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

Abstract

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.

Delayed Impact of 2-Oxoadipate Dehydrogenase Inhibition on the Rat Brain Metabolism Is Linked to Protein Glutarylation

“…In our samples, glutarylation of the low molecular mass proteins is negligible, compared to the major glutarylated protein bands of 130 and 70 kDa, responding to the TMAP treatment. This is in line with an earlier comparative study of the lysine glutarylation, succinylation, and acetylation detectable by specific antibodies in the brain cortex homogenates: Only the acetylation band is visible in the region of the protein molecular masses below 20 kDa ( 72 ). Hence, further studies are required for assessment of the OADH-dependent glutarylation of histones after their enrichment, not compatible with the experimental design of the current work.…”

“…These molecular masses accurately match the apparent molecular masses of the previously characterized mammalian isoforms of OADH ( 26 ). Remarkably, in homogenates of liver, where the OADH expression is an order of magnitude higher than in the brain ( 34 ), the glutarylated proteins of the same molecular masses are stained by anti-glutaryllysine antibodies with a much higher intensity than in the brain homogenates ( 72 ). Moreover, the same protein bands are also detected as major glutarylated proteins in the fraction of liver mitochondria ( 72 ).…”

“…Remarkably, in homogenates of liver, where the OADH expression is an order of magnitude higher than in the brain ( 34 ), the glutarylated proteins of the same molecular masses are stained by anti-glutaryllysine antibodies with a much higher intensity than in the brain homogenates ( 72 ). Moreover, the same protein bands are also detected as major glutarylated proteins in the fraction of liver mitochondria ( 72 ). After chemical glutarylation of the liver mitochondrial proteins, many additional protein bands become reactive to anti-glutaryl-lysine antibodies.…”