In this study we investigated to see whether or not a shortened MUC1 mucin peptide epitope with the sequence GVTSAPD containing a single prolyl residue would still bind specific monoclonal antibody as its native sequence (e.g., PDTRP), known to be the specific recognition site on the Variable Number Tandem Repeat (VNTR) region of MUC1 mucin by the immune system. The affinity of GVTSAPD peptide to a mouse Muc1 mucin specific monoclonal antibody (clone 6A4, IgG1 isotype) was investigated by Saturation Transfer Difference NMR spectroscopy (STD NMR). Results showed that the shortened mucin epitope GVTSAPD still retained affinity to Muc1 specific monoclonal antibody (mAb) while one that lacks the prolyl residue at position 6 lost its affinity, which suggests that P6 is necessay for antibody binding. The interactions observed by STD NMR occurred strongest at the P6 side chain 1 H’s (bH and gH); the P6 Ha showed lower degree of saturation transfer effect. Minor interactions also occurred at the methyl groups of V2 , T3 and A5 . Mucin peptides derived from the VNTR region have been the target of cancer vaccine research, thus properties associated with mucin peptide structure, conformation and antibody interaction are central to peptide design or engineering towards that end

MUC1 antibody recognition epitope;Mucin peptide;Saturation Transfer Difference NMR Spectroscopy (STD 
NMR)

Her C, Westler WM, Yang T (2013) Significance of Proline Residue on Short Mucin Peptide Interactions with Mouse MUC1 Monoclonal Antibody Studied by Saturation Transfer Difference NMR Spectroscopy. JSM Chem 1(1): 1004.

DCM: Dichloromethane; DIPEA: Diisopropylethylamine; DMF: Dimethylformamide; Fmoc: N-α-9-fluorenylmethoxycarbonyl; Fmoc-Asp(OtBu)-Wang resin (100-200 mesh): N-α-Fmoc-Laspartic acid β-t-butyl ester-Wang resin; HB: Hydrogen Bond; HBTU : O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; HOBt: N-hydroxybenzotriazole; mAb : monoclonal antibody; SDS-PAGE: Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis; Wang resin: p-alkoxybenzyl alcohol-linked Wang resin

