The Nature and Sequence of the Amino Acid Aglycone Strongly Modulates the Conformation and Dynamics Effects of Tn Antigen's Clusters

Abstract

Synthetic oligosaccharide vaