Isoaspartate (isoAsp) formation, through deamidation of asparaginyl residues or isomerization of aspartyl residues, constitutes a large proportion of spontaneous protein damage observed both in vitro and in vivo.
1 –6 Generation of isoAsp sites is initiated by nucleophilic attack on the side-chain carbonyl of aspartate (Asp) or asparagine (Asn) by the C-flanking amide bond nitrogen, resulting in an intermediate succinimide (
Fig. 1). Protein L-isoaspartate O-methyltransferase (PIMT; EC 2.1.1.77) selectively methylates the α-carboxyl group of L-isoaspartyl residues, and the isoAsp methyl ester formed spontaneously demethylates to reform a succinimide that can restore a normal α-linked Asp-Xaa bond.
7–11 Continuing cycles of PIMT action efficiently repair L-isoAsp sites, as has been demonstrated in vitro with a number of peptides and proteins.
12–15 A repair function for PIMT in vivo is supported by the observation that reduction of PIMT activity in cultured cells or PIMT knockout (KO) mice dramatically increases the level of isoAsp-containing proteins.
16–19 A critical need for PIMT action in the brain is evident by its high specific activity in this tissue
20,21 as well as the overt neurological phenotype of PIMT KO mice: increased brain size, abnormal neuroanatomical and electrophysiological properties of hippocampal cells, atypical open-field behavior, and fatal epileptic seizures beginning at 4 weeks of age.
17,18,22–24 Perhaps because of its small size, the retina of the PIMT KO mouse has apparently received no attention with regard to alterations in its morphology, physiology, biochemistry, or function. Remarkably, no published studies to date mention any evidence for a vision deficit in the PIMT KO mouse. Such a deficit could be masked by (or consistent with) their atypical behavioral characteristics and the fact that olfactory and tactile functions serve them well in a caged environment.
In mammalian tissues, the specific activity of PIMT varies over a 10-fold range, with highest levels to date found in the brain and testis.
17,18,20 Given that the retina is an extension of the central nervous system, one would expect that it might also exhibit relatively high PIMT activity and potential for isoaspartyl protein damage. Modest PIMT activity has been reported in homogenates of whole eye and lens
18,25 ; but to our knowledge, PIMT activity in the isolated vertebrate retina has never been investigated. This is unfortunate because isoaspartyl damage not only has the capacity to disrupt protein function, but also can trigger an immune reaction to self-proteins.
26,27 Either of these effects could potentially play a role in the development or exacerbation of eye diseases such as macular degeneration, autoimmune retinopathies, and retinal uveitis.
Because of their high metabolic demands and photosensitivity, retinal cells are subject to a high burden of oxidative stress that promotes oxidative damage of proteins and lipids.
28,29 A number of recent studies suggest that protein oxidation and isoAsp formation interact synergistically. Oxidation of purified hemoglobin with acetylphenylhydrazine produces a rapid increase in its isoAsp content, and exposure of erythrocytes to hydrogen peroxide has been found to induce isoAsp formation in membrane proteins.
30,31 Ultraviolet A radiation has been found to trigger isoAsp formation in melanoma cells, while natural antioxidants such as hydroxytyrosol (found in olive oil) protect melanoma cells against both oxidative damage and isoAsp accumulation.
32 Cellular levels of PIMT can greatly affect the susceptibility of cells to apoptosis induced by oxidative stress, as observed in cultured human endothelial cells and the nematode
C. elegans .
33,34 This synergism between two common forms of protein damage creates a strong rationale for investigating the potential role of isoAsp formation as a contributor to retinal dysfunction.
We report here the use of PIMT +/+ (WT, wild type), ± (HZ, heterozygote), and −/− (KO) mice to investigate the expression and activity of PIMT in isolated retina, as well as the propensity of the retina to accumulate isoAsp-damaged protein when PIMT activity is absent. Our results confirm the expectation that mouse retina exhibits both a robust PIMT activity and a propensity to accumulate isoaspartyl proteins that rivals that of brain. This suggests that isoaspartyl protein damage, like protein oxidation, should be considered as a possible factor in one or more retinal diseases. It also raises the possibility that diminished visual function may be an unrecognized facet of the PIMT KO mouse phenotype.