A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.

中文翻译：

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天然抗体的位点特异性缀合：转谷氨酰胺酶介导的保守谷氨酰胺修饰，同时通过糖苷内切酶修剪维持一级序列和核心 Fc 聚糖

转谷氨酰胺酶（TGase，EC 2.3.2.13）是一种用于位点特异性修饰天然（非工程化）抗体的多功能化学酶工具。该酶以各种胺作为共底物，将肽和蛋白质中谷氨酰胺（Gln 或 Q）的未取代侧链酰胺转化为取代酰胺（即缀合物）。令人惊喜的是，IgG Fc 区中只有一个保守的谷氨酰胺 (Gln295) 被微生物 TGase (mTGase，EC 2.3.2.13) 修饰，从而提供了一种高度特异性且普遍适用的缀合方法。然而，在转酰胺基化（通过 mTGase 接触谷氨酰胺残基）之前，必须减少附近保守 N-聚糖（IgG1 中的 Asn297）的空间位阻。在之前的方法中，使用酰胺酶（PNGase F，EC 3.5.1.52）来完全去除 N-聚糖。然而，PNGase F 还将净中性天冬酰胺 (Asn297) 转化为带负电的天冬氨酸 (Asp297)。这种电荷改变可能会显着改变 IgG 抗体的结构、功能和免疫原性。相比之下，在我们本文提出的新方法中，N-聚糖被糖苷内切酶（EndoS2，EC 3.2.1.96）修剪，因此保留了核心N-乙酰葡糖胺（GlcNAc）部分和中性天冬酰胺酰胺。修剪后的聚糖还减少或消除了 Fc 受体介导的功能，从而通过减少与其他细胞（例如免疫细胞）的非特异性结合来产生更好的成像剂。此外，剩余的核心聚糖允许进一步衍生化，例如聚糖重塑和双重缀合。我们的方法实用且稳健，以接近定量的产率生成缀合物，并且两种酶均可在市场上购买。