Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study
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- 作者：Jeffrey S. Grinstead ; Jason T. Schuman ; and A. Patricia Campbell
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：December 9, 2003
- 年：2003
- 卷：42
- 期：48
- 页码：14293 - 14305
- 全文大小：281K
- 年卷期：v.42,no.48(December 9, 2003)
- ISSN：1520-4995
文摘
MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which theextracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH.In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed.Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state,as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previousstudy [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlatethe effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognitionand binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presentedhere, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probethe recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides werestudied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeatMUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxationparameters were measured for both peptides free in solution and bound to Fab B27.29. The 13C
T1 valuesbest represent changes in the local correlation time of the peptide epitope upon binding antibody, anddemonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result isalso reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of thesame peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptideepitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J.S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMRexperiments. The implications of these results are discussed within the context of MUC1 breast cancervaccine design.
T1 valuesbest represent changes in the local correlation time of the peptide epitope upon binding antibody, anddemonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result isalso reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of thesame peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptideepitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J.S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMRexperiments. The implications of these results are discussed within the context of MUC1 breast cancervaccine design.