The antibody binding study of a shortened MUC1 mucin peptide epitope derived from the Variable Tandem Repeat domain (VNTR) of MUC1 mucin to a mouse Muc1 mucin specific monoclonal antibody (isotype IgG1, clone 6A4) was investigated by Saturation Transfer Difference NMR (STD NMR) spectroscopy. MUC1 mucin is a large molecular weight (> 400 kDa) glycosylated protein tethered to a transmembrane C-terminal domain (MUC1-C) on the cell surface expressed by glandular and ductal epithelial cells [1,2]. Most human adenocarcinoma epithelial cells, including those from breast and ovary overexpress a form of MUC1 mucin that is aberrantly glycosylated causing exposure of peptide backbone and cryptic oligosaccharide chains that exhibit antigenic properties, which make them novel target species for cancer therapy [1,3]. Some hematopoietic cells also produce MUC1 mucin, for further review see references 4 and 5 [4,5]. The role of MUC1 mucin is diverse, ranging from lubrication and/or hydration of epithelial cell surfaces, protection against invasion of pathogens and acidity, involved in embryonic implantation, epithelial sheet formation and morphogenesis, cell-cell interactions, intracellular cell signaling that promote growth and transformation, to immune regulation [6-9]. MUC1 mucin in tumor cells is immunosuppressive, the overproduction of it masses tumor cell surfaces, inhibiting cell lysis, promoting their growth and metastatic progression [10,11], thus affirming an evidence for rendering the immune system ineffective in countering against tumor growth. Cryptic peptide epitopes dispersed into the circulation or microenvironment of tumor have been known to induce cellular and humoral immune responses to MUC1 mucin [12,13]. A well known 20-amino acid (20-aa) MUC1 epitope residing at the VNTR domain which comprises the sequence GVTSAPDTRPAPGSTAPPAH is an immunodominant sequence recognized by the immune system either as naked peptide or glycosylated peptide [14-16]. The x-ray crystal structure of a breast cancer specific monoclonal antibody (clone SM3) that recognizes the immunodominant sequence (a 13-aa peptide, TSAPDTRPAPGST) has been determined [17]. The peptide is bound in an extended structure with most of the interactions (vdW contacts, direct HB or water mediated HB) between the antigen peptide and the antibody occurring at the N-terminal end located at the APD region [17]; most interactions are of hydrophobic nature. Several MUC1 mucin peptide - antibody binding studies in solution by NMR had been carried out, for further review see [18-24]. Of direct relevance to this investigation are the studies carried out by Grinstead et al. [21] and Möller et al. [22]. Grinstead and coworkers carried out a binding study of a 16-aa and a 20-aa MUC1 mucin peptides and the antibody Fab fragment by 15N and 13C NMR relaxations of the peptides. These workers showed that the peptide domain that was immobilized was located at the entire domain PDTRPAP within the 20-aa VNTR domain, and that the preceding and subsequent sequences of the MUC1 mucin peptides were unbound and flexible in solution. The investigation by Möller et al. [22] using a 5-aa MUC1 mucin peptide PDTRP and a glycopteptide PDT(O-αD-GalNAc)RP binding to antibody SM3 by STD NMR showed that the interactions between the PDTRP peptide and the antibody was strongest at the P1 residue and dropped off sequentially at residues 2-5; similarly the P1 residue had the strongest binding effect in the PDT(O-α-D-GalNAc)RP peptide, in contrast, residues 2-5 had similar binding strength. In addition, the GalNAc residue showed a strongest binding at the methyl group comparable to that of the P1 residue, whereas the rest of the GalNAc ring protons (CH protons) showed considerable binding effects but lower in degree of saturation effect compared to those observed for residues 2-5.

The single and main epitope sequence that is well-defined and accepted to be that which is recognized by monoclonal antibody is the 5-aa sequence PDTRP [17,21]. Therefore, most mucin peptideantibody binding cases have focused on that main epitope. In this investigation we are interested to probe the binding activity of MUC1 mucin peptide with sequence that lies slightly outside of the main epitope (i.e. PDTRP). The Muc1 antibody (mouse IgG1 monoclonal antibody, clone 6A4 from Genway Biotech) is an antibody that recognizes the sequence SAPDTRPA. We chose to use the 7-aa sequence GVTSAPD that resides at the N-terminal region of the main epitope to see if it would still be recognized by the specific monoclonal antibody expressed against the main binding epitope. In addition, we wanted to evaluate the critical groups on a particular residue or a residue necessary for binding. Thus, position 6 was selected to be the mutation point. The first mutant peptide used has P6 substituted by D6 (i.e., GVTSADD). It is logical that in order to be successful in developing a mucin peptide-based vaccine, the antigenic agent must be able to induce humoral responses to such agent. The mere binding of mucin peptide to antibody does not ensure that an effective immune response against tumor mucin or tumor cells will develop. Hence, it would not be illogical to explore variety of antigenic agents, including peptides with altered sequences, or cyclic peptides, glycopeptides, glyco-lipopeptide complex [25,26], and combination of multivalency glycoconjugates, which constitute the latest antigenic agents being designed [27]. Undertanding which amino acid residue or which group of protons on the mucin peptide is essential for binding antibody will be one of the crucial bases for designing antigenic agents. Herein, we report the interactions of a shortened MUC1 mucin peptide GVTSAPD to a specific Muc1 monoclonal antibody versus one that lacks binding (e.g. GVTSADD).

