Molecular basis of Fab-dependent IgA antibody recognition by gut-associated metallopeptidases

Immunoglobulin A (IgA) is essential for mucosal immunity and is implicated in autoimmune diseases like IgA nephropathy (IgAN). Certain pathogenic and commensal bacteria produce IgA proteases (IgAPs) that selectively cleave IgA, potentially aiding bacterial colonization and offering therapeutic avenues for IgAN. Here, we investigate the substrate specificity of M64 family peptidases, focusing on the IgAP ThomasA from Thomasclavelia ramosa and BF3526 from Bacteroides fragilis. Structural, biochemical, and mutagenesis data demonstrate that ThomasA cleaves IgA through exclusive recognition of the Fab region—a mechanism distinct from other antibody-specific peptidases, which typically require engagement of the Fc region. In contrast, X-ray crystal structures of BF3526 in complex with substrate and product peptides, together with enzymology assays, show that this enzyme targets the N-terminus of predigested proteins. These findings reveal divergent substrate recognition strategies within the M64 family and provide structural insight into their conserved catalytic mechanism. Together, they enhance our understanding of bacterial adaptation and support the rational design of enzymes for IgA-mediated autoimmune diseases. The LC-MS data associated with the project is included in the current dataset.免疫球蛋白A（Immunoglobulin A, IgA）是黏膜免疫不可或缺的关键分子，同时与IgA肾病（IgA nephropathy, IgAN）等自身免疫病密切相关。部分致病菌与共生菌可分泌IgA蛋白酶（IgA proteases, IgAPs），通过选择性切割IgA促进细菌定植，也为IgAN的治疗提供了潜在方向。 本研究聚焦M64家族肽酶的底物特异性，以来自分枝托马斯菌（Thomasclavelia ramosa）的IgAP ThomasA以及脆弱拟杆菌（Bacteroides fragilis）的BF3526为研究对象。结构生物学、生物化学与诱变实验数据表明，ThomasA仅通过识别抗体的抗原结合片段（Fab region）完成IgA切割——这一机制与其他抗体特异性肽酶截然不同，后者通常需要结合可结晶片段（Fc region）方可发挥催化活性。与之相对，BF3526与底物、产物肽复合物的X射线晶体结构，结合酶学活性检测结果显示，该酶靶向预消化蛋白质的N末端。本研究揭示了M64家族肽酶间各异的底物识别策略，并为其保守的催化机制提供了结构层面的解析。 上述成果不仅加深了我们对细菌适应性机制的认知，也为靶向IgA介导自身免疫病的酶类理性设计提供了理论支撑。本数据集包含该研究项目关联的液相色谱-质谱联用（LC-MS）实验数据。