1. Results Cheng Her and Thao Yang  Celebration of Excellence in Research and Creative Activity, May 1-2, 2013 Department of Chemistry, University of Wisconsin – Eau Claire Synthesis and Antibody Binding Study of a Cyclic Dimer MUC1 Mucin Peptide Acknowledgments We would like to thank the UWEC Office of Research and Sponsored Programs for providing grants for this research. We would also like to thank the U.S. Dept. of Education McNair Achievement Program for funding this project. Lastly, we would like to thank the UWEC Chemistry Department for providing the equipment and materials necessary to make this project possible. Acknowledgments We would like to thank the UWEC Office of Research and Sponsored Programs for providing grants for this research. We would also like to thank the U.S. Dept. of Education McNair Achievement Program for funding this project. Lastly, we would like to thank the UWEC Chemistry Department for providing the equipment and materials necessary to make this project possible. Conclusions The synthesis of the head-to-tail mucin cyclic peptide with sequence GVTSAPD was successful through SPPS, though two different cyclic products were isolated. 2D NMR analysis was sufficient to confirm the cyclic nature of the products (observed a NMR cross-peak between glycine of chain 1 and aspartic acid of chain 2). 2D NMR analysis suggest that the dimer peptide consists of two identical halves (no difference in NMR chemical shift). Similar to its linear counterpart, STD-NMR analysis showed that the cyclic dimer mucin peptide binds 6A4 monoclonal antibody (mAb). In fact, STD NMR data suggest stronger binding of mAb to the cyclic dimer peptide compared to the linear peptide. The methyl groups of valine, threonine and alanine showed STD-NMR signals indicating antibody interactions. The degree of saturation transfer is highest (strongest binding) with the 1 H’s of the proline residues thus proline may be evaluated as a critical residue required for binding to antibody. The STD-NMR analysis suggest that the peptide-antibody interface is hydrophobic in nature. Conclusions The synthesis of the head-to-tail mucin cyclic peptide with sequence GVTSAPD was successful through SPPS, though two different cyclic products were isolated. 2D NMR analysis was sufficient to confirm the cyclic nature of the products (observed a NMR cross-peak between glycine of chain 1 and aspartic acid of chain 2). 2D NMR analysis suggest that the dimer peptide consists of two identical halves (no difference in NMR chemical shift). Similar to its linear counterpart, STD-NMR analysis showed that the cyclic dimer mucin peptide binds 6A4 monoclonal antibody (mAb). In fact, STD NMR data suggest stronger binding of mAb to the cyclic dimer peptide compared to the linear peptide. The methyl groups of valine, threonine and alanine showed STD-NMR signals indicating antibody interactions. The degree of saturation transfer is highest (strongest binding) with the 1 H’s of the proline residues thus proline may be evaluated as a critical residue required for binding to antibody. The STD-NMR analysis suggest that the peptide-antibody interface is hydrophobic in nature. Aim In general, the introduction of conformational constraints to biomolecules (cyclization, attachment to molecular scaffold) often lead to increased biological activity (4). In a previous study we have determined that the shortened mucin peptide with sequence GVTSAPD possesses SM3 monoclonal antibody binding capability, suggesting that this antibody may not be as selective as previously thought as its normal recognition site was accepted to be PDTRP. The results of that study suggest flexibility in terms of the antigen composition and structure. In an attempt to engineer an optimal peptide antigen we synthesized and studied the structure and monoclonal antibody binding ability of a cyclic dimer form of the 7-residue mucin peptide with sequence GVTSAPD. Aim In general, the introduction of conformational constraints to biomolecules (cyclization, attachment to molecular scaffold) often lead to increased biological activity (4). In a previous study we have determined that the shortened mucin peptide with sequence GVTSAPD possesses SM3 monoclonal antibody binding capability, suggesting that this antibody may not be as selective as previously thought as its normal recognition site was accepted to be PDTRP. The results of that study suggest flexibility in terms of the antigen composition and structure. In an attempt to engineer an optimal peptide antigen we synthesized and studied the structure and monoclonal antibody binding ability of a cyclic dimer form of the 7-residue mucin peptide with sequence GVTSAPD. Introduction MUC-1 mucins are attracting interests as study targets in the development of vaccines