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Published on: March 22, 2015
Isoaspartyl protein damage and repair in mouse retina
Zhenxia Qin1, Jing Yang, Henry J Klassen
1Department of Molecular Biology and Biochemistry, University of California, Irvine, California.
Purpose:
To determine the propensity of retinal proteins for spontaneous damage via formation of isoaspartyl sites, a common type of protein damage that could contribute to retinal disease.
Methods:
Tissue extracts were obtained from retinas and brains of control mice and from mice in which the gene for protein L-isoaspartate O-methyltransferase (PIMT; an enzyme that repairs isoaspartyl protein damage) was knocked out. PIMT expression in these extracts was measured by Western blot, and its specific activity was assayed by monitoring the rate of [(3)H]methyl transfer from S-adenosyl-[methyl-(3)H]L-methionine to γ-globulin. Isoaspartate levels in extracts were measured by their capacity to accept [(3)H]methyl groups via the PIMT-catalyzed methylation reaction. To compare molecular weight distributions of isoaspartyl-rich proteins in retina versus brain, proteins from PIMT knockout (KO) and control mice were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Isoaspartyl proteins were (3)H-labeled on-blot using a PIMT overlay and imaged by autoradiography.
Results:
When normalized to the β-actin content of each tissue, retina was found to be nearly identical to brain with regard to expression and activity of PIMT and its propensity to accumulate isoaspartyl sites when PIMT is absent. The two tissues show distinct differences in the molecular weight distribution of isoaspartyl proteins.
Conclusions:
The retina is rich in PIMT activity and contains a wide range of proteins that are highly susceptible to this type of protein damage. Recoverin may be one such protein. Isoaspartate formation, along with oxidation, should be considered as a potential source of protein dysfunction and autoimmunity in retinal disease.
Insights
The retina has high protein L-isoaspartate O-methyltransferase (PIMT) activity, indicating susceptibility to protein damage. This damage, along with oxidation, may contribute to retinal diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Ophthalmology
Background:
- Protein damage via isoaspartyl site formation is a common aging process.
- Protein L-isoaspartate O-methyltransferase (PIMT) is an enzyme that repairs isoaspartyl damage.
- Dysfunctional PIMT may contribute to age-related diseases.
Purpose of the Study:
- To investigate the propensity of retinal proteins for spontaneous damage through isoaspartyl site formation.
- To compare retinal protein damage to that of brain tissue.
- To assess the role of PIMT in retinal protein integrity.
Main Methods:
- Compared PIMT expression and activity in retinal and brain tissue extracts from control and PIMT knockout mice.
- Quantified isoaspartate levels using a PIMT-catalyzed methylation assay.
- Analyzed molecular weight distribution of isoaspartyl proteins using SDS-PAGE and on-blot PIMT labeling.
Main Results:
- Retina exhibited similar PIMT expression and activity to brain tissue.
- In the absence of PIMT, both tissues showed comparable accumulation of isoaspartyl sites.
- Distinct differences were observed in the molecular weight profiles of isoaspartyl-rich proteins between retina and brain.
Conclusions:
- The retina possesses significant PIMT activity and numerous proteins susceptible to isoaspartyl damage.
- Recoverin is a potential candidate protein for isoaspartyl modification in the retina.
- Isoaspartate formation and oxidation are potential contributors to retinal dysfunction and autoimmunity in disease.
