ปฏิกิริยาสัมพันธ์ระหว่าง chimeric TTR กับโปรตีนใน CSF และผลกระทบต่อความสามารถของ chimeric TTR ในการยับยั้งและสลาย amyloid B

Abstract

Alzheimer's disease (AD), one of the most frequent types of amyloidosis, is commonly associates with dementia in human. The extracellular deposition as amyloid plaque and toxicity to cells of amyloid B (AB) are common causes. Thus, removal of AB is the most important therapeutic strategy for AD. Besides function as a distributor for thyroid hormones (THS) and retinol, transthyretin (TTR) contains a proteolytic activity and it is known as a major AB sequestering protein in human brain. Recently, chimeric TTRs with higher proteolytic activity than wild type TTR were constructed in our laboratory. To ensure these TTRs can be used as potent therapeutic agents, we investigated their proteolytic activities both in vitro and in vivo. In addition, to explore a possible effect of particular proteins in the CSF such as metallothinein (MT) on the degradation of AB, the experiment was also carried with the presence of the CSF protein. The recombinant chimeric TTRs including xenoN/croc TTR and pigC/croc TTR were successfully synthesized and secreted by using the heterologous gene expression system of Pichia pastoris. The purification by preparative discontinuous native-PAGE showed only a single band with a migration to the position corresponding to TTR. The synthesized TTRs contain the physicochemical characteristics similar to those observed in the native TTRs. By using FITC-casein as substrate, the results showed that crocTTR with longer N- or C-terminal sequence had higher catalytic activity than wild type crocTTR, indicating to the effect of N- and C-terminal sequences on the activity of TTR. In addition, neither the presence of metallothionein 1 nor metallothionein 2 (MT1 and MT2) changed the AB1-42 degradation activity of human TTR. On the other hand, the degradation of AB 142 by the two chimeric TTRs was enhanced with the presence of either MTI or MT2. We also determined whether the two chimeric TTRs toxic to cells and/or have the protective effect on the toxicity of AB. The cytotoxicity assay was conducted using neuroblastoma as cell target and the cell viability was determined by MTT assay. The results showed that the viability of cells with the presence of TTR was significantly higher than the control in which cell was treated with AB alone. In comparison with human TTR, crocTTR and chimeric TTR (either xenoN/crocTTR or pigC/crocTTR) showed more protective effect. However, there was no significantly difference of the effect between xenoN/crocTTR and pigC/crocTTR. This indicated to non-toxic but ability to protect cell of the chimeric TTRS.