The STD NMR results showed that the protons of the methyl groups of V2 , T3 and A5 have interactions with the antibody, but other protons of these three residues have essentiall no binding effect. Significant interactions are centered at the protons of the P6 residue. Based on the fact that all its protons received saturation transfer effect unlike other residues, it may be concluded that the P6 residue is a critical residue in the peptide’s structure and ability for interacting with the antibody.

The antibody binding study of a shortened MUC1 mucin peptide epitope derived from the Variable Tandem Repeat domain (VNTR) of MUC1 mucin to a mouse Muc1 mucin specific monoclonal antibody (isotype IgG1, clone 6A4) was investigated by Saturation Transfer Difference NMR (STD NMR) spectroscopy. MUC1 mucin is a large molecular weight (> 400 kDa) glycosylated protein tethered to a transmembrane C-terminal domain (MUC1-C) on the cell surface expressed by glandular and ductal epithelial cells [1,2]. Most human adenocarcinoma epithelial cells, including those from breast and ovary overexpress a form of MUC1 mucin that is aberrantly glycosylated causing exposure of peptide backbone and cryptic oligosaccharide chains that exhibit antigenic properties, which make them novel target species for cancer therapy [1,3]. Some hematopoietic cells also produce MUC1 mucin, for further review see references 4 and 5 [4,5]. The role of MUC1 mucin is diverse, ranging from lubrication and/or hydration of epithelial cell surfaces, protection against invasion of pathogens and acidity, involved in embryonic implantation, epithelial sheet formation and morphogenesis, cell-cell interactions, intracellular cell signaling that promote growth and transformation, to immune regulation [6-9]. MUC1 mucin in tumor cells is immunosuppressive, the overproduction of it masses tumor cell surfaces, inhibiting cell lysis, promoting their growth and metastatic progression [10,11], thus affirming an evidence for rendering the immune system ineffective in countering against tumor growth. Cryptic peptide epitopes dispersed into the circulation or microenvironment of tumor have been known to induce cellular and humoral immune responses to MUC1 mucin [12,13]. A well known 20-amino acid (20-aa) MUC1 epitope residing at the VNTR domain which comprises the sequence GVTSAPDTRPAPGSTAPPAH is an immunodominant sequence recognized by the immune system either as naked peptide or glycosylated peptide [14-16]. The x-ray crystal structure of a breast cancer specific monoclonal antibody (clone SM3) that recognizes the immunodominant sequence (a 13-aa peptide, TSAPDTRPAPGST) has been determined [17]. The peptide is bound in an extended structure with most of the interactions (vdW contacts, direct HB or water mediated HB) between the antigen peptide and the antibody occurring at the N-terminal end located at the APD region [17]; most interactions are of hydrophobic nature. Several MUC1 mucin peptide - antibody binding studies in solution by NMR had been carried out, for further review see [18-24]. Of direct relevance to this investigation are the studies carried out by Grinstead et al. [21] and Möller et al. [22]. Grinstead and coworkers carried out a binding study of a 16-aa and a 20-aa MUC1 mucin peptides and the antibody Fab fragment by 15N and 13C NMR relaxations of the peptides. These workers showed that the peptide domain that was immobilized was located at the entire domain PDTRPAP within the 20-aa VNTR domain, and that the preceding and subsequent sequences of the MUC1 mucin peptides were unbound and flexible in solution. The investigation by Möller et al. [22] using a 5-aa MUC1 mucin peptide PDTRP and a glycopteptide PDT(O-αD-GalNAc)RP binding to antibody SM3 by STD NMR showed that the interactions between the PDTRP peptide and the antibody was strongest at the P1 residue and dropped off sequentially at residues 2-5; similarly the P1 residue had the strongest binding effect in the PDT(O-α-D-GalNAc)RP peptide, in contrast, residues 2-5 had similar binding strength. In addition, the GalNAc residue showed a strongest binding at the methyl group comparable to that of the P1 residue, whereas the rest of the GalNAc ring protons (CH protons) showed considerable binding effects but lower in degree of saturation effect compared to those observed for residues 2-5.

