Effect of D-amino acid substitution in a mucin 2 epitope on mucin-specific monoclonal antibody recognition.
We have studied the influence of D-amino acid substitution in the flanking region on the antibody recognition of the 19TGTQ22 epitope core in the tandem repeat of mucin 2 (MUC2) glycoprotein. Analogue peptides corresponding to the optimal epitope sequence (16PTPTGTQ22) have been prepared by the replacement of single or multiple L-amino acid residues at the N-terminal part of the molecule. According to previous studies, this portion of the all-L 16PTPTGTQ22 peptide possesses a beta-turn secondary structure important for efficient monoclonal antibody interaction. The binding properties of sequentially modified peptides (pTPTGTQ, ptPTGTQ, ptpTGTQ, and ptptGTQ) have been analyzed by a MUC2 glycoprotein specific monoclonal antibody (MAb 996) using RIA inhibition assay and characterized by IC50 values. At the same time, we have investigated the secondary structure of the compounds by circular dichroism and Fourier transform infrared spectroscopy in solution. Our data showed that the presence of D-amino acid residue(s) at position(s) 16P, 16PT17, or 16PTP18 resulted in gradually decreasing antibody binding, but the replacement of the L-Thr at position 19 almost abolished activity. Parallel with this reduction, changes in the conformer population have been detected. The propensity of the pTPTGTQ peptide to adopt folded, most probably beta-turn, structure in water can be in correlation with its essentially preserved antibody recognition. After further substitution, the peptide still contained beta- and/or gamma-turn folded secondary structural elements. The conformation of peptide ptptGTQ could be characterized mostly by semiextended (polyproline II) and probably classic gamma-turn conformers built up from D residues.
Uray
K
K
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest.
Kajtár
J
J
Vass
E
E
Price
M R
MR
Hollósi
M
M
Hudecz
F
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D017550:Spectroscopy, Fourier Transform Infrared
D000911:Antibodies, Monoclonal; D000939:Epitopes; C524625:MUC2 protein, human; D055262:Mucin-2; D009077:Mucins