Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.
Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.
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DOI：
10.1016/s0021-9258(19)50702-5
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发表时间：
1992-02
期刊：
影响因子：
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通讯作者：
M. K. Lee;R. Milne;A. Z. Ge;Donald H. Dean
中科院分区：
文献类型：
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作者：
M. K. Lee;R. Milne;A. Z. Ge;Donald H. Dean
用~(125)I标记的苏云金芽孢杆菌胰酶激活的杀虫毒素CryIA(A)和CryIA(C)与家蚕幼虫中肠的刷状缘膜泡(BBMV)进行了受体结合研究。通过用稀释的毒素轻轻强迫家蚕饲养来进行生物测定。CryIA(A)毒素(LD50；0.002微克)对家蚕幼虫的杀灭活性是CryIA(C)毒素(LD50，0.421微克)的200倍，并表现出高亲和力饱和结合。CryIA(A)毒素的Kd值为3.5 nM，结合部位浓度为7.95pmol/mg。CryIA(C)毒素(Kd，50.35 nM；Bmax，2.85pmol/mg)与家蚕BBMV无高亲和力结合。与CryIA(A)和CryIA(C)毒素的对照实验表明，CryIA(A)和CryIA(C)毒素不与小鼠小肠BBMV结合，与猪肾BBMV非特异性结合。这些数据表明，与中肠上皮细胞膜上特定受体的结合是杀虫晶体蛋白杀虫谱差异的重要决定因素。为了定位CryIA(A)毒素上的家蚕白僵菌受体结合区，用一组突变蛋白进行了同源和异源竞争结合研究(GE，A.Z.，Shivarova，N.I.和Dean，D.H.(1989)Proc)。娜塔莉。阿卡德。SCI。美国86,4037-4041)。这些突变蛋白的基因区域与CryIA(C)基因相互交换。CryIA(A)毒素上的家蚕白僵菌受体结合区包括高变区的氨基末端部分，即332-450个氨基酸，这与先前描述的家蚕白僵菌特异性决定区完全相同。这些数据提供了直接证据，证明德尔塔-内毒素含有一段组成结合区的氨基酸，并因此决定了毒素的特异性。
Receptor binding studies were performed with 125I-labeled trypsin-activated insecticidal toxins, CryIA(a) and CryIA(c), from Bacillus thuringiensis on brush-border membrane vesicles (BBMV) prepared from Bombyx mori larval midgut. Bioassays were performed by gently force feeding B. mori with diluted toxins. CryIA(a) toxin (LD50; 0.002 micrograms) was 200 times more active against B. mori larvae than CryIA(c) toxin (LD50; 0.421 micrograms) and showed high-affinity saturable binding. The Kd and the binding site concentration for CryIA(a) toxin were 3.5 nM and 7.95 pmol/mg, respectively. CryIA(c) toxin (Kd, 50.35 nM; Bmax, 2.85 pmol/mg) did not demonstrate high-affinity binding to B. mori BBMV. Control experiments with CryIA(a) and CryIA(c) toxins revealed no binding to mouse small intestine BBMV and nonspecific binding to pig kidney BBMV. These data provide evidence that binding to a specific receptor on the membrane of midgut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of insecticidal crystal proteins. To locate a B. mori receptor binding region on the CryIA(a) toxin, homologous and heterologous competition binding studies were performed with a set of mutant proteins which had previously been used to define the B. mori “specificity domain” on this toxin (Ge, A. Z., Shivarova, N. I., and Dean, D. H. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041). These mutant proteins have had regions of their genes reciprocally exchanged with the cryIA(c) gene. A B. mori receptor binding region on CryIA(a) toxin includes the amino-terminal portion of the hypervariable region, amino acids 332-450, which is identical to the previously described B. mori specificity determining region. These data provide direct evidence that delta-endotoxins contain a tract of amino acids that comprise a binding region and as a results determines the specificity of a toxin.