The single and main epitope sequence that is well-defined and accepted to be that which is recognized by monoclonal antibody is the 5-aa sequence PDTRP [17,21]. Therefore, most mucin peptideantibody binding cases have focused on that main epitope. In this investigation we are interested to probe the binding activity of MUC1 mucin peptide with sequence that lies slightly outside of the main epitope (i.e. PDTRP). The Muc1 antibody (mouse IgG1 monoclonal antibody, clone 6A4 from Genway Biotech) is an antibody that recognizes the sequence SAPDTRPA. We chose to use the 7-aa sequence GVTSAPD that resides at the N-terminal region of the main epitope to see if it would still be recognized by the specific monoclonal antibody expressed against the main binding epitope. In addition, we wanted to evaluate the critical groups on a particular residue or a residue necessary for binding. Thus, position 6 was selected to be the mutation point. The first mutant peptide used has P6 substituted by D6 (i.e., GVTSADD). It is logical that in order to be successful in developing a mucin peptide-based vaccine, the antigenic agent must be able to induce humoral responses to such agent. The mere binding of mucin peptide to antibody does not ensure that an effective immune response against tumor mucin or tumor cells will develop. Hence, it would not be illogical to explore variety of antigenic agents, including peptides with altered sequences, or cyclic peptides, glycopeptides, glyco-lipopeptide complex [25,26], and combination of multivalency glycoconjugates, which constitute the latest antigenic agents being designed [27]. Undertanding which amino acid residue or which group of protons on the mucin peptide is essential for binding antibody will be one of the crucial bases for designing antigenic agents. Herein, we report the interactions of a shortened MUC1 mucin peptide GVTSAPD to a specific Muc1 monoclonal antibody versus one that lacks binding (e.g. GVTSADD).

The STD NMR results showed that the protons of the methyl groups of V2 , T3 and A5 have interactions with the antibody, but other protons of these three residues have essentiall no binding effect. Significant interactions are centered at the protons of the P6 residue. Based on the fact that all its protons received saturation transfer effect unlike other residues, it may be concluded that the P6 residue is a critical residue in the peptide’s structure and ability for interacting with the antibody.

Peptide synthesis, HPLC purification and mass spectral analysis

Peptide was synthesized by the solid-phase technique via usual Fmoc-chemistry. The crude yield is 70 - 85%. Peptide was purified to ≥ 95% by HPLC prior to use. LC-MS analysis of GVTSAPD peptide showed an average isotopic mass of [M + H]+ of 646.54 u compared to 646.66 u, theoretical mass, with the [M + Na]+ of 668.39 u and the [M + K]+ of 684.36 u (data not shown); for the GVTSADD peptide a mass of [M + H]+ of 664.28 u was observed versus a theoretical mass of 664.63 u, with the [M + Na]+ of 686.27 u (data not shown).

NMR results

The TOCSY 2D-NMR data were used for 1 H assignments, and the ROESY 2D-NMR data, for detection of spatial interproton distances (dH-H ≤ 5 Å). Assignments for the protons were achieved completely, except for the degenerate geminal αH’s of G1 , and βH’s of S4 ; no stereospecific assignments were made. Table 1

Table 1: 1 H chemical shifts (ppm) of mucin peptide GVTSAPD (3 mM) acquired in 20 mM phosphate, 5 mM NaCl, 90% H2 O, 10% D2 O, pH 5.0, at 7 ?C in the absence of monoclonal antibody.

Chemical shift values of second row under each residue were acquired in 20 mM phosphate, 5 mM NaCl, 100% D2 O, pH 7, at 7 ?C in the presence of monoclonal antibody

shows the 1 H chemical shifts assigned for GVTSAPD; and (Table 2), the 1 H chemical shifts assigned for GVTSADD.

