探討Rhodostomin 及Trimucin 中連接區域對於整合蛋白辨識所扮演的角色

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.