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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/3753


    Title: 鑑定第四型與第九型雙胜?蛋白水解?之生物化學特性;Biochemical Characterization of Dipeptidyl Peptidase IV (DPP-IV) and DPP9
    Authors: 唐弘寬;Hung-Kuan Tang
    Contributors: 化學工程與材料工程研究所
    Keywords: 第四型雙胜肽蛋白水解酶;生物化學;第二型糖尿病;脯胺酸雙胜肽蛋白水解酶;蛋白質結構;DPP-IV;DPP8;DPP9;protein structure;type II diabetes;toxicology
    Date: 2009-07-16
    Issue Date: 2009-09-21 12:21:46 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 脯胺酸雙胜肽蛋白水解酶 (prolyl dipeptidase) 家族,具有從胜肽上移除N端第二個脯胺酸殘基的功能。家族成員包括有第二型、第四型、第八型、第九型雙胜肽蛋白水解酶 [DPPII (E.C. 3.4.14.2)、DPP-IV (EC 3.4.14.5)、DPP8及DPP9] 及纖維母細胞激活蛋白(fibroblast activation protein,FAP)。其中第四型雙胜肽蛋白水解酶已有廣泛的研究。然而,其餘的家族成員之生物功能至今仍尚未被解析。其中,第八型及第九型雙胜肽蛋白水解酶在其胺基酸序列有58%相同。 首先,本論文著重於鑑定第九型雙胜肽蛋白水解酶的生物化學特性,包括其酵素活性、四級結構、受質特性選擇 (substrate profile) 及酸鹼值最適範圍。第九型雙胜肽蛋白水解酶的表現是利用昆蟲細胞表現系統,在Strep?TactinTM的純化系統中純化。在此表現系統中,我們所得到的產率相較於文獻中所發表的數值,有約100倍的提升。生物化學鑑定的結果顯示,第九型雙胜肽蛋白水解酶在其酵素活性、受質特性選擇和酸鹼值最適範圍,皆類似於第八型雙胜肽蛋白水解酶。且其四級結構皆為雙聚體 (homodimer)。第九型雙胜肽蛋白水解酶位於雙聚體組合的接合面 (dimer interface) 之定點突變株 (F842A),經表現純化後,發現其四級結構亦為二聚體,但其酵素活性相較於野生型減低了約300倍。根據我們實驗室先前的研究成果,這結果顯示出第九型雙胜肽蛋白水解酶的二聚化(dimerization) 的形式是近似於第八型雙胜肽蛋白水解酶。因此,第八型及第九型雙胜肽蛋白水解酶的生物化學特性是相當類似。我們推論第八型及第九型雙胜肽蛋白水解酶在生物體內可能扮演著同樣的角色。 另外,在纖維母細胞、表皮細胞及血球細胞中,我們發現第八型及第九型雙胜肽蛋白水解酶皆有表現,且相互之間的表現量並無顯著差異。而且,和文獻中的所發表的不同的地方在於,我們的研究發現第八型與第九型雙胜肽蛋白水解酶的表現,在T細胞活化前與活化後,其表現量沒有顯著的差異。 第四型雙胜肽蛋白水解酶為第二型糖尿病之藥物標的(drug target)。文獻中指出,藥物在抑制第四型雙胜肽蛋白水解酶時,若有第八型及第九型雙胜肽蛋白水解酶的酵素活性同時被抑制,會有動物體內毒性的問題。然而,目前並無確切的證據,指示出第八型及第九型雙胜肽蛋白水解酶的抑制為導致動物體內毒性的原因。我們利用對第八型及第九型雙胜肽蛋白水解酶具有高度選擇性的抑制劑 (名為1G244),於大鼠 (Sprague-Dawley rat) 內進行實驗。結果顯示,1G244的Ki值對於第八型雙胜肽蛋白水解酶,相較於文獻中所使用的結抗劑 (DPP8/9 selective),其值小了15倍;而對於第九型雙胜肽蛋白水解酶,1G244的Ki值相較於DPP8/9 selective小了8倍。顯示出1G244較DPP8/9 selective有更強的抑制效果。另外,我們的結果發現,1G244很快的被細胞所吸收。相對的,DPP8/9 selective幾乎無法進入細胞。在動物實驗中,實驗結果指出1G244在大鼠以靜脈注射的方式給藥14天,經檢測血液學及血清學的參數後,僅發現輕微的副作用。綜合以上結果,顯示出第八型及第九型雙胜肽蛋白水解酶的抑制,對動物並不會產生嚴重的毒性。而先前文獻中的結果,可能是由於所謂的”off-target”的現象。因為所使用的結抗劑 (DPP8/9 selective) 無法進入細胞,進而達到抑制第八型及第九型雙胜肽蛋白水解酶。而其所觀察到的毒性反應,很可能是因為抑制了其它未知的酵素或蛋白質所導致。 最後,我們研究propeller環,對第四型與第九型雙胜肽蛋白水解酶的活性及結構上所扮演的角色。Propeller環為propeller功能性區塊上所延伸出來的小片段,座落在雙聚體組合的接合面 (dimer interface) 上。對脯胺酸雙胜肽水解酶而言,propeller環所扮演的角色至今仍未明瞭。由於第九型雙胜肽蛋白水解酶的結構至今仍未被解析,我們根據其胺基酸序列和其它脯胺酸雙胜肽水解酶的序列排比,以及文獻中與第八型雙胜肽水解酶的電腦模擬,推估其propeller環的位置。我們針對酵素演化保留殘基 (conserved residue) 做四個定點突變,包括V319A、E325A、K328A及Y334A,以及將propeller環去除的突變 (DEL)。經由表現及純化後,結果發現在V319A、E325A及K328A的突變株中,酵素活性及雙聚體的四級結構皆和野生型的第九型雙胜肽水解酶相似。相對的,Y334A及DEL的突變株中,其酵素活性相較於野生型有所減低 (動力學常數kcat值下降,Km值上升)。 而對第四型雙胜肽水解酶,propeller環相較於第九型雙胜肽水解酶,對其酵素活性及四級結構有不同的影響。根據解析出的結構,我們將propeller環分為兩個部份探討:intermolecular及intramolecular interaction,其中Y248為intermolecular interaction,而Y238及Y256為intramolecular interaction。在intermolecular interaction方面,我們將Y248做三個定點突變,包含Y248F、Y248T及Y248A,以及將propeller環去除的突變 (Del)。結果顯示出Y248上的phenyl group對雙聚體的形成相當重要。在沒有phenyl group的突變株中,包括Y248T、Y248A及Del,四級結構皆為單聚體而非雙聚體,且活性相較於雙聚體有下降的情形,或無法測得。其中Y248A及Del的單聚體其活性無法測得,而Y248T的雙聚體其動力學常數kcat值下降,Km值不變。不同的Km值影響效應,顯示出hydroxyl group可能對第四型雙胜肽蛋白水解酶與受質的黏合,扮演重要的角色。在intramolecular interaction方面,Y238A及Y256A的突變株同時有雙聚體及單聚體產生。值得一提的是,其單聚體的酵素活性和雙聚體相似。此一結果和我們先前所發表的結果不同。先前的研究中皆指出單聚體的第四型雙胜肽蛋白水解酶,酵素活性相較於雙聚體,都有下降的情形。未來,我們會針對有活性的單聚體,深入的了解其機轉。總而言之,我們的結果顯示出propeller環,對第四型及第九型雙胜肽蛋白水解酶的結構及酵素活性有著不同的影響。