For the GVTSAPD peptide, the amide temperature coefficients of all residues (Table 1), except that of G1 which could not be estimated, were all more negative than -4.6 ppb/K (a value indicative of a buried NH involved in hydrogen bonding) [40,41]; the values suggest all NH’s are accessible to solvent and that the peptide has no unique signature conformation, but an extended structure. For the GVTSADD peptide, all amide temperature coefficients were also more negative than -4.6 ppb/K (Table 2)

Table 2: 1 H chemical shifts (ppm) of mucin peptide GVTSADD (3 mM) acquired in 20 mM phosphate, 5 mM NaCl, 90% H2 O, 10% D2 O, pH 5.0, at 7 ?C in the absence of monoclonal antibody.

except that of D7 , in addition a medium nOe was observed at D6 NH-D7 NH, which may indicate a turn-like structure and inaccessibility to solvent at D7 NH.

The STD NMR results showed that the linear peptide GVTSAPD has binding to the Muc1 monoclonal antibody 6A4 despite it containing only one prolyl residue and being an upstream sequence of the normal binding epitope PDTRP in the VNTR domain. (Figure 1)

Figure 1: STD 1D 1 H NMR data showing the protons from the linear GVTSAPD peptide directly binding to monoclonal antibody (mAb) 6A4 in 20 mM phosphate, 5 mM NaCl, pH 7, 100% D2 O at 7 °C. The traces are: A). 1D 1 H NMR spectrum of linear peptide GVTSAPD in the absence of antibody (6A4, mAb); B). STD NMR spectrum of mAb in the absence of peptide showing broad-hump unresolved resonances indicative of efficient saturation transfer effect throughout the protein as a control spectrum; C). 1D 1 H NMR spectrum of a mixture of mAb and linear peptide GVTSAPD showing the 1 H resonances of the peptide riding on top of the unresolved resonances (broad-hump) of mAb (16 µM mAb, 1000 µM peptide); D). STD NMR spectrum of GVTSAPD plus mAb mixture showing mostly the STD effect of 1 Hs of P6 residue that are directly bound to the mAb and some STD effect from the -CH3 groups of V2 , T3 , and A5 on top of the broadhump mAb resonances; no protein background suppression on this spectrum; huge impurities resonances that do not bind to mAb do not show up as STD effect; spectrum was processed with a line broadening of 1.0 Hz and intensities were enhanced for visibility; E). Same STD NMR spectrum as in (D) but ran with protein background subtraction in order to see the STD effect better; note the huge contaminants in (C) do not bind to the mAb, thus are all subtracted out (in spectra D and E); dashed lines signify STD peaks; spectrum was processed with a line broadening of 1.0 Hz and intensities were enhanced for visibility; F). STD NMR spectrum of GVTSAPD peptide in the absence of mAb as a control spectrum; there is no mAb-peptide interactions, so no STD peaks resulted.

shows the STD NMR spectra of GVTSAPD peptide-Muc1 antibody mixture (traces D and E) compared to control spectra (Traces B and F). The STD NMR data showed that there are saturation transfer effect to the methyl groups of V2 , T3 , and A5 . The βH-region showed saturation transfer effect to the side chain protons of P6 and a minor effect at D7 (Figure 1, Trace E). The STD peak intensities of the rest of the βH’s (V2 , T3 and S4 ) were essentially zero and their percent STD were not estimated.

The STD NMR peak intensity is strongest at P6 Hδ (Traces D and E). The αH-region showed bigger saturation transfer effect to the P6 and minor effect to the D7 .

