Fighting isoaspartate

Isoaspartate (IsoAsp) is a damaging amino acid residue generated either from asparagine (Asn) deamidation or aspartate (Asp) isomerization. Both reactions are spontaneous in physiological conditions and require no enzymes. In isoAsp, a CH2 group is rearranged from the side chain and extends the polypeptide backbone. The accumulation of isoAsp disrupts protein structures and functions, making them prone to aggregation, and eventually contributes to the onset of Alzheimer’s disease (AD) and other neurodegenerative diseases (NDDs). Since the mass of isoAsp is the same as Asp, and only +0.98 Da different than Asn, the quantification of isoAsp requires high sensitivity and specificity of the analysis. Moreover, it is very difficult to develop a monoclonal antibody against isoAsp with high specificity. Although Asn deamidation is considered irreversible, there is an enzyme protein L-isoaspartyl methyltransferase (PIMT) that mitigates its damaging effect. PIMT methylates isoAsp using S-adenosylmethionine (SAM) as a methyl donor. Upon methanol loss, methylated isoAsp spontaneously becomes normal L-Asp in a minority (< 25%) of cases or more probably isoAsp again (≥ 75%). However, with age SAM production declines, rendering repair insufficient. This may result in isoAsp accumulation in long-lived proteins, including human serum albumin (HSA), the most abundant protein in blood. In the present thesis, we depict different ways to “fight” isoAsp, either via its early detection or exploring the repair mechanism, which we believe render avenues to fighting aging and age-related diseases.

In Paper I, we developed a monoclonal antibody (mAb) specific to isoAsp in an important domain of HSA, and characterized it via DNA/amino acid sequencing, kinetic analysis, paratope mapping, glycosylation analysis, etc. We also quantified the isoAsp level in HSA of the blood of 100 healthy donors, the histogram of which resembled the normal distribution. In Paper II, for the first time we demonstrated that isoAsp-containing HSA forms aggregates with reduced binding capacity toward amyloid beta (Aß) peptide and phosphorylated tau (p- Tau) protein. Using the mAb raised in Paper I and size exclusion chromatography, we found a significant increase of isoAsp level in HSA and its aggregates of the patients with AD compared with controls. We also discovered in AD group a significantly decrease of antibodies against isoAsp in HSA, as well as an increase of free Aß not bound with HSA. Based on the findings, we updated the isoAsp hypothesis of AD, supporting the role of isoAsp accumulation as a triggering factor in AD. In Paper III, we validated the results in Paper II, and further explored the capacity of isoAsp in diagnostics of other NDDs. The most significant finding was the best performance of isoAsp-related biomarkers in mild cognitive impairment (MCI) detection compared with other blood biomarkers. In addition, the levels of isoAsp in HSA and its antibodies significantly correlated with cognitive decline. In Paper IV, we discussed theoretical considerations supporting the possibility of a full repair of isoAsp to Asn and reported the first experimental evidence on the reversibility of isoAsp formation via protein succinimide/isoaspartate ammonia ligase (PSIAL) activity. We also discovered PSIAL activity in the recombinant cytoplasmic human aspartate aminotransferase (GOT1).

List of scientific papers

I. Wang J, Lundström SL, Seelow S, Rodin S, Meng Z, Astorga-Wells J, Jia Q, Zubarev RA. First immunoassay for measuring isoaspartate in human serum albumin. Molecules. 2021; 26(21):6709.
https://doi.org/10.3390/molecules26216709

II. Wang J, Guo C, Meng Z, Zwan MD, Chen X, Seelow S, Lundström SL, Rodin S, Teunissen CE*, Zubarev RA*. Testing the link between isoaspartate and Alzheimer’s disease etiology. Alzheimer’s and Dementia. 2022;1-12. *These authors contributed equally.
https://doi.org/10.1002/alz.12735

III. Wang J*, Zhang Y*, Shen X, Han J, Cui M, Tan L, Dong Q, Zubarev RA*, Yu J*. Isoaspartate-related blood biomarkers show promise for early diagnostics of neurodegeneration. *These authors contributed equally. [Manuscript]

IV. Wang J, Rodin S, Dibavar AS, Zhang X, Zubarev RA. First experimental evidence for reversibility of ammonia loss from asparagine. International Journal of Molecular Sciences. 2022; 23(15):8371.
https://doi.org/10.3390/ijms23158371