trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation

• Published in: Biochemical and Biophysical Research Communications Vol. 534; pp. 261 - 265
• Main Authors: Young, Rebecca, Jones, Dylan E., Diacovich, Lautaro, Witkowski, Andrzej, Ryan, Robert O.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021; Elsevier BV
• Subjects: 3-METHYLGLUTACONIC ACIDURIA; Acylation; anhydrides; animal models; Animals; ANTIBODY; antiserum; blood serum; bovine serum albumin; catalytic activity; Coenzyme A; Coenzyme A - metabolism; detection limit; Dicarboxylic Acids; Dicarboxylic Acids - analysis; Dicarboxylic Acids - immunology; ENZYME LINKED IMMUNOSORBANT ASSAY; Glutarates; Glutarates - analysis; Glutarates - chemistry; Glutarates - immunology; HAPTEN; Haptens; Haptens - immunology; Hemocyanins; Hemocyanins - immunology; Hemocyanins - metabolism; Hot Temperature; https://purl.org/becyt/ford/1; https://purl.org/becyt/ford/1.6; https://purl.org/becyt/ford/3; https://purl.org/becyt/ford/3.2; https://purl.org/becyt/ford/3.3; hydrolysis; immune response; Immune Sera; Immune Sera - immunology; Immunoglobulin G; Immunoglobulin G - immunology; Isomerism; isomerization; keyhole limpet hemocyanin; ORGANIC ACIDURIA; Rabbits; Serum Albumin, Bovine; Serum Albumin, Bovine - immunology; urine; Western blotting; LOD; BSA; AUH; Antibody; ETC; Organic aciduria; 3-Methylglutaconic aciduria; HMG CoA; 3MCCCase; Hapten; 3MGC; Enzyme linked immunosorbant assay
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2020.11.100

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. •A polyclonal antibody directed against trans-3MGC acid has been generated.•In ELISA assays, the antibody displayed much greater specificity for cis-3MGC acid.•The trans-3MGC hapten isomerized during or after conjugation to KLH.•trans-3MGC CoA undergoes nonenzymatic isomerization to a reactive cyclic anhydride.•Cyclic anhydride reaction with albumin led to protein 3MGCylation.