Figure 2

Figure 2: STD 1D 1 H NMR data showing the result of saturation transfer effect from mAb (6A4, mAb) onto the linear peptide GVTSADD in 20 mM phosphate, 5 mM NaCl, pH 7, 100% D2 O at 7 °C. The traces are: A). 1D 1 H NMR spectrum of a mixture of mAb and linear peptide GVTSADD showing the 1 H resonances of the peptide riding on top of the unresolved resonances (broad-hump) of mAb; B). STD NMR spectrum of GVTSADD and mAb mixture (16.4 µM mAb, 1000 µM peptide) showing no STD peaks on top of the broad-hump protein resonances because the protons of the peptide do not have any interactions with the antibody; C). 1D 1 H NMR spectrum of linear peptide GVTSADD in the absence of antibody; D). STD NMR spectrum of GVTSADD peptide in the absence of mAb as a control spectrum; there is no mAb-peptide interactions, so no STD peaks resulted. Sharp peaks marked with stars (*) are contaminants.

shows the STD NMR results for the GVTSADD peptideMuc1 monoclonal antibody binding study. The spectrum showed broad humps indicative of the saturation transfer effect on the antibody only (Figure 2, Trace B); no STD peaks were observed corresponding to any of the resonances of the peptide, which is indicative of no interaction between the protons of the peptide and the antibody. A separate STD NMR spectrum ran with the spoil pulse sequence using excitation sculpting with gradients and spinlock (30 ms) to suppress the broad protein background resulted in a flat line (data not shown); thus, no interaction occurred between GVTSADD and the antibody (mAb 6A4).

Table 3

Table 3: The percent areas of STD peaks relative to the corresponding nonsaturated transferred resonance peak areas. The values represent percent area of STD peaks to the corresponding peaks without saturation.

shows the percent STD peak area for each type of side chain protons (Figure 1, Trace E) extracted relative to the peak area of the corresponding protons on a control spectrum (Figure 1, Trace C) for GVTSAPD. The values represent percent areas of each STD peak relative to the corresponding peak area without the STD effect. Most significant saturation transfer effect occurred on the protons of P6 . For P6 residue, the percent STD of the γH and βH peak overlapping together is 7.1%; for a second βH, 4.5%; and for the αH, 1.6%. The percent STD of the δH’s of P6 was not estimated due to overlapping with the huge contaminant peaks from the antibody sample sitting on top of them. The percent STD of CH3 -groups of A5 , T3 and V2 were estimated to be 1.9%, 1.2% and 1.3%, respectively. Saturation transfer effect to the protons of S4 Hβ was too small and not estimated, but for G1 Hα and D7 Hβ , their effects were 1.1% and 2.0%, respectively.

Several protons on the GVTSAPD peptide showed interactions with the Muc1 monoclonal antibody 6A4 as indicated by the rising STD peaks corresponding to the individual peaks on the peptide, whereas the peptide lacking the prolyl residue at position 6 showed no proton interactions with the antibody. However, the STD peak intensities observed for the α-protons were very weak, except for the peak where the αH of P6 is centered, which showed stronger intensity but is still overlapped with the αH’s of S4 and T3 . The STD peak intensities of the βH’s that were observed were also very weak compared to those of P6 Hβ . A STD peak where the γH’s of P6 are centered at showed the largest percent STD (7.1%), though this peak is also overlapped with one of the βH of P6 . According to the saturation transfer difference NMR theory, when a ligand has a binding strength to a protein within the range of Kd = 10-2-103 μM [17,22,35], the saturation transfer effects on ligand protons directly interacting with the protein are detectable, and that the higher the intensity of the STD peak for a particular ligand proton, the stronger that proton has interaction with the protein that the ligand binds to [35-37]. In the case of the mucin peptide GVTSADD lacking the prolyl residue, we interpreted the result as having no binding protons since there is no STD peak arose corresponding to any of its proton resonances. In tight ligandreceptor binding domain (Kd in nM range) saturation transfer effect is not detected due to slow-exchange of the bound ligand on the NMR time scale which rendered it obsolete of magnetization transfer to the free ligand [42]. The condition is however usually accompanied with proton resonance shifts resulting in spectral characteristic differences between the bound-ligand and freeligand [42], and in the present case (for GVTSADD) we detected no spectral differences between the free peptide and the peptide mixed with mAb.

