Brain fingerprinting blotted science tab

The upper chamber of the electrophoresis apparatus was filled with 10 m m H 3PO4 solution, and the lower chamber was filled with degassed cathode solution (20 m m NaOH). The solubilized samples were centrifuged for 5 min at 14,000 rpm, and the clear supernatant (в€ј50 Ојl) was applied to the tube gel, overlaid with 20 Ојl of NEPHGE sample buffer, and filled with anode solution (10 m m H 3PO4). в€ј160 Ојg of mouse brain extract was solubilized in NEPHGE sample buffer (9 m urea, 0.8% Bio-Lyte 5/8, and 0.2% Bio-Lyte 3/10). First dimension gels were polymerized in 13.5 Г— 0.45-cm glass tubes containing 4% acrylamide/bisacrylamide (29:1), 9.2 m urea, 2% (w/v) Nonidet P-40, 0.4% Bio-Lyte 3/10, 1.6% Bio-Lyte 5/8. Peptide mass fingerprinting of these spots allowed us to identify 22 proteins that are major endogenous substrates for PIMT. Autofluorography of the membrane provided information on which Coomassie-stained spots on a parallel two-dimensional gel corresponded to major methyl acceptors. To circumvent this problem, we separated the unmethylated proteins by two-dimensional-gel electrophoresis, transferred them to a polyvinylidene difluoride (PVDF) membrane, and then 3H-methylated them on the membrane with an overlay of recombinant PIMT and AdoMet.

Unfortunately, until now, the low chemical stability of the methyl esters has made it difficult to employ the high resolution separation techniques needed to identify the individual radiolabeled proteins. When extracts of the KO mouse brain are 3H-methylated by PIMT in vitro and separated by SDS-PAGE under acidic conditions, it is apparent that a diverse group of proteins are acting as endogenous substrates.

Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. Fluorography of the blot revealed 30-35 3H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. Isoaspartyl proteins were radiolabeled on-blot using S-adenosyl- l-methionine and recombinant PIMT. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures.

Protein l-isoaspartyl methyltransferase (PIMT) catalyzes repair of l-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions.

Glycobiology and Extracellular Matrices.

Brain fingerprinting blotted science tab