Serine phosphate group in casein phosphopeptide is crucial for IgE binding to αs1-casein
Serine phosphate group in casein phosphopeptide is crucial for IgE binding to αs1-casein
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酪蛋白磷酸肽中的丝氨酸磷酸基对于 IgE 与 αs1-酪蛋白的结合至关重要
DOI：
10.1016/j.alit.2023.02.001
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发表时间：
2023
影响因子：
6.8
通讯作者：
Izumi Hidehiko
中科院分区：
文献类型：
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作者：
Naito Michihiro;Matsui Teruaki;Ito Komei;Izumi Hidehiko
酪蛋白磷酸肽 (CPP) 的簇序列被称为 as1-、as2- 和 b-酪蛋白 (CN) 的胰蛋白酶降解产物，由三个磷酸化的丝氨酸残基 (Ser) 和两个谷氨酸残基“SSSEE”组成。在此，我们将该簇序列周围的氨基酸序列命名为“CPP 核心基序”。尽管as1-CN 和b-CN 之间的同源性较低（4%），2 as1-、as2- 和b-CN 中存在的四个CPP 基序彼此显示出较高的序列同源性（图1A）。尽管 CN 中的大多数 IgE 结合表位在胰蛋白酶消化后被破坏，但据报道，极少量的 CPP（实际上是 CPP 与无定形磷酸钙结合）会引起牛奶 (CM) 过敏患者的过敏症状。 3 尽管有这些发现，但使用合成重叠肽对 CN 进行的表位作图研究很少鉴定出 CPP 核心基序中的 IgE 结合表位。 4 e8 因此，我们假设由于合成肽中缺乏 Ser 的磷酸基团，天然 CN 和 CPP 的 IgE 结合位点在之前的研究中被忽视。 9 为了验证这一假设，我们评估了 IgG 和 IgE 与 as1-CN 和 CPP 的结合能力是否受到去磷酸化处理的影响。我们从 12 名 CM 过敏患者那里获得了血清，这些患者是通过口服食物激发试验确诊的（补充表 1）。在所有患者中，使用ImmunoCAP®（Thermo Fisher Scientific，Uppsala，Sweden）检测针对CM（> 50 kUA/L）和CN（> 50 kUA/L）的sIgE。使用碱性磷酸酶处理 1 或 24 小时（分别为 D1-或 D24-）对 as1-CN 和 CPP 进行去磷酸化，并使用 SDS-PAGE 和 Phos-tag® SDS-PAGE 进行确认（补充方法和图 1 B）。使用 as1-CN 的抑制 ELISA 评估 IgG 和 IgE 与样品的结合。 2 微量滴定板涂有 10 mg/mL as1-CN，并用含有 0.05% Tween 20 (PFBB-T) 的无蛋白封闭缓冲液封闭。 as1-CN-sIgG 或 PFBB-T 中的患者血清与样品一起预孵育。然后将预孵育的一抗添加到每个孔中并孵育。当D24-CN与as1-CN-sIgG预孵育时，as1-CN包被的ELISA抑制曲线较NeCN右移，且D24-CN的IC50（0.898 mg/mL）高于NeCN（0.385 mg/mL）（图1 C，D）。 CPP在去磷酸化处理下也使抑制曲线右移，D1-和D24-CPP的IC50分别为327.9和>1000 mg/mL，高于N-CPP的值（206.5 mg/mL）。这些结果表明Ser中的磷酸基团在as1-CN-sIgG的抗原识别中发挥一定的作用。在as1-CN-sIgE的抑制ELISA中，as1-CN和CPP的去磷酸化处理降低了对as1-CN-sIgE的抑制活性（P<0.01，图1E）。 D24-CN (0.302 mg/mL) 的 IC50 高于 NeCN (< 0.1 mg/mL)，D24-CPP (250.5 mg/mL) 高于 NCPP (32.1 mg/mL，图 1 F)。这些发现表明，as1-CN 和 CPP 的 Ser 中的磷酸基团在 CMA 患者 IgE 抗体的结合中发挥着重要作用。相反，胰蛋白酶的降解产物CPP也降解了as1-CN的一些IgE表位，这表明它并没有完全抑制as1-CN-sIgE。 as1-CN-sIgE 抑制 ELISA 的个体结果如补充图 1 所示。尽管任何患者的去磷酸化抑制率均未增加，但 CPP 的抑制功效存在个体差异……
The cluster sequence of casein phosphopeptide (CPP), known as the tryptic degradative product of as1-, as2-, and b-casein (CN), consists of three serine residues (Ser), which are phosphorylated (SerP), and two glutamic acid residues “SSSEE.“1 Herein, we named the amino acid sequence around this cluster sequence as “CPP core motif.” Despite the low homology (4%) between as1-CN and b-CN, 2 the four CPP motifs present in as1-, as2-, and b-CN show high sequence homology with each other (Fig. 1 A). Although most of the IgE-binding epitopes in CNs are supposed to be destroyed after tryptic digestion, a very small amount of the CPP (in fact, CPP coupled with amorphous calcium phosphate) reportedly induced allergic symptoms in patients with cow's milk (CM) allergy. 3 Despite these findings, epitope mapping studies of CNs using synthetic overlapping peptides have rarely identified IgE-binding epitopes in the CPP core motif. 4 e8 Consequently, we hypothesized that the IgE-binding sites of the natural CN and CPP were overlooked in previous studies owing to the lack of phosphate group of Ser in the synthetic peptides. 9 To verify this hypothesis, we evaluated whether the binding ability of IgG and IgE to as1-CN and CPP is affected by the dephosphorylation treatment. We obtained sera from 12 patients with CM allergy, who had been definitively diagnosed using an oral food challenge test (Supplementary Table 1). In all patients, the sIgE against CM (> 50 kUA/L) and CN (> 50 kUA/L) was detected using ImmunoCAP®(Thermo Fisher Scientific, Uppsala, Sweden). Dephosphorylation of as1-CN and CPP was performed using alkaline phosphatase treatment for 1 or 24 h (D1-or D24-, respectively) and confirmed using SDS-PAGE and Phos-tag® SDS-PAGE (Supplementary Methods and Fig. 1 B). IgG and IgE binding to the samples was evaluated using inhibition ELISA for as1-CN. 2 The microtiter-plates were coated with 10 mg/mL as1-CN and blocked with protein-free blocking buffer containing 0.05% Tween 20 (PFBB-T). as1-CN-sIgG or patient sera in PFBB-T were preincubated with the samples. The pre-incubated primary antibodies were then added to each well and incubated. When D24-CN was pre-incubated with as1-CN-sIgG, the inhibition curve for the as1-CN-coated ELISA shifted to the right from that of NeCN, and the IC50 of D24-CN (0.898 mg/mL) was higher than that of NeCN (0.385 mg/mL)(Fig. 1 C, D). CPP also shifted the inhibitory curve to the right under the dephosphorylation treatment and the IC50 of D1-and D24-CPP was 327.9 and> 1000 mg/mL, respectively, which were higher than the value of N-CPP (206.5 mg/mL). These results suggest that the phosphate groups in Ser play a certain role in the antigen recognition of as1-CN-sIgG. In the inhibition ELISA of as1-CN-sIgE, the dephosphorylation treatment of as1-CN and CPP decreased the inhibitory activity against as1-CN-sIgE (P< 0.01, Fig. 1 E). The IC50 of D24-CN (0.302 mg/mL) was higher than that of NeCN (< 0.1 mg/mL), and that of D24-CPP (250.5 mg/mL) was higher than the value of NCPP (32.1 mg/mL, Fig. 1 F). These findings suggest that the phosphate groups in the Ser of as1-CN and CPP play a major role in the binding of IgE antibodies in patients with CMA. Contrarily, CPP, a degradation product of trypsin, also degraded some IgE epitopes of as1-CN, suggesting that it did not completely inhibit as1-CN-sIgE. The individual results of the inhibition ELISA of as1-CN-sIgE are shown in Supplementary Figure 1. Although the inhibition rate did not increase with dephosphorylation in any of the patients, there were individual differences in the inhibitory efficacy of CPP …