Enzymatic synthesis of poly(α-ethyl ß-aspartate) by poly(ethylene glycol) modified poly(aspartate) hydrolase-1.

We recently discovered that poly(aspartate) (PAA) hydrolase-1 from Pedobacter sp. KP-2 has a unique property of specifically cleaving the amide bond between ß-aspartate units in thermally synthesized PAA (tPAA). In the present study, the enzymatic synthesis of poly(α-ethyl ß-aspartate) (ß-PAA) was performed by taking advantage of the substrate specificity of PAA hydrolase-1. No polymerization of diethyl L-aspartate by native PAA hydrolase-1 occurred because of the low dispersibility of the enzyme in organic solvent. Poly(ethylene glycol) (PEG) modification of the enzyme improved its dispersibility and enabled it to polymerize the monomer substrate. MALDI-TOF MS analysis showed that the synthesized polymer was observed in the range of m/z = 750-2 500. This analysis also revealed that the polymer was composed of ethyl aspartate units, containing either an ethyl ester or a free carboxyl end group at its carboxyl terminus. (1) H NMR analysis demonstrated that the synthesized polymer consisted of only ß-amide linkages. Thus, the present results indicate that PAA hydrolase-1 modified with PEG is useful for the synthesis of ß-PAA due to its unique substrate specificity and good dispersibility in organic solvent.

Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain.

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. 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. 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. 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. 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. 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.