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trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation
Rebecca Young1, Dylan E Jones1, Lautaro Diacovich2
1Department of Biochemistry and Molecular Biology, Mail Stop 0330, 1664 N. Virginia Street, University of Nevada, Reno, NV, 89557, USA.
Biochemical and Biophysical Research Communications
|December 7, 2020
Summary
Antibody detection of 3-methylglutaconic (3MGC) aciduria revealed unexpected cis-isomerization during protein conjugation. This isomerization explains 3MGCylated proteins in models and suggests a source for urinary cis-3MGC acid.
Area of Science:
- Biochemistry
- Immunology
- Metabolic Disorders
Background:
- 3-methylglutaconic (3MGC) aciduria encompasses various inborn errors of metabolism.
- Accurate detection and quantification of 3MGC acid are crucial for diagnosing these disorders.
Purpose of the Study:
- To develop an antibody-based method for detecting and quantifying 3-methylglutaconic acid.
- To investigate the unexpected isomerization of trans-3MGC acid during protein conjugation.
Main Methods:
- Conjugation of trans-3-methylglutaconic acid to keyhole limpet hemocyanin (KLH) for antibody generation in rabbits.
- Western blot and competitive enzyme-linked immunosorbent assays (ELISAs) to assess antibody specificity and detection limits.
- Incubation of trans-3MGC CoA with bovine serum albumin (BSA) to study isomerization and acylation reactions.
Main Results:
- Immune serum recognized 3MGC-conjugated BSA, indicating successful antibody production.
- Antibodies showed higher sensitivity to cis-3MGC acid (limit of detection ~0.1 mg/ml) than trans-3MGC acid (~1.0 mg/ml).
- Evidence of non-enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA, forming cis-3MGC anhydride, which then acylated BSA.
Conclusions:
- Trans-3MGC acid isomerizes to its cis-form during conjugation or in the presence of BSA, explaining the observed antibody reactivity.
- This isomerization and subsequent anhydride formation provide a mechanism for 3-methylglutaconylation of proteins.
- Non-enzymatic hydrolysis of cis-3MGC anhydride may contribute to urinary cis-3MGC acid levels in patients.
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