整合蛋白們（integrins）是一群位於細胞表面的、異質雙體，其功能經啟動訊息傳遞而與細胞的生長、死亡、黏著、移動等有關。截至目前為止，哺乳類中已發現有18種α次單元及8種β次單元經非共價鍵組成24種不同的異質雙體。整合蛋白常
藉由配體 (ligand)上的RGD (Arg-Gly-Asp)序列能與配體做結合，而RGD序列也存在許多蛇毒的去整合蛋白中，這些去整合蛋白能與整合蛋白作結合、進而抑制其功能；由於相較於胞外基質，這些去整合蛋白對整合蛋白有較佳的結合能力，因此可用來治療整合蛋白造成的相關疾病且被認為是具發展潛力的整合蛋白結抗劑。之前研究指出去整合蛋白與整合蛋白的結合能力會受到RGD區域周圍兩側胺基酸及C端區域組成不同所影響。此外，最近有研究指出去整合蛋白elegantin的連接區域 (41KKKR45T)能取代Fibronectin的Synergy site進而增加與整合蛋白 51的結合。在本實驗中，利用擁有不同連接區域序列的去整合蛋白Rhodostomin (39SRAG43K)及Trimucin (41KKKR45T) 探討連接區域對於整合蛋白 IIb3、V3、51辨識所扮演的角色。本實驗中利用Pichia pastoris系統表現表現出十六株Rho及一株Tmu的突變蛋白，並均質純化。利用血小板凝集抑制試驗及細胞黏著抑制實驗作為這些序列的功能性分析。我們發現當去整合蛋白中連接區域的序列是39KKKR43T時對整合蛋白IIb3、V3、51的活性分別有0.8-3.0、3.0-12.0、3.0-5.0倍的增加，而增加情形不一致，可能與39KKKR43T linker region 會與RGD loop、C端區域有合作關係、進而影響整個去整合蛋白與整合蛋白的結合有關。這樣的結果由Docking結構方法推測可能是由於這些帶正電的連接區域序列39KKKR43T 可能與帶負電的次單元上鄰近金屬依賴型的附著部位作用，進而經由丙胺酸突變分析發現K40A及T43A突變株對整合蛋白IIb3、V3、51的活性分別有2.0、5.0、5.0倍及2.5、5.0、5.0倍的下降，另外K39A突變株則造成對整合蛋白V3、51的活性分別有2.0、5.0倍的下降；而41K及42R的突變則無造成太大影響，顯示39K 、40K與43T可能是參與整合蛋白結合的重要胺基酸。另一方面，我們也發現I47R突變株包含連接區域序列39KKKR43T時會幫助去整合蛋白對整合蛋白51的活性有5.0-7.0倍的上升，若配合連接區域序列39SRAG43K則對整合蛋白IIb3、V3、51的活性分別有2.3、9.2、24.0倍的增加。分析不同去整合蛋白的連接區域序列（39IEEG43T、39KGAG43K、39LKEG43T及 39MKKG43T）發現連接區域序列對整合蛋白的辨識的確扮演重要角色。綜合而言，Rho突變蛋白包含序列39KKKR43T-48PRGDM53P-67Y68H、39MKKG43T-48ARGDN53P-67NGLY71G 及39KAKR43A-48ARGDN53P-67NGLY71 G可以特異性地抑制表現整合蛋白V3細胞的黏結；而含有序列39KKKRTICR47 I ARGDN53P-67NGLY71G39、
39KKKRTICR47RARGDN53P-67NGLY71G 39及SRAGKICR47RARGDN53P-67NGLY71G的突變蛋白則同時對整合蛋白V3及51有特異性。這些結果將來可作為我們探索整合蛋白與去整合蛋白複合體間的結構與活性關係的基礎，並可以幫助整合蛋白相關的藥物設計。

Integrins are a family of heterodimeric receptors, which modulate many cellular processes including growth, death, adhesion, migration, and invasion by activating several signaling pathways. Until now, 18α and 8β subunits are known in mammals to noncovalently associate and form 24 integrin heterodimers. Integrin-binding site, the RGD (arginine-glycine-aspartate) motif, is found from several important extracellular matrix proteins, which serve as adhesive integrin ligands. The RGD motif has also been found from snake venom toxins, which inhibit integrin-binding function and serve as potent integrin antagonists. Many of these proteins are potential therapeutic agents in the treatment of integrin-realted diseases because they have higher affinity than extracellular matrix proteins. Although the RGD motif is crucial for their binding to integrins, the selectivity depends on the composition of the amino acid flanking the RGD motif and C-terminal region. Recently, a region (41KKKR45T) of the disintegrin elegantin termed the “linker region” has been shown to exhibit the inhibitory activity against the synergy site of fibronectin in promoting α5β1 integrin-mediated cell adhesion. In this study we used rhodostomin (Rho), a disintegrin with a 39SRAG43K linker sequence, and trimucin (Tmu), a disintegrin with a 41KKKR45T linker sequence, as the scaffolds to study the role of different linker regions in recognizing integrins α5β1, αIIbβ3, and αvβ3. I have expressed sixteen Rho mutant proteins and one Tmu mutant protein and purified them to homogeneity. Analysis of platelet aggregation and cell adhesion assays showed that disintegrins with 41KKKR45T linker sequence exhibited 0.8-3, 3-12, and 3-5 folds increases in inhibitory activity to integrins IIb3, v3, and 51, respectively. This is consistent with our docking structure of the Rho-integrin complex that the linker region may interact with an adjacent metal-ion-dependent adhesion site of subunit through ionic interaction. Using alanine scanning mutation on the 39KKKR43T region, we found that the K40A, and T43A mutants caused 2-, 5-, and 5-, and 2.5-, 5-, and 5-folds decreases in inhibitory activity to integrins IIb3, v3, and 51, respectively. The K39A also caused 2- and 5-folds decreases in inhibitory activity to integrins v3 and 51. In contrast, the mutations on 41K and 42R have little effect on their inhibitory activity, suggesting that the 39K, 40K, and 43T residues in linker region of disintegrin may interact with integrins. We also found that the I47R mutant with 39KKKR43T exhibited 5.0-7.0-folds increases in inhibiting integrin 51, and the I47R mutant with 39SRAG43K exhibited 2.3-, 9.2-, and 24-folds-increase in inhibitory activity to integrins IIb3, v3, and 51, respectively. The analysis of the mutants containging the linker region sequences (39IEEG43T, 39KGAG43K, 39LKEG43T, and 39MKKG43T) from other disintegrins showed that the linker region sequences play an important role in their binding to integrins. Taken together, Rho mutants containing 39KKKR43T-48PRGDM53P-67Y68H, 39MKKG43T-48ARGDN53P-
67NGLY71G, and 39KAKR43A-48ARGDN53P-67NGLY71G have higher affinity for integrins V3. In addition, the mutants containing 39KKKRTICR47IARGDN53P-67NGLY71G, 39KKKRTICR47RARGD N53P-67NGLY71G, and 39SRAGKICR47RARGDN53P-67NGLY71G have higher activitiy in inhibiting integrins 51 and V3. The results of this study will serve as the basis to design potent integrins-specific disintegrins.

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