Assay Truncation continued (BDD update 202404, Kahn 2024 #2973 LC/MS isoforms in cingulate cortex), SNCA truncation isoforms (-126/-112/-98 with theoretical MW/aa), Bluhm 2021 #2157 schematic protease cleavage map (trypsin/MMP-3/Calpain-1/Cathepsin D/Neurosin/casp1), Sorrentino 2020 schematic, binding site 102-130 abundance table
Assay Truncation (continued)
| citation / source | method | region | fraction | findings |
|---|---|---|---|---|
| Antibodies generated with epitopes of aa 1-40 and 60-95 | ||||
|
[BDD monthly update 202404] • Affinity maturation of ADx clone recognizing N-term region of α-syn was initiated. It will take approximately 2 months to have the first screening data. • Due to low affinity, affinity maturation is on-going. • MJFF and 4 commercial antibodies were selected as candidates for full-length α-syn epitope. (Yasuko Matsui) | ||||
| Randall | current standard assay; MSD assay; epitope; Washington University School of Medicine, St. Louis, MO, USA | |||
| (Kahn, 2024 #2973) Eli Lilly | LC/MS | Bilateral cortex | SDS soluble and insoluble |
FL acetylated 1-140 form is the most abundant species of α-syn in all disease cases and does not differ significantly between groups. In order of abundance: 5-140, 1-122, 1-119, 1-125, 39-140, 68-140; Found in SDS insoluble fractions: 1-119, 1-125, 1-122, 5-140, 1-140; we found evidence of ~20 modified forms, including N-terminal acetylation and C-terminal truncation at 119. We did not detect any nonacetylated N-terminal α-syn; User trend of increased α-syn levels in the LB-enriched (detergent-insoluble fraction) of PD cases vs controls; Ac-α-syn1-9, α-syn5-21, α-syn35-43, α-syn80-58, α-syn80-63 to α-syn80-90 and α-syn80-119 were significantly increased in PD coagulate region compared to controls in the Lewy body-enriched α-syn fraction. In the soluble fraction, only Ac-α-syn 1-9 was significantly increased in PD compared to controls. |
| (Bhattacharjee, 2019 #2237) | 2019 Zetterberg Henrik parallel reaction monitoring liquid chromatography-tandem mass spectrometry (PRM LC-MS/MS) | cingulate cortex (affected) and occipital cortex (unaffected) | soluble & detergent-soluble (detergent-insoluble fractions, Carrie body-) | |
| (Sorrentino 2020) | Good review | |||
Question: 어들 truncation 이 정량적으로 intracellular 단무가 양만가 또시카가? 그게 어디서 TAK-341가 자료요… 어떤 것 이런 듯 (Olga 가 CALPAIN MUF 보고 있네)
SNCA truncation isoforms
| Theoretical MW (kDa) | aa | aa positions / truncation | In SN, Wandt H #1423 | RNA in cb | RNA in cb (TAK-341 epitope cover) | Crisha-2014 #1423 | (Murtana 2012 #2304) WB | |
|---|---|---|---|---|---|---|---|---|
| 4 major isoforms | (Sorrentino 2020), it is difficult to recreate | resulting changes in α-syn properties due to the low abundance of the proteoforms in CNS, particularly in HC or HC tissue with a sphere of natural physiology of expression. (Murtana, 2014 #2304); Additionally, the majority of truncated forms often isolated from intact extracts contain formidable cellular fronts, presenting alterations in spliceosome (RNA) machinery not throughputs (purine, otravarin in disease (47); aSyn protein produced by alternative splicing (RNA: SNCA: variants 50-1 ?, 11) | ||||||
| SNCA | (no values shown) | |||||||
| SNCA-126 | lacks residues 47-54 due to loss of exon 3 | Not increased | SNCA 126 deletion in HC vs PD compared | |||||
| SNCA-112 | lacks residues 103-130 due to loss of exon 5 | 1 (but Murtana 2014 #2304 may not detected in HC or PD) | X | X | O | (In HC or PD 다 검출됨 - C-term truncated -15 kDa N-term truncated -16 kDa truncated -11 kDa N or other aberrant aSyn species in N-terminal fragments) | ||
| SNCA-98 | Lacks both exons 3 & 5 | |||||||
|
12.16 kDa (amino acids 14-133) 10.44 kDa aa6-140 7.27 kDa (72-140) (2019 Wargi cytokins) | ||||||||
proteolytic cleavage
pSyn or pSyn STAR A toxic form of pSyn 5y truncated, adamant, and reactive, pSyn, which lacks N and C terminal ends and is mistreted (Grassi et al, 2018, 2018) | ||||||||
(Bluhm, 2021 #2157)
Schematic presentation of αSyn species within the α-synuclein molecule generated by trypsin (purple), MMP-3 (blue), calpain-1 (orange), cathepsin D (red), and neurosin (orange). Cathepsin D produces the formation of t-N40s and t-N40s α-synuclein fragments NfM/NS specifically.
trypsin, MMP-3, CALPAIN-1, Cathepsin D, Neurosin and casp1 → aggregation
(Sorrentino 2020) RKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAOKTVE α-syn schematic
Various truncation products (including the ones missing 96-140 epitopes) identified in both soluble and insoluble protein fractions
Unknown what species are present in the CSF
Table Z dependence of derived from from the SDS-soluble fraction of human aSyn isoforms 가게 specifies fewer have noted
(4.1 binding site 102-130) — abundance table
| isoform | HC | PD | PD+AD | Averaged for detection events only and normalized to Ac 1-140 response | significant | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| T-119 | 0.18 ± 0.04 | 0.16 ± 0.05 | 10/10 | 0.26 ± 0.20 | 4/6 | 0.21 | 1 | No | ||
| Ac 1-122 (deamidation N122) | 0.29 ± 0.16 | 0.19 ± 0.05 | 10/10 | 0.21 ± 0.05 | 6/6 | 0.4 | 1 | No | ||
| Ac 1-125 | 0.24 ± 0.05 | 0.21 ± 0.07 | 10/10 | 0.21 ± 0.03 | 6/6 | 0.81 | 1 | No | ||
| Ac 1-125 (alt) | 0.21 ± 0.04 | 0.17 ± 0.02 | 16/16 | 0.19 ± 0.03 | ||||||
| 5-140 | 0.43 ± 0.05 | 0.31 ± 0.40 | 11/16 | 0.04 ± 0.02 | 2/10 | 0.013 | 0.65 | Yes | ||
| Ac 1-140 | 1 | 16/16 | 1 | 10/10 | 1 | 6/6 | 0.37 | 1 | No | |
Total enough detection events for valid statistical comparison
HC = Normal Control; PD = Parkinson’s Disease; PD + AD = Parkinson’s Disease with Alzheimer’s Disease