A Comparative Analysis of the Immunological Evolution of Antibody 28B4
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- 作者：Jun Yin ; Emily C. Mundorff ; Priscilla L. Yang ; K. Ulrich Wendt ; Denise Hanway ; Raymond C. Stevens ; and Peter G. Schultz
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：September 11, 2001
- 年：2001
- 卷：40
- 期：36
- 页码：10764 - 10773
- 全文大小：693K
- 年卷期：v.40,no.36(September 11, 2001)
- ISSN：1520-4995
文摘
In an effort to gain greater insight into the evolution of the redox active, catalytic antibody28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, andthe X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 weredetermined. Comparison with the previously determined structures of the unliganded and hapten-boundaffinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad.Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in anorientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonatemoiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has moreelectrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the haptenin a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticityin the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G.,and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differentialbinding affinities of the germline Fab (Kd = 25 
M) and 28B4 Fab (Kd = 37 nM) to hapten 3. Ninereplacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to itsgermline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 werecharacterized in detail in the contexts of both the germline and the affinity-matured antibodies. One ofthe mutations, Asp95HTrp, leads to a change in the orientation of the bound hapten, and its presence isa prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody forhapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwisemanner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germlineantibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities werefound to be highly homologous to the germline antibody of 28B4, consistent with the notion that certaingermline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg,F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificityof the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somaticmutation. Insights into the evolution of antibody-combining sites provided by this and other structuralstudies are discussed.
M) and 28B4 Fab (Kd = 37 nM) to hapten 3. Ninereplacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to itsgermline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 werecharacterized in detail in the contexts of both the germline and the affinity-matured antibodies. One ofthe mutations, Asp95HTrp, leads to a change in the orientation of the bound hapten, and its presence isa prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody forhapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwisemanner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germlineantibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities werefound to be highly homologous to the germline antibody of 28B4, consistent with the notion that certaingermline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg,F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificityof the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somaticmutation. Insights into the evolution of antibody-combining sites provided by this and other structuralstudies are discussed.