Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts the secondary and higher order structures of proteins, damaging their functions and making them prone to aggregation. An association has been suggested between isoAsp and Alzheimer's Disease (AD). Here we strengthened the link between isoAsp and AD by novel approaches to isoAsp analysis in blood human serum albumin (HSA), the most abundant blood protein, a major carrier of amyloid beta (Aβ) peptide and phosphorylated tau (pTau) protein in blood and a key participant in their clearance pathway. We discovered a reduced amount of anti-isoAsp antibodies (P < .0001), an elevated isoAsp level in HSA (P < .001), more HSA aggregates (P < .0001) and increased levels of free Aβ (P < .01) in AD blood compared to healthy controls. We also found that deamidation significantly reduces HSA capacity to bind with Aβ and pTau (P < .05). These findings support the presence in AD of a bottleneck in clearance of Aβ and pTau leading to their increased concentrations in brain and facilitating their aggregations there.

doi: bioRxiv preprint RESEARCH IN CONTEXT1. Systematic review: We reviewed the evidence that associates isoaspartate (isoAsp) residue in blood proteins with the etiology of Alzheimer's disease (AD). However, the link between isoAsp in blood and aggregation of amyloid beta (Aß) peptide and phosphorylated tau (pTau) protein in brain remained unclear. Interpretation:For the first time we demonstrate that isoAsp-containing human serum albumin (HSA) forms aggregates with reduced binding capacity toward Aß peptide and pTau protein. Using a novel ELISA, we discovered in AD blood elevated levels of isoAsp in HSA, together with reduced endogenous anti-isoAsp antibody levels, suggesting hampered Aß and pTau clearance in AD. Future directions:As degradation of the innate anti-isoAsp defenses may take years to develop, investigation of the isoAsp role in early stages of AD is warranted. And enrollment of different neurodegenerative disease cohorts will illustrate if isoAsp is AD-specific or universal to diseases related to aging.

Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins mostly as a result of spontaneous deamidation of asparaginyl residues. An association has been found between isoAsp in human serum albumin (HSA) and Alzheimer’s disease (AD). Here we report on a novel monoclonal antibody (mAb) 1A3 with excellent specificity to isoAsp in the functionally important domain of HSA. Based on 1A3 mAb, an indirect enzyme-linked immunosorbent assay (ELISA) was developed, and the isoAsp occupancy in 100 healthy plasma samples was quantified for the first time, providing the average value of (0.74 ± 0.13)%. These results suggest potential of isoAsp measurements for supplementary AD diagnostics as well as for assessing the freshness of stored donor blood and its suitability for transfusion.