了解propeller環對脯胺酸雙胜肽蛋白水解酶家族的結構及酵素活性的影響,將會幫助了解脯胺酸雙胜肽蛋白水解酶在biogenesis及雙聚體的形成。 The family of prolyl dipeptidases has attracted extensive investigation in recent years because of their unique ability to cleave the peptide bond after a penultimate proline residue. It includes dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), FAP (fibroblast activation protein), DPP2 (E.C. 3.4.14.2), DPP8 and DPP9. DPP-IV is the most extensively studied, and the functions for other members are not known so far. DPP9 and DPP8 are highly homologous proteases with 58% sequence identity at the amino acid level. Both the structure and function of these two proteases are not known. In this thesis, we first characterized the biochemical property of DPP9 including its enzymatic activity, quaternary structure, substrate specificity and pH optimum. DPP9 was expressed and purified from the baculovirus-infected insect cells using Strep?TactinTM purification system. The yield is significantly higher than what was reported in the literature. DPP9 has similar enzymatic activity, substrate specificity and pH optimum as DPP8. Both of them are homodimeric. Single site mutation at the C-terminal loop (F842A), one of the dimer interfaces, results in dimeric DPP9 with little enzymatic activity. The results indicate that the interaction mode of dimerization is similar to that of DPP8, reported previously from our lab. Therefore, the biochemical property of DPP9 we discovered so far is almost identical to that of DPP8. We speculate that DPP9 and DPP8 carry out overlapping functions in vivo. We have also determined the expression profile of DPP9. DPP9 is ubiquitously expressed in different cell types including fibroblasts, epithelial and blood cells. Surprisingly, contrary to previous report, we found that the expression levels of DPP8 and DPP9 do not change upon the activation of T-cells such as PBMC and Jurkat cells. Because DPP8 and DPP9 are ubiquitously expressed, whether they involve in immunological function as speculated awaits further studies. The characterization of DPP9 reported here lays the foundation for revealing its function in the future DPP-IV is a validated drug target for human type II diabetes. DPP-IV inhibitors without DPP8/9 inhibitory activity have been sought because a possible association was reported between a “DPP8/9 inhibitor” and severe toxicity in animals. However, at present, it is not known whether the observed toxicity is associated with DPP8/9 inhibition, or an off-target effect induced by the compound. We investigated whether the inhibition of DPP8/9 is the cause of the severe toxicity in animals using a very potent and selective DPP8/9 inhibitor with different pharmacophore, 1G244. By Ki measurement, 1G244 is 15- and 8-fold more potent against DPP8 and DPP9, respectively, than the “DPP8/9 inhibitor”. Strikingly, the “DPP8/9 inhibitor” does not penetrate the plasma membrane but remains outside the cells, whereas 1G244 readily enters the cells, even at low doses. By repeatedly exposing Strague-Dawley rats to 1G244 by intravenous injection for a period of 14 days, we observed no significant toxicological symptoms associated with 1G244. Blood and serum chemistry parameters were all within the normal ranges for the treated animals. Because of the high potency, good