Effect of core epitope modification on the antibody recognition of a MUC2 mucin peptide

Abstract

Identification of protein epitopes via combinatorial chemistry was one of the most important discoveries of the past three decades. Mapping of linear antibody epitopes can be achieved rapidly and cost-effectively by the polymer pin-bound peptide approach. In this article, the determination of the fine epitope structure of MUC2 mucin glycoprotein is described by using specific monoclonal antibody. We have used positional scanning combinatorial approach, and also parallel synthesis. The residues within the MUC2 epitope (18)PTGTQ(22) of MAb 996 were replaced by all other proteinogenic amino acids on pin-bound peptide libraries, and their antibody binding was studied in modified ELISA. Thr(19) was the least important of the residues in antibody recognition; most of the other amino acids could be replaced, except Pro. The other residues cannot be replaced without loss of antibody binding, where both the size and character of the amino acids were important. The significance of the non-chiral Gly(20) residue was further studied by competitive ELISA of parallelly synthesized soluble peptides containing L - or D-Ala instead of Gly residue. However, the D-Ala-containing oligopeptides showed no antibody binding; therefore, the backbone conformation is characteristic of that of L-amino acid containing peptides in this position as well. With the combinatorial approach we obtained relevant information about the contribution of individual amino acid side chains to the MAb 996 antibody binding within the PTGTQ predominant MUC2 mucin epitope. These results could be utilized for the design of synthetic antigens for detection of MUC2 protein core-specific antibodies related to carcinoma(s).

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope

“…The effect of the position and number (1 vs. 3 units) of monosaccharide, N ‐acetyl‐galactoseamine (Tn) unit present on the MAb 996 antibody binding of the 16 PTPTGTQ 22 epitope peptide was analyzed. Even a single N ‐acetyl‐galactoseamine on Thr21 completely hindered the MAb 996 recognition, in accordance with our previous findings on pin‐attached peptides, as in this position only a limited number of small residues was able to replace the Thr to a measurable antibody binding. Glycosylation on Thr17, which is not part of the epitope (although the presence of this residue is important for efficient antibody binding), had no effect on the antibody binding.…”

“…To our surprise, 16 PTPT(GalNacα)GTQ 22 peptide bound to MAb 996 significantly more strongly than the unglycosylated peptide, although Thr19 is part of the epitope. On the other hand, as we have shown previously, it is interesting to note that this residue can be replaced by almost all amino acids (except Pro) without significant loss of antibody binding, or even with enhanced binding. In this position not the side chain, but the backbone configuration is the most important factor, on which glycosylation seems to have an ameliorating effect.…”

“…Position 19 had significant effect on the binding, but contrarily to our expectations the glycosylation on Thr19 of the epitope sequence significantly increased the antibody binding (IC 50 = 23 μmol/L). We have shown before in a different experimental setting (immobilized peptides) that the backbone is more important in this position than the side chain, and in this position almost any amino acid residue, with the exception of Pro, is able to effectively substitute Thr, certain residues even enhanced the binding, apparently Thr(GalNacα) as well. Peptides with N ‐acetyl‐galactoseamine on Thr21 ( 16 PTPTGT(GalNacα)Q 22 and 16 PT(GalNacα)PT(GalNacα)GT(GalNacα)Q 22 ) did not inhibit the binding of the MAb 996 antibody to the target in the concentrations applied in our experiments, which was expected, having shown before that Thr21 can be modified only with the small Ala and Ser residues, with limited binding ability …”

“…Using the approach of pin‐attached peptides, we have also shown the importance of individual residues within the 19 TGTQ 22 sequence. Thr19 was interchangeable with almost all residues except Pro, while the other residues were more important …”

The mapping of linear B‐cell epitope regions in the extracellular parts of the desmoglein 1 and 3 proteins: recognition of immobilized peptides by pemphigus patients' serum autoantibodies

“…The fine epitope structure (the amino acids participating in the antibody binding) of a protein can be determined exclusively by monoclonal antibodies; for this several further attempts can be performed. The epitope region localized by the methods summarized earlier can be systematically shortened on both termini to identify the epitope sequence . After the epitope sequence has been localized, the fine epitope structure can be identified by methods such as single amino acid deletion sets , positional scanning (Ala‐scan) , or positional scanning in which each of the amino acids in the appropriate peptide are replaced, one at a time, by the other 19 natural amino acids, whereas the rest of the sequence remains the same , mixture of peptide libraries with positional scanning .…”