The Role Of Transthyretin Modifition In Senile System Amyloidosis Formation Mechanism

Objective:Transthyretin(TTR), a protein synthesized and secreted by liver and choroid plexus that functioning in transportation of thyroxine and retinol, was proved to be the amyloid precursor protein in senile systemic amyloidosis(SSA) and familial amyloidotic polyneuropathy(FAP). It was demonstrated that amyloid content was composed of non-branched fiber. Nevertheless, it was still unclear how amyloid precursor protein was polymerized into amyloid fiber and deposited in targeted organs. The aim of this study, through the relation between TTR chemical modification and amyloid fiber forming, investigate the role of TTR protein metabolism in SSA amyloidosis formation.Methods:(1) According to the SSA diagnostic criteria, collected suspected4SSA patients, TTR gene mutation was analyzed by TTR exon sequencing after PCR amplification with specific primers designed from TTR gene bank. Serum was collected from those patients whose TTR gene was non-mutated, then subjected to TTR separation.(2) Using of ProteomeLab PF-2D system, protein was collected from elution of Proteomelab PF-2D systemin different pH, with elution buffer pH ranging from4to8, an increase of0.3pH as an unit.(3) Protein concentration was measured by Bicinchoninic Acid method.(4) Thioflavin T(ThT), a fluorescein which can specially combined with amyloid contents, was used to measure each eluted part of TTR fluorescence intensity.(5) Collected TTR was detected by Dot blot with anti-human TTR antibody.(6) Ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry(UPLC/Q-TOF MS) was used to analyze the TTR metabolism modification in each eluted part.Results:(1) TTR gene in all of SSA patients was wild type as shown by PCR amplification and DNA sequencing.(2) Serum proteins were separated to46parts by Proteomelab PF-2D system, applying a continuous pH gradient elution, in which there were3main protein peaks in both the SSA patients and healthy control groups.(3) BCA quantitative analysis found no significant differences between the two groups.(4) In ThT fluorescence assay, there was greatly increased fluorescence intensity in pH7.8-6.7and pH5.6-4.8part,in the same time, TTR immune reaction is positive indicated the existence of amyloidosis fluorescent component.(5) Using Dot blot identified the scope of the TTR protein, it was found that TTR from SSA patients was mainly in pH7.83-7.61,but in the control group was pI7.95-7.72, suggesting that the chemical modification differences of TTR protein between them.(6) Ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry(UPLC/Q-TOF MS) analyzed the TTR chemical modification found that the modification type of SSA patients was significantly reduced which included Dehydration ST、Phosphoryl STY、Hydroxyl DKNP、Carbamidomethyl C、 Phosphopantetheine S andO-GlcNac ST.Conclusions:(1) Firstly demonstrated PF-2D used to separate and enrich TTR from SSA patients was established, based on pH gradient,building models for TTR related amyloidsis, without impact the structure of amyloidosis fiber, lay the foundation for further study of the metabolism modification of TTR protein and amyloidosis fiber.(2) The modification type of SSA patients was significantly reduced (Dehydration ST、Phosphoryl STY、Hydroxyl DKNP、Carbamidomethyl C、Phosphopantetheine S andO-GlcNac ST) which suggested the reduction of TTR chemical modification lead to TTR molecule surface polar molecules reduced, increasing hydrophobic property, enhanced TTR intermolecular polymerization ability, maybe have a correlation with SSA amyloidosis formation.