membrane penetration and adequate tissue distribution of 1G244, the mild symptoms observed are probably associated with DPP8/9 inhibition. Our results demonstrate that there is no direct causal effect of DPP8/9 inhibition with toxicity in animals. Finally, we have examined the contribution of the propeller loop to the enzymatic activity and quaternary structure of DPP9 and DPP-IV. The propeller loop is one of the two dimer interfaces and its function for the structure and activity of prolyl dipeptidase family is not known. Because the crystal structure of DPP9 has not been resolved, we have identified the sequence corresponding to the propeller loop of DPP9 based on the sequence alignment with other members of prolyl dipeptidases, and computer modeling of DPP8 reported in the literature. The conserved residues or the corresponding residues whose mutations have drastic effects on DPP-IV structure and activity were chosen to be mutated. The mutant proteins were expressed and purified from insect cells. For DPP9, five mutations located on the propeller loop were generated, which include a complete deletion of the propeller loop (DEL), V319A, E325A, K328A and Y334A. Among them, the dimeric structure and enzymatic activity of V319A, E325A and K328A mutant proteins were similar to those of wild type DPP9. In contrast, Y334A and DEL fail to disrupt DPP9 dimers to monomers. However, the mutant dimers are inactive with kcat significantly decreased and Km increased. Interestingly, differential effects on the structure and activity of DPP-IV were discovered with mutations on the propeller loop. Based on the crystal structure of DPP-IV, we have identified two groups of residues on the propeller loop that are involved in inter-molecular and intra-molecular interactions, Y248 involved in intermolecular interaction, and Y238 and Y256 involved in intramolecular interaction. We have introduced single site mutation to these residues resulting in Y248F, Y248A, Y248T, Y238A and Y256A, respectively. We also generated a deletion mutation, called Del, by deleting the whole propeller loop. We demonstrated that phenyl group of Y248 is essential for dimer formation. Lack of phenyl group, such as Y248T, Y248A and Del, results in monomeric DPP-IVs with very low or no activities. Specifically, Y248A and deletion mutants result in monomers with no activity detectable, while monomeric Y248T has a low kcat and an unchanged Km. Difference on Km effects suggests that the hydroxyl group might be important for the integrity of the substrate binding pocket. Y238A and Y256A mutations result in a mixture of monomers and dimers. Intriguingly, the monomers of Y238A and Y256A were fully active as the dimers. This is drastically different from all the monomeric mutations we generated previously. Further work will be required to fully understand the underlying mechanism of these active monomeric DPP-IVs. In summary, our results demonstrate that the propeller loop exerts differential effect on the structure and activity of DPP-IV and DPP9. Understanding how propeller loop affects the structure and activity of prolyl dipeptidases will help the understanding of the biogenesis and folding of homomeric proteins in the future.
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