Calcium-induced Tetramerization and Zinc Chelation Shield Human Calprotectin from Degradation by Host and Bacterial Extracellular Proteases.
Calcium-induced Tetramerization and Zinc Chelation Shield Human Calprotectin from Degradation by Host and Bacterial Extracellular Proteases.
复制标题
钙诱导的四聚化和锌螯合可保护人钙卫蛋白免受宿主和细菌细胞外蛋白酶的降解。
DOI：
10.1039/c5sc03287c
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发表时间：
2016
期刊：
影响因子：
8.4
通讯作者：
Nolan,ElizabethM
中科院分区：
文献类型：
--
作者：
Stephan,JulesR;Nolan,ElizabethM
钙卫蛋白（CP，S100 A8/S100 A9寡聚体，MRP-8/14寡聚体，钙颗粒蛋白A和B）是先天免疫系统的蛋白质组分，其通过在感染部位螯合生物可利用的过渡金属离子而有助于金属抑制应答。人CP利用Ca（II）离子来调节其四级结构、过渡金属结合特性和抗微生物活性。在这项工作中，我们报告的发现，钙（II）诱导的自协会的人CP提供异源四聚体保护的蛋白质支架从宿主丝氨酸蛋白酶降解。我们提出了两种新的人CP-Ser变体S100 A8（C42 S）（I60 E）/S100 A9（C3 S）和S100 A8（C42 S）（I60 K）/S100 A9（C3 S）的设计和表征，其表现出缺陷的四聚化特性。分析性尺寸排阻色谱和分析性超离心显示，两种变体（下文称为I60 E和I60 K）在仅Ca（II）存在下作为异二聚体持续存在，并且在仅Mn（II）存在下以及在Ca（II）和Mn（II）两者存在下形成异四聚体。与Fe（II）的配位也导致I60 E和I60 K在不存在和存在Ca（II）的情况下形成异源四聚体。Ca（II）结合的I60 E和I60 K异二聚体容易被胰蛋白酶、胰凝乳蛋白酶和人嗜中性粒细胞弹性蛋白酶降解，而Ca（II）结合的CP-Ser异四聚体和Ca（II）和Mn（II）结合的I60 E和I60 K异四聚体对这些宿主蛋白酶的降解具有抗性。葡萄球菌胞外蛋白酶GluC在残基89的C-末端切割CP-Ser的S100 A8亚基以提供ΔSHKE变体。GluC切割位点非常接近His 3Asp金属结合位点，其以高亲和力与Zn（II）配位，并且Zn（II）螯合保护S100 A8亚基免受GluC切割。两者合计，这些结果提供了新的见解如何钙（II）离子和过渡金属调节CP的化学和生物学，并表明，协调二价阳离子转化为抗蛋白酶的形式，使人类CP的先天免疫功能在敌对条件下的感染部位。
Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/14 oligomer, calgranulins A and B) is a protein component of the innate immune system that contributes to the metal-withholding response by sequestering bioavailable transition metal ions at sites of infection. Human CP employs Ca(II) ions to modulate its quaternary structure, transition metal binding properties, and antimicrobial activity. In this work, we report the discovery that Ca(II)-induced self-association of human CP to afford heterotetramers protects the protein scaffold from degradation by host serine proteases. We present the design and characterization of two new human CP-Ser variants, S100A8(C42S)(I60E)/S100A9(C3S) and S100A8(C42S)(I60K)/S100A9(C3S), that exhibit defective tetramerization properties. Analytical size exclusion chromatography and analytical ultracentrifugation reveal that both variants, hereafter I60E and I60K, persist as heterodimers in the presence of Ca(II) only, and form heterotetramers in the presence of Mn(II) only and both Ca(II) and Mn(II). Coordination to Fe(II) also causes I60E and I60K to form heterotetramers in both the absence and presence of Ca(II). The Ca(II)-bound I60E and I60K heterodimers are readily degraded by trypsin, chymotrypsin, and human neutrophil elastase, whereas the Ca(II)-bound CP-Ser heterotetramers and the Ca(II)- and Mn(II)-bound I60E and I60K heterotetramers are resistant to degradation by these host proteases. The staphylococcal extracellular protease GluC cuts the S100A8 subunit of CP-Ser at the C-terminal end of residue 89 to afford a ΔSHKE variant. The GluC cleavage site is in close proximity to the His3Asp metal-binding site, which coordinates Zn(II) with high affinity, and Zn(II) chelation protects the S100A8 subunit from GluC cleavage. Taken together, these results provide new insight into how Ca(II) ions and transition metals modulate the chemistry and biology of CP, and indicate that coordination to divalent cations transforms human CP into a protease-resistant form and enables innate immune function in the hostile conditions of an infection site.