Frankel A E, Burbage C, Fu T, Tagge E, Chandler J, Willingham M C
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Biochemistry. 1996 Nov 26;35(47):14749-56. doi: 10.1021/bi960798s.
Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, consists of a cell binding lectin subunit (RTB) disulfide linked to an rRNA N-glycosidase protein synthesis inactivating subunit (RTA). While X-ray crystallography and equilibrium dialysis suggested two sugar-combining sites located in subdomains 1 alpha and 2 gamma, biochemical and mutational analyses suggested the existence of a third lectin site. We performed oligonucleotide-directed mutagenesis on RTB cDNA to create mutants with modifications in subdomains 1 alpha, 2 gamma, and either 1 beta or 2 alpha. The triple-site mutant RTBs were expressed in insect cells. Partially purified recombinant proteins obtained from infected cell extracts and cell supernatants were characterized for asialofetuin and cell binding, immunoreactivites, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Yields of both triple-site mutants were similar to the parent double-site mutant. Both mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. The triple-site mutant with modification of amino acid residues in subdomains 1 alpha, 2 alpha, and 2 gamma bound asialofetuin and cells similarly to the parent 1 alpha, 2 gamma, subdomain mutant. In contrast, the 1 alpha, 1 beta, 2 gamma subdomain triple-site mutant had a one and one-half log decrease in asialofetuin and cell binding relative to the parent double-site mutant. The 1 alpha, 2 alpha, 2 gamma triple-site mutant and 1 alpha, 2 gamma parent protein had sugar binding which was inhibited by 3-27-fold by lactose and asialofetuin. Both triple-site mutants reassociated well with RTA. The 1 alpha, 2 alpha, 2 gamma triple-site mutant-RTA was equally cytotoxic to mammalian cells as the double-site mutant-RTA heterodimer. In contrast, the 1 alpha, 1 beta, 2 gamma triple-site mutant-RTA was 25 times less toxic than the double mutant and 20 times more toxic than RTA alone. These data support a model for at least three lectin-binding subdomains in RTB.
蓖麻毒素是一种在蓖麻籽中合成的65 kDa异源二聚体糖蛋白，由一个细胞结合凝集素亚基（RTB）和一个rRNA N - 糖苷酶蛋白合成失活亚基（RTA）通过二硫键连接而成。虽然X射线晶体学和平衡透析表明在亚结构域1α和2γ中有两个糖结合位点，但生化和突变分析表明存在第三个凝集素位点。我们对RTB cDNA进行了寡核苷酸定向诱变，以创建在亚结构域1α、2γ以及1β或2α中具有修饰的突变体。三重位点突变体RTB在昆虫细胞中表达。从感染细胞提取物和细胞上清液中获得的部分纯化重组蛋白，针对去唾液酸胎球蛋白结合、细胞结合、免疫反应性、与RTA重新结合的能力以及重组异源二聚体细胞毒性进行了表征。两个三重位点突变体的产量与亲本双位点突变体相似。两个突变体都与一组抗RTB单克隆和多克隆抗体表现出免疫反应性。在亚结构域1α、2α和2γ中具有氨基酸残基修饰的三重位点突变体与去唾液酸胎球蛋白和细胞的结合情况与亲本1α、2γ亚结构域突变体相似。相比之下，1α、1β、2γ亚结构域三重位点突变体相对于亲本双位点突变体，去唾液酸胎球蛋白和细胞结合能力降低了一个半对数。1α、2α、2γ三重位点突变体和亲本1α、2γ蛋白的糖结合受到乳糖和去唾液酸胎球蛋白3 - 27倍的抑制。两个三重位点突变体都能很好地与RTA重新结合。1α、2α、2γ三重位点突变体 - RTA对哺乳动物细胞的细胞毒性与双位点突变体 - RTA异源二聚体相同。相比之下，1α、1β、2γ三重位点突变体 - RTA的毒性比双突变体低25倍，比单独的RTA高20倍。这些数据支持了RTB中至少存在三个凝集素结合亚结构域的模型。
