Sehr P, Joseph G, Genth H, Just I, Pick E, Aktories K
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Germany.
Biochemistry. 1998 Apr 14;37(15):5296-304. doi: 10.1021/bi972592c.
We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and -37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPgammaS release from Rho proteins was increased 3-6-fold by glucosylation. ADP ribosylation decreased the rate of GTPgammaS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3-7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity ( approximately 2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminished its ability to support the activation of the superoxide-generating NADPH oxidase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxidase activation by unmodified Rac1, even when present in marked molar excess, indicating that it was incapable of competing for a common effector. The data indicate that the functional inactivation of small GTPases by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.
我们研究了艰难梭菌毒素B对RhoA、Rac1和Cdc42苏氨酸-35和-37位点的糖基化作用对核苷酸结合、GTP酶活性及效应器偶联的影响，并将这些结果与肉毒杆菌C3转移酶催化的RhoA天冬酰胺-41位点的ADP核糖基化作用进行了比较。虽然糖基化和ADP核糖基化对RhoA、Rac1和Cdc42释放GDP没有主要影响，但糖基化使Rho蛋白释放GTPγS的速率提高了3至6倍。ADP核糖基化使GTPγS释放速率降低了约50%。糖基化使GTP酶的内在活性降低了3至7倍，并完全阻断了Rho-GAP对GTP酶的刺激。相反，ADP核糖基化使GTP酶活性略有增加（约2倍），对GAP刺激GTP酶没有主要影响。虽然ADP核糖基化不影响RhoA与蛋白激酶N结合结构域的相互作用，但糖基化抑制了这种相互作用。Rac1的糖基化显著削弱了其支持吞噬细胞超氧化物生成NADPH氧化酶激活的能力。糖基化的Rac1即使以明显过量的摩尔数存在，也不会干扰未修饰的Rac1对NADPH氧化酶的激活，这表明它无法竞争共同的效应器。数据表明，小GTP酶通过糖基化的功能失活主要是由GTP酶-效应器蛋白相互作用的抑制引起的。
