性，因为细胞表达S100 A9 E36 Q，E78 Q与S100 A8不能抵抗细菌入侵。预测S100 A9 E36 Q、E78 Q中的突变会导致钙卫蛋白中钙诱导的阳性面丧失，减少与微管的相互作用，并且似乎对角质形成细胞抵抗细菌入侵至关重要。
Epithelial cells expressing calprotectin, a heterodimer of S100A8 and S100A9 proteins, are more resistant to bacterial invasion. To determine structural motifs that affect resistance to bacterial invasion, mutations were constructed in S100A9 targeting the calcium-binding loops I and II (E36Q, E78Q, E36Q, E78Q) and the Cterminus (S100A91-99 and S100A91-112), which contains putative antimicrobial zinc-binding and phosphorylation sites. The S100A8 and mutated S100A9 encoding plasmids were transfected into calprotectin-negative KB carcinoma cells. All transfected cells (except KB-sham) expressed 27E10-reactive heterodimers. In bacterial invasion assays with Listeria monocytogenes and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), cell lines expressing S100A8 in complex with S100A9E36Q, S100A9E78Q, S100A91-99, or S100A91-112 mutants or the S100A91-114 (full-length) calprotectin resisted bacterial invasion better than KB-sham. When compared with KB-S100A8/A91-114, cells expressing truncated S100A91-99 or S100A91-112 with S100A8 also showed increased resistance to bacterial invasion. In contrast, glutamic acid residues 36 and 78 in calcium-binding loops I and II promote resistance in epithelial cells, because cells expressing S100A9E36Q, E78Q with S100A8 were unable to resist bacterial invasion. Mutations in S100A9 E36Q, E78Q were predicted to cause loss of the calcium-induced positive face in calprotectin, reducing interactions with microtubules and appearing to be crucial for keratinocyte resistance to bacterial invasion.