Department of Biochemistry and Molecular Biology, Mail Stop 0330, 1664 N. Virginia Street, University of Nevada, Reno, NV, 89557, USA.
Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
Biochem Biophys Res Commun. 2021 Jan 1;534:261-265. doi: 10.1016/j.bbrc.2020.11.100. Epub 2020 Dec 4.
3-methylglutaconic (3MGC) aciduria is associated with a growing number of discrete inborn errors of metabolism. Herein, an antibody-based approach to detection/quantitation of 3MGC acid has been pursued. When trans-3MGC acid conjugated keyhole limpet hemocyanin (KLH) was inoculated into rabbits a strong immune response was elicited. Western blot analysis provided evidence that immune serum, but not pre-immune serum, recognized 3MGC-conjugated bovine serum albumin (BSA). In competition ELISAs using isolated immune IgG, the limit of detection for free trans-3MGC acid was compared to that for cis-3MGC acid and four structurally related short-chain dicarboxylic acids. Surprisingly, cis-3MGC acid yielded a much lower limit of detection (∼0.1 mg/ml) than trans-3MGC acid (∼1.0 mg/ml) while all other dicarboxylic acids tested were poor competitors. The data suggest trans-3MGC- isomerized during, or after, conjugation to KLH such that the immunogen was actually comprised of KLH harboring a mixture of cis- and trans-3MGC haptens. To investigate this unexpected isomerization reaction, trans-3MGC CoA was prepared and incubated at 37 °C in the presence of BSA. Evidence was obtained that non-enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA precedes intramolecular catalysis to form cis-3MGC anhydride plus CoASH. Anhydride-dependent acylation of BSA generated 3MGCylated BSA, as detected by anti-3MGC immunoblot. The results presented provide an explanation for the unanticipated detection of 3MGCylated proteins in a murine model of primary 3MGC aciduria. Furthermore, non-enzymatic hydrolysis of cis-3MGC anhydride represents a potential source of cis-3MGC acid found in urine of subjects with 3MGC aciduria.
3-甲基戊烯二酸（3MGC）酸尿症与越来越多的离散先天性代谢错误有关。在此，我们采用了一种基于抗体的方法来检测/定量 3MGC 酸。当反式 3MGC 酸与血蓝蛋白（KLH）偶联后，兔子会产生强烈的免疫反应。Western blot 分析表明，免疫血清而非预免疫血清可识别 3MGC 结合的牛血清白蛋白（BSA）。在使用分离的免疫 IgG 的竞争 ELISA 中，比较了游离反式 3MGC 酸的检测限与顺式 3MGC 酸和四种结构相关的短链二羧酸的检测限。令人惊讶的是，顺式 3MGC 酸的检测限（约 0.1mg/ml）远低于反式 3MGC 酸（约 1.0mg/ml），而测试的所有其他二羧酸均为较差的竞争物。数据表明，在 KLH 偶联过程中或之后，反式 3MGC 发生了异构化，使得免疫原实际上由 KLH 组成，其中包含 cis-和 trans-3MGC 半抗原的混合物。为了研究这种意外的异构化反应，制备了反式 3MGC CoA 并在 37°C 下与 BSA 孵育。获得的证据表明，反式 3MGC CoA 向顺式 3MGC CoA 的非酶异构化先于形成顺式 3MGC 酐和 CoASH 的分子内催化。顺式 3MGC 酐依赖性酰化生成 3MGC 酰化的 BSA，如抗 3MGC 免疫印迹所示。所提出的结果为在原发性 3MGC 酸尿症的小鼠模型中检测到 3MGC 酰化蛋白提供了一种解释。此外，顺式 3MGC 酐的非酶水解代表了在 3MGC 酸尿症患者尿液中发现的顺式 3MGC 酸的潜在来源。
