甘藷塊根質體型澱粉磷解酶所形成之蛋白質複合體之鑑定與功能研究

Abstract

蛋白質的轉譯後修飾調控了許多重要的生理機制。早期研究顯示質體型澱粉磷解酶 (L-SP) 可能受到蛋白質降解性後修飾作用所調控。本實驗室在純化L-SP的過程中，發現一高分子量、且具有L-SP活性之色帶 (簡稱HX)。本論文進一步利用免疫共沉澱與免疫螢光組織定位等方法，證明HX由L-SP與20S proteasome所組成。並且在45°C熱處理下，HX會立即消失，可觀察到L-SP隨著熱處理的時間增加，發生階段性降解的現象，這個降解作用可隨著20 proteasome的活性受到proteasome專一性抑制劑 (MG132) 的抑制，而減緩L-SP之降解，顯示20S proteasome可能參與此降解作用。以酵素動力學分析降解前後L-SP之生化性質差異，發現降解後的L-SP對於Glc-1-P的親和力下降，進而降低澱粉合成方向的活性。因此我們推測20S proteasome可能會受到熱逆境的刺激，進而以降解機制修飾L-SP，來調控L-SP催化方向之活性。 另一方面，以L-SP單株抗體進行免疫共沉澱時，意外地發現一個分子量約65 kDa的蛋白質可能也與L-SP互相結合；經LC-MS/MS定序，此蛋白質為DPE1 (D-enzyme, disproportionating enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25)。DPE1催化可逆性的 alpha-1,4鏈結葡聚醣之裂解與轉移反應，改變寡糖之鏈長分布。過去的研究顯示，在E coli中，malQ (DPE1同源基因) 和malP (L-SP同源基因) 位於相同的malA操作組，故推測兩者有類似的功能，可能共同作用。進一步，本論文以二維電泳 (native PAGE/SDS-PAGE)、GST pull-down assay、以及FRET-confocal microscopy為工具，證明L-SP與DPE1互相結合，形成蛋白質複合體 (SP-DPE complexes)。此外，膠體過濾法與二維電泳之結果顯示，SP-DPE complexes可能是由四個L-SP單元體與四個DPE1單元體，結合為一個分子量約為700 kDa之蛋白質複合體。以酵素動力學比較SP-DPE complexes與DPE1之間的差異，顯示SP-DPE complexes對於麥芽三糖 (maltotriose) 具有較高的親和力，而對於麥芽四糖 (maltotetraose) 則有較高的催化效率；而在SP-DPE complexes的酵素催化作用中，則觀察到基質快速轉移的現象。另外SP-DPE complexes在直徑15-20 mm大小之甘藷塊根中含量最多，顯示其與澱粉快速累積有重要的關聯性。這部份的結果顯示，在甘藷塊根的造粉體中，L-SP與DPE1可能形成蛋白質複合體，以幫助澱粉的快速累積，其生理作用可能扮演有效地回收再利用短鏈麥芽寡糖，或直接作用在短鏈分支之澱粉結構中，正確決定澱粉的結構。

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidal starch phosphorylase (L-SP) may be regulated by proteolytic modification. During the purification of L-SP from sweet potato roots, an unknown high molecular weight complex (HX) showing L-SP activity was constantly observed. Its mobility was significantly slower than the typical L-SP on native PAGE. We utilized mass spectrometry, coimmunoprecipitation, Ouchterlony double immunodiffusion, two-dimensional gel electrophoresis, and confocal microscopy as tools to demonstrate that HX was composed of L-SP and the 20S proteasome. Furthermore, we found that the amount of HX decreased immediately after 45°C heat treatment, which caused stepwise degradation of L-SP in a time-dependent mode. This degradation process was strongly inhibited by MG132, suggesting that the 20S proteasome might be involved in L-SP degradation. In addition, kinetic studies indicated that the proteolytic modification of L-SP caused it to decrease the binding affinity toward Glc-1-P and subsequently reduced its starch-synthesizing activity. This work demonstrates the role of the 20S proteasome as a regulator of L-SP activity, which may be controlled by stressful condition. On the other hand, immunoprecipitation experiments with L-SP mAbs showed that another protein might associate with L-SP. This protein was identified as DPE1 (D-enzyme, disproportionating enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25) by LC/MS/MS. DPE1 catalyses the cleavage and transfer reactions involving alpha-1,4 linked glucans and alters the chain length distribution of oligosaccharides. Previous studies suggested that DPE1 might work in conjunction with L-SP. Furthermore, we utilized 2-DE (native PAGE/SDS-PAGE), GST pull-down assay, and confocal microscopy as tools to demonstrate that L-SP might interact with DPE1, suggesting that these enzymes may form protein complexes (SP-DPE complexes). The results from gel-filtration chromatography and 2-DE (native PAGE/SDS-PAGE) indicated that SP-DPE complexes might be composed of four L-SP subunits and four DPE1 subunits with molecular weight around 700 kDa. In addition, protein complex forms of DPE1 showed a higher affinity toward maltotriose and a higher catalytic activity toward maltotetraose than DPE1 monomers. The efficient passage of the product of one enzyme to the next enzyme in SP-DPE complexes was also be observed. Moreover, the protein levels of SP-DPE complexes were shown to become higher in the middle stage of sweet potato root development where starch accumulated fast. These results suggest that SP-DPE complexes may either efficiently recycle short chain malto-oligosaccharides to produce Glc-1-P for starch synthesis, or may specifically edit short-chain amylopectin, thus resulting in the formation of correct starch structure.