against cancer. MUC-1 mucins are large transmembrane glycoproteins that consist of a 20 amino acid tandem repeat unit (GVTSAPDTRPAPGSTAPPAH) on its extracellular domain (1,2). Healthy epithelial cells express mucin proteins that are heavily coated with complex carbohydrate structures attached at serine and threonine residues of the repeating sequence (1-3). In addition, the location on the cell surface of the expression is regulated and limited to the apical side (2). On the other hand, tumor-associated cells have unregulated expression of the mucin core protein and display aberrant carbohydrate patterns (1-3). In such cases the glycosylation is greatly reduced, resulting in the exposure of the mucin protein core that is known to induce low levels of immunologic responses. The released mucin specific monoclonal antibody is known to recognize the sequence PDTRP (bold above) in the tandem repeat domain. Introduction MUC-1 mucins are attracting interests as study targets in the development of vaccines against cancer. MUC-1 mucins are large transmembrane glycoproteins that consist of a 20 amino acid tandem repeat unit (GVTSAPDTRPAPGSTAPPAH) on its extracellular domain (1,2). Healthy epithelial cells express mucin proteins that are heavily coated with complex carbohydrate structures attached at serine and threonine residues of the repeating sequence (1-3). In addition, the location on the cell surface of the expression is regulated and limited to the apical side (2). On the other hand, tumor-associated cells have unregulated expression of the mucin core protein and display aberrant carbohydrate patterns (1-3). In such cases the glycosylation is greatly reduced, resulting in the exposure of the mucin protein core that is known to induce low levels of immunologic responses. The released mucin specific monoclonal antibody is known to recognize the sequence PDTRP (bold above) in the tandem repeat domain. Method Synthesis of Peptide Synthesized cyclic peptide through Solid-Phase Peptide Synthesis (SPPS) using Fmoc-Chemistry and Wang resin. Peptide purification and analysis using HPLC and LC-MS. 1 H assignment achieved by 2D NMR (TOCSY, 400 MHz Bruker Spectr.) at 3-5mg peptide in 20 mM phosphate, 5mM NaCl, pH5 and 7˚C. STD-NMR analysis for antibody binding experiment. Method Synthesis of Peptide Synthesized cyclic peptide through Solid-Phase Peptide Synthesis (SPPS) using Fmoc-Chemistry and Wang resin. Peptide purification and analysis using HPLC and LC-MS. 1 H assignment achieved by 2D NMR (TOCSY, 400 MHz Bruker Spectr.) at 3-5mg peptide in 20 mM phosphate, 5mM NaCl, pH5 and 7˚C. STD-NMR analysis for antibody binding experiment. Figure 1. a) Difference between glycosylation pattern of tandem repeat domain of normal (left) and tumor (right) associated mucin protein. b) structure of 20 amino acid tandem repeat found on extracellular domain of MUC1 mucin proteins. Red, orange and green circles represent sugar units attached to the protein core (blue). (http://ars.els-cdn.com/content/image/1- s2.0-S1357431098013227-gr1.jpg) References 1.Grinstead, J.S., Koganty, R.R., Krantz, M.J., Longenecker, M.B., and Campbell, P.A. (2002) Biochemistry, 41, 9946-9961. 2.Singh, R. and Bandyopadhyay, D. (2007) Cancer. Biol. Ther., 6, 481-486. 3.Hollingsworth, M.A., and Swanson, B.J. (2004) Nature, 4, 45-60. 4.Demmer, O., Frank, A. O. and Kessler, H. (2009) Design of Cyclic Peptides, in Peptide and Protein Design for Biopharmaceutical Applications (ed K. J. Jensen), John Wiley & Sons, Ltd, Chichester, UK. 5.Alcaro, M. C., Sabatino, G., Uziel, J., Chelli, M., Ginanneschi, M., Rovero, P., and Papini, A. M. (2004) On-resin Head-to-tail Cyclization of Cyclotetrapeptides: Optimization of Crucial Parameters. J. Peptide Sci., 10, 218 - 228. 6.Chan, W. C., and White, P. D., (ed.), (2000) in “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” Basic Procedures, Oxford University Press, p. 41 - 74. 7.Her, C., and Yang, T., (2012) Antibody Binding Study of Mucin Peptide Epitopes. Division of Biological Chemistry, 243 rd ACS meeting, San Diego, CA., March 25-29. 8.Chan, W. C. and White, P. D., (ed.), (2000) in “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” RP-HPLC using lipophilic chromatography probes, Oxford University Press, p. 269 - 276. 9.Berger, S. and Braun, S., (ed.), (2004) in "200 and More NMR Experiments, A Practical Course," WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, p. 422 - 425. 10.Mayer, M. and Meyer, B. (2001) Group Epitope Mapping by Saturation Transfer Difference NMR To Identify Segment of a Ligand in Direct Contact with a Protein Receptor. J. Am. Chem. Soc., 123, 6108 – 6117. References 1.Grinstead, J.S., Koganty, R.R., Krantz, M.J., Longenecker, M.B., and Campbell, P.A. (2002) Biochemistry, 41, 9946-9961. 