trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation

Abstract

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.

Keywords: 3-Methylglutaconic aciduria; Antibody; Enzyme linked immunosorbant assay; Hapten; Organic aciduria.

Figure 1.. Antibody characterization

Panel A) Unmodified BSA and 3MGC-conjugated BSA (25 ng) were electrophoresed on a 4–20% polyacrylamide gradient SDS gel and transferred to a PVDF membrane. The membrane was probed with rabbit immune serum (1:40,000 dilution) and antibody binding detected with HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution) and TMB substrate. Lane 1) molecular weight standards; Lane 2) unmodified BSA; Lane 3) 3MGC-BSA. Panel B) Isolated immune IgG (2 μg/mL) was coated onto the wells of a microtiter plate. Biotinylated 3MGC-BSA was incubated with indicated concentrations of free trans-3MGC acid for 1 h and then added to wells of the plate. After washing, HRP streptavidin (1:5000 dilution) and TMB substrate reagent were used for detection. Data are presented as a histogram of the normalized OD₄₅₀ as a percentage of maximum signal of the non-competition control (B/B₀ × 100 versus concentration). B =OD₄₅₀ of the sample with competitor and B₀ =OD₄₅₀ with no competitor. Values reported are the mean ± standard deviation (n=3). A 75% arbitrary threshold was used to determine sensitivity.

Figure 2.. ELISA evaluation of competitor specificity.

Panel A) Competition ELISA inhibition histograms were generated for trans-3MGC acid, cis-3MGC acid and four other different short chain dicarboxylic acids. Values plotted correspond to the mean of duplicate determinations with error bars representing the range of the replicates. HMG=3-hydroxy-3-methylglutaric acid, 3MG=3-methylglutaric acid. Panel B) Inhibition histograms were generated for trans-3MGC acid following 16 h pre-incubation at 22°C or 90°C. Values reported are the mean ± standard deviation (n=3).

Figure 3.. Immunoblot analysis of non-enzymatic protein 3MGCylation.

Isolated recombinant 3MCCCase was incubated with ATP, 3-methylcrotonyl CoA and HCO₃⁻ to produce trans-3MGC CoA. BSA was added to the 3MCCCase reaction product mixture prior to incubation at 4°C or 37°C for 24 h. Following incubation, an aliquot of each sample was subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with immune IgG and HRP conjugated secondary antibody with TMB used as substrate for color development. Lane 1) 4°C incubation; Lane 2) 37°C incubation.

Figure 4.. Proposed non-enzymatic reaction sequence from trans-3MGC CoA to protein 3MGCylation / cis-3MGC acid formation.

In subjects with either primary or secondary 3MGC aciduria, as trans-3MGC CoA is produced, isomerization to cis-3MGC CoA proceeds. Upon formation, cis-3MGC CoA undergoes intramolecular catalysis to form cis-3MGC anhydride. cis-3MGC anhydride has two possible fates; 1) protein 3MGCylation via reaction with lysine side chain amino groups or 2) reaction with H₂O to generate cis-3MGC acid.