Schildbach J F, Near R I, Bruccoleri R E, Haber E, Jeffrey P D, Ng S C, Novotny J, Sheriff S, Margolies M N
Program on Immunology, Harvard University Graduate School of Arts and Sciences, Cambridge, Massachusetts 02138.
J Biol Chem. 1993 Oct 15;268(29):21739-47.
Antibody produced by a variant of the murine antidigoxin hybridoma 26-10 has reduced affinity for digoxin but enhanced recognition of the digoxin 12-hydroxyl due to a Tyr to His substitution at heavy chain position 50 (Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E., Haber, E., and Margolies, M. N. (1991) J. Biol. Chem. 266, 4640-4647). Consistent with these data, the 26-10 Fab-digoxin x-ray crystal structure (Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y., Campbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N., and Sheriff, S. (1993) Proc. Natl. Acad. Sci. U. S. A., in press) reveals that Tyr-50 contacts a region of digoxin that includes the hapten-12 carbon. To determine the effects of other heavy chain position 50 substitutions, mutant antibodies were engineered, and their affinities for digoxin and digoxin analogues were measured. The affinity of the mutant antibodies for digoxin roughly correlates with the size of the position 50 side chain. Substitutions of Trp or Phe have no effect on affinity, whereas substitutions of Asn, His, Leu, Ala, Gly, and Asp confer progressively lower affinities. Although Trp and Phe mutants exhibit wild-type specificity, Asn and Asp mutants have improved affinity for digoxin relative to digitoxin (12-deshydroxydigoxin). Leu, Ala, and Gly mutants have improved affinity for 12-acetyldigoxin relative to digoxin as compared with 26-10. These results indicate that position 50 is a determinant of both antibody affinity and fine specificity for antibody 26-10 and that single-amino acid substitutions can alter antibody fine specificity. Models of the mutants were computationally constructed, and haptens were docked into the modeled binding sites. The results suggest that 12-acetyldigoxigenin occupies different orientations in the 26-10 and in the Ala mutant binding sites, resulting in altered binding.
由鼠抗地高辛杂交瘤26 - 10的变体产生的抗体，对洋地黄毒苷的亲和力降低，但由于重链第50位的酪氨酸被组氨酸取代，对洋地黄毒苷12 - 羟基的识别增强（席尔德巴赫，J.F.，潘卡，D.J.，帕克斯，D.R.，雅格，G.C.，诺沃特尼，J.，赫岑伯格，L.A.，穆吉特 - 亨特，M.，布鲁科勒里，R.E.，哈伯，E.，和马戈利斯，M.N.（1991）《生物化学杂志》266，4640 - 4647）。与这些数据一致，26 - 10 Fab - 洋地黄毒苷的x射线晶体结构（杰弗里，P.D.，斯特朗，R.K.，西克，L.C.，张，C.Y.，坎贝尔，R.L.，佩茨科，G.A.，哈伯，E.，马戈利斯，M.N.，和谢里夫，S.（1993）《美国国家科学院院刊》，即将发表）表明，酪氨酸50接触洋地黄毒苷的一个区域，该区域包括半抗原12 - 碳。为了确定重链第50位其他取代的影响，构建了突变抗体，并测量了它们对洋地黄毒苷和洋地黄毒苷类似物的亲和力。突变抗体对洋地黄毒苷的亲和力大致与第50位侧链的大小相关。色氨酸或苯丙氨酸的取代对亲和力没有影响，而天冬酰胺、组氨酸、亮氨酸、丙氨酸、甘氨酸和天冬氨酸的取代导致亲和力逐渐降低。虽然色氨酸和苯丙氨酸突变体表现出野生型特异性，但天冬酰胺和天冬氨酸突变体对洋地黄毒苷的亲和力相对于洋地黄毒苷（12 - 去氢洋地黄毒苷）有所提高。与26 - 10相比，亮氨酸、丙氨酸和甘氨酸突变体对12 - 乙酰洋地黄毒苷相对于洋地黄毒苷的亲和力有所提高。这些结果表明，第50位是抗体26 - 10的抗体亲和力和精细特异性的决定因素，并且单氨基酸取代可以改变抗体的精细特异性。通过计算构建了突变体的模型，并将半抗原对接至模拟的结合位点。结果表明，12 - 乙酰洋地黄毒苷元在26 - 10和丙氨酸突变体的结合位点中占据不同的取向，从而导致结合改变。