2.Singh, R. and Bandyopadhyay, D. (2007) Cancer. Biol. Ther., 6, 481-486. 3.Hollingsworth, M.A., and Swanson, B.J. (2004) Nature, 4, 45-60. 4.Demmer, O., Frank, A. O. and Kessler, H. (2009) Design of Cyclic Peptides, in Peptide and Protein Design for Biopharmaceutical Applications (ed K. J. Jensen), John Wiley & Sons, Ltd, Chichester, UK. 5.Alcaro, M. C., Sabatino, G., Uziel, J., Chelli, M., Ginanneschi, M., Rovero, P., and Papini, A. M. (2004) On-resin Head-to-tail Cyclization of Cyclotetrapeptides: Optimization of Crucial Parameters. J. Peptide Sci., 10, 218 - 228. 6.Chan, W. C., and White, P. D., (ed.), (2000) in “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” Basic Procedures, Oxford University Press, p. 41 - 74. 7.Her, C., and Yang, T., (2012) Antibody Binding Study of Mucin Peptide Epitopes. Division of Biological Chemistry, 243 rd ACS meeting, San Diego, CA., March 25-29. 8.Chan, W. C. and White, P. D., (ed.), (2000) in “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” RP-HPLC using lipophilic chromatography probes, Oxford University Press, p. 269 - 276. 9.Berger, S. and Braun, S., (ed.), (2004) in "200 and More NMR Experiments, A Practical Course," WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, p. 422 - 425. 10.Mayer, M. and Meyer, B. (2001) Group Epitope Mapping by Saturation Transfer Difference NMR To Identify Segment of a Ligand in Direct Contact with a Protein Receptor. J. Am. Chem. Soc., 123, 6108 – 6117. Figure 2. Steps involved in SPPS of linear GVTSAPD peptide using Fmoc-Chemistry. Figure 3. Cyclization scheme. Solid line is pathway to the cyclic monomer GVTSAPD peptide with mass of 627.31 Da (left). Dotted line is pathway to cyclic dimer peptide with mass of 1254.60 Da (right). a b c Figure 5. HPLC chromatogram of crude peptide sample. Chromatogram was obtained from a 500 µL concentrated sample injection. Acquisition time was 40 minutes with a 5 minute post equilibrium/hold and monitored with UV light. Spectrum was extracted from wavelength of 220nm. a) peak corresponding to the cyclic monomer peptide of interest. b) peak corresponding to the dimer cyclic peptide of interest. c) a contaminant found in the crude peptide sample and does not contain any product of interest. Figure 4. LC-MS TIC chromatogram of crude peptide sample immediately following completion of peptide synthesis and extraction. Analysis was conducted with ESI-Positive Mode under 0.5ml/min flow rate using 99% ACN as the organic buffer. Acquisition time was set at 20 minutes with a 4 minute post run. Peaks of interest were a) monomer product of mass [M + H + ] = 628.31 Da. and b) dimer product of mass [M + H + ] = 1255.60 Da. a b Future Directions Study binding of mutant peptides in which proline is replaced by another non-polar side chain containing amino acid. Study glycosylated linear and cyclic mucin peptides Couple mucin peptide to carrier protein to study antigenic effect (ability to evoke antibody production) in animal subjects. Future Directions Study binding of mutant peptides in which proline is replaced by another non-polar side chain containing amino acid. Study glycosylated linear and cyclic mucin peptides Couple mucin peptide to carrier protein to study antigenic effect (ability to evoke antibody production) in animal subjects. GlyαH-NH AspαH-NH AspαH - GlyNH Figure 6. 2D ROESY spectrum of CH-NH region of cyclic dimer peptide. Interactions between alpha-carbon protons and amide protons of glycine and aspartic acid residues are indicated. In addition, a unique NOE peak bridging the interaction between adjacent Asp (chain 1) and Gly (chain 2) residues confirms the cyclic nature of this molecule. Figure 7. STD 1D NMR data indicating what 1 H’s on the cyclic dimer peptide are directly involved in the binding to monoclonal antibody (mAb, clone 6A4) from mice cell in phosphate buffer, pH 7 at 7 °C. The traces are: A) 1D 1 H NMR spectrum of a mixture of mAb and cyclic dimer peptide showing the 1 H resonances of the peptide on top of the unresolved resonances of the mAb (10µM mAb, 1mM peptide). B) STD spectrum of cyclic dimer peptide plus mAb mixture showing what 1 H’s directly bind to mAb. Protein background subtracted in this spectrum. C) 1D 1 H NMR spectrum of a mixture of mAb and linear peptide showing the 1 H resonances of the peptide on top of the unresolved resonances of the mAb (10µM mAb, 1mM peptide). D) STD spectrum of linear peptide plus mAb mixture showing what 1 H’s directly bind to mAb. Protein background subtracted in this spectrum. E) Control STD spectrum of mAb in the absence of any peptide; only observed broad hump from the resonances of the mAb. F) Control STD spectrum of peptide in absence of mAb. There are no mAb-peptide interactions, thus no STD peaks observed. * = contaminant. * * A B C D E F Results