腺嘌呤核苷二磷酸核糖化相似因子4A (ARL4A)與其結合蛋白GCC185之特性探討

Abstract

腺嘌呤核苷二磷酸核糖化相似因子4A (ARL4A) 是一個22 kDa GTPase，多量存在於青春期的老鼠與成鼠，先前的研究指出在胚胎發育時ARL4A mRNA 的出現時間與體節形成與腦部分區發生的時間相當。為了找出ARL4A可能的結合蛋白，我運用酵母菌雙雜交系統(Yeast Two-Hybrid System)以ARL4A-Q79L為誘餌來篩選人類胚胎時期腦的cDNA library。 我選擇GCC185來進行後續的研究，在酵母菌雙雜交系統，GCC185 (a.a. 500-733) 會與野生型的ARL4A與ARL4A-Q79L有交互作用，Pull down assay顯示，GCC185 (a.a. 500-733)比較偏好與MBP-ARL4A-Q79L-His結合。我們也製備抗GCC185的抗體，其所辨認的於TGN位置的訊號與p230相近，而在GCC185降解(knockdown)的COS細胞所作的免疫染色也顯示出此抗體能辨認內生的GCC185。 GCC185參與志賀毒素(Shiga Toxin)與甘露糖-6-磷酸受器(Mannose 6-phosphate receptor)的胞內運輸，為研究ARL4A與GCC185結合的生物意義，我想評估變異型的ARL4A是否會影響志賀毒素(Shiga Toxin)與甘露糖-6-磷酸受器(Mannose 6-phosphate receptor)的胞內運輸，我純化志賀毒素並將之接上Cy3螢光分子以進行志賀毒素胞內運輸的實驗，初步實驗結果並不顯示表現變異型的ARL4A會影響志賀毒素(Shiga Toxin)與甘露糖-6-磷酸受器(Mannose 6-phosphate receptor)的胞內運輸，但是，未來關於以RNAi降解ARL4A的實驗將會提供明確的結論。

ARL4A is a 22 kDa GTPase which showed to be abundant in testes of pubertal and adult rodents. In addition, previous study also showed that the appearance of mouse ARL4A mRNA during embryonic development coincided temporally with the sequential formation of somites and the establishment of brain compartmentation (Lin et al., 2000). To identify potential ARL4A effectors, Yeast Two Hybrid Screening was conducted. I used pBTM116-ARL4A-Q79L as a bait in a yeast-two hybrid screen of human fetal brain cDNA library. A putative effector of ARL4A, golgin GCC185, was chosen for further study. Yeast two-hybrid analysis showed that GCC185 (a.a. 500-733) interacted with wild type ARL4A and ARL4A-Q79L, but not with ARL4A-T34N. Pull down assay demonstrated preferential binding of GCC185 (a.a. 500-733) to MBP-ARL4A-Q79L-His. Polyclonal antisera against GCC185 were raised, and the data of immunostaining using the antisera suggested the antisera can recognize endogenous GCC185. Putative endogenous GCC185 signal recognized by the antisera was supported by the data that this signal colocalized with trans Golgi marker p230/Golgin245. By immunostaining of COS cells depleted of GCC185, a diminish of the TGN signal can be observed using the antiserum, further supporting that the antiserum can recognize endogenous GCC185. GCC185 was known to be involved in retrograde transport of Shiga Toxin subunit B (STxB) and mannose 6-phosphate receptors (MPRs). To investigate biological relevance of the interaction between ARL4A and GCC185, we attempted to evaluate whether mutant ARL4A could affect the retrograde transport of STxB and MPRs. I purified STxB and conjugated it with fluorescent dye Cy3 in order to study STxB internalization. Preliminary data did not suggest overexpression of ARL4A mutant could affect the STxB internalization and retrograde transport of MPRs. The experiments on RNAi gene silencing of ARL4A will provide a more definite conclusion.