The STD data showed that the prolyl residue P6 is a residue on the peptide GVTSAPD that has stronger interactions with Muc1 monoclonal antibody 6A4 based on STD peak intensities of its protons that were observed, in which P6 may be viewed as being the critical residue in making contact with Muc1 monoclonal antibody at the antigen binding interface. Furthermore, the interactions between the side chain protons of P6 and the antibody would be of hydrophobic nature. It is not known what residues line the surface of the antigen binding pocket of mouse Muc1 monoclonal antibody 6A4 used in this study; nonetheless, in a crystal structure of a monoclonal antibody (clone SM3) complexed with a 13-residue peptide TSAPDTRPAPGST, the residue P4 , which would correspond to P6 on the peptide tested, has its prolyl ring buried in a hydrophobic pocket formed by Y32 and W91 of the light chain and W33 and Q97 of the heavy chain of the SM3 antibody [17]. Figure 3

Figure 3: Model of P4 residue in the peptide TSAPDTRPAPGST binding to monoclonal antibody SM3 in a cleft that is lined with hydrophobic residues, adopted from reference 17. The prolyl ring of P4 stacks against W91 of light chain, while being surrounded by Y32 (light chain), W33 and Q97 of heavy chain. The residue P6 of GVTSAPD would correspond to the P4 residue (white dotted arrow) of the peptide on this figure

shows the four residues Y32 and W91 of light chain, and W33 and Q97 of heavy chain at the antigen binding pocket of SM3 antibody complexed with the 13-residue mucin peptide, taken from Dokurno et al., 1998 [17], as a model for mucin peptide antigen binding to antiobody. According to this model, the prolyl ring stacks directly against the indole ring of W91 (light chain) while being surrounded by Y32 (light chain), W33 and Q97 (heavy chain). The prolyl ring has several van der Waals contacts (~ 4 Å) on either side of it; its αH would be closest to the two indole rings of W91 and W33; the βH’s closest to the Q97Hγ ’s; the γH’s closest to the Y32 phenol ring and Q97Hγ ’s; the δH’s closest to the Y32 phenol ring and W91 indole ring [17]. Here, hydrophobic interactions form critical element in mucin peptideantibody binding, especially, for mucin peptide derived from the VNTR region. The data in this study suggest that the prolyl residue within the sequence tested is a critical residue necessary for antibody binding. We are testing this hypothesis further by substituting P6 with other hydrophobic residues and aromatic residues on this peptide and conduct further peptide-antibody binding properties of those peptides; their results will be published elsewhere. When designing antigenic peptide model based on the VNTR domain, prolyl residue should be considered an important element for binding. The Muc1 mucin monoclonal antibody 6A4-mucin peptide binding study has implication and relevance in the biological activity of mucin peptides that are to be employed as possible antigenic agents for potential induction of immune responses against them.

The data showed that a shortened upstream mucin peptide epitope GVTSAPD, even though lacking the full binding sequence PDTRP and contained only one proline in the peptide sequence GVTSAPDTRPAPGSTAPPAH found on the VNTR domain could still bind mAb as revealed by the more pronounced saturation transfer effects (e.g., STD NMR peaks) observed on the side chain protons of P6 residue and the methyl groups of V2 , T3 and A5 .

The relatively strong STD NMR peak intensities of the αH, βH’s and γH’s of P6 indicate that they interacted stronger with Muc1 monoclonal antibody 6A4 at the antigen binding interface. The same peptide with proline at position 6 substituted for aspartate, more than likely, has no interactions with the antibody. Thus, the P6 residue on the sequence studied may be evaluated as a critical residue essential for binding. The interactions via the methyl groups of residues V2 , T3 and A5 were weaker compared to those found for the γH’s of P6 .

This work was supported by Faculty/Student Collaborative grants and Minority Mentoring grants from the University of Wisconsin-Eau Claire (UWEC) Office of Research and Sponsored Program. We thank the UWEC Chemistry Department for generous support of countless of hours on NMR spectrometer and LC-MS time, and supplies of chemicals.

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