| 1. |
- Wang, Jijing
(författare)
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Fighting isoaspartate
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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).
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| 2. |
- Wang, Jijing, et al.
(författare)
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First Experimental Evidence for Reversibility of Ammonia Loss from Asparagine
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:15
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Tidskriftsartikel (refereegranskat)abstract
- Ammonia loss from (L)-asparaginyls is a nonenzymatic reaction spontaneously occurring in all proteins and eventually resulting in damaging isoaspartate residues that hamper protein function and induce proteinopathy related to aging. Here, we discuss theoretical considerations supporting the possibility of a full repair reaction and present the first experimental evidence of its existence. If confirmed, the true repair of (L)-asparaginyl deamidation could open new avenues for preventing aging and neurodegenerative diseases.
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| 3. |
- Wang, Jijing, et al.
(författare)
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First Immunoassay for Measuring Isoaspartate in Human Serum Albumin
- 2021
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 26:21
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Wang, Jijing, et al.
(författare)
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SpotLight Proteomics Identifies Variable Sequences of Blood Antibodies Specific Against Deamidated Human Serum Albumin
- 2023
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Ingår i: Molecular & Cellular Proteomics. - : Elsevier. - 1535-9476 .- 1535-9484. ; 22:7
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Tidskriftsartikel (refereegranskat)abstract
- Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in the blood of patients with Alzheimer's disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.
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| 5. |
- Wang, Jijing, et al.
(författare)
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Testing the link between isoaspartate and Alzheimer's disease etiology
- 2023
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Ingår i: Alzheimer's & Dementia. - : John Wiley & Sons. - 1552-5260 .- 1552-5279. ; 19:4, s. 1491-1502
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Tidskriftsartikel (refereegranskat)abstract
- Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts protein structures, making them prone to aggregation. Here we strengthened the link between isoAsp and Alzheimer's disease (AD) by novel approaches to isoAsp analysis in human serum albumin (HSA), the most abundant blood protein and a major carrier of amyloid beta (A beta) and phosphorylated tau (p-tau) in blood. We discovered a reduced amount of anti-isoAsp antibodies (P < 0.0001), an elevated isoAsp level in HSA (P < 0.001), more HSA aggregates (P < 0.0001), and increased levels of free A beta (P < 0.01) in AD blood compared to controls. We also found that deamidation significantly reduces HSA capacity to bind with A beta and p-tau (P < 0.05). These suggest the presence in AD of a bottleneck in clearance of A beta and p-tau, leading to their increased concentrations in the brain and facilitating their aggregations there.
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