Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues

Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in...

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Bibliographic Details
Published in:Biomolecules (Basel, Switzerland) Vol. 11; no. 8; p. 1168
Main Authors: Artiukhov, Artem V., Kolesanova, Ekaterina F., Boyko, Aleksandra I., Chashnikova, Anastasiya A., Gnedoy, Sergei N., Kaehne, Thilo, Ivanova, Daria A., Kolesnichenko, Alyona V., Aleshin, Vasily A., Bunik, Victoria I.
Format: Journal Article
Language:English
Published: Basel MDPI AG 07-08-2021
MDPI
Subjects:
Acetylation
Acylation
Affinity
Affinity chromatography
Amino acids
Antibodies
antibodies against ε-glutaryl-lysine residues
Antigens
Dehydrogenases
Enzymes
Gene expression
glutarylation
Immunization
Immunoblotting
Laboratories
Ligands
Lysine
Medical research
Metabolism
Mitochondria
Peptides
Polyclonal antibodies
Proteins
succinylation
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Russia
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Summary:The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.
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ISSN:2218-273X
2218-273X
DOI:10.3390/biom11081168