α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10⁻⁹ m), which is nearly identical to that of the full‐length enzyme (K_D=16×10⁻⁹ m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.

中文翻译：

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亲和质谱揭示人α-半乳糖苷酶A的抗体表位：逆转法布里氏病的酶替代疗法中的免疫反应性的基础。

α-半乳糖苷酶（αGal）是一种溶酶体酶，可水解糖鞘脂的末端α-半乳糖基部分。αGal编码基因的突变会导致酶缺陷或折叠错误，从而导致底物积累和随后的器官功能障碍。由人类α-半乳糖苷酶A缺乏引起的代谢性疾病被称为法布里病或法布里-安德森氏病，它属于较大的一类，称为溶酶体贮积病。通过酶替代疗法（ERT）已开发出一种有效的法布里病治疗方法，该疗法涉及输注纯化的重组酶以增加酶水平并减少积累的底物量。然而，免疫反应性和IgG抗体的形成是ERT的主要，局限性的，最终威胁生命的并发症。本研究集中于针对其形成的抗体的人α-半乳糖苷酶A的表位测定。在这里，我们报告了固定化免疫复合物的蛋白水解切除与表面等离振子共振生物传感质谱法的结合，鉴定了由人类单克隆抗αGal抗体识别的人类αGal（309–332）表位。表位肽αGal（309–332）是通过固相肽合成法合成的。通过表面等离振子共振分析确定其亲和力显示出对抗体的高结合亲和力（使用固定化免疫复合物的蛋白水解切除和表面等离振子共振生物传感质谱联用。表位肽αGal（309–332）是通过固相肽合成法合成的。通过表面等离振子共振分析确定其亲和力显示出对抗体的高结合亲和力（使用固定化免疫复合物的蛋白水解切除和表面等离振子共振生物传感质谱联用。表位肽αGal（309–332）是通过固相肽合成法合成的。通过表面等离振子共振分析确定其亲和力表明对抗体具有高结合亲和力（K _D = 39×10 ^-9  m），与全长酶（K _D = 16×10 ^-9  m）几乎相同。蛋白水解切除亲和质谱法在此显示是一种用于鉴定免疫原性溶酶体酶的表位的有效工具。由于全长αGal和抗体表位显示相似的结合亲和力，因此可通过以下步骤为ERT逆转免疫原性：1）用表位肽治疗患者以中和抗体，或2）通过单采血液分离术去除抗体，因此大大改善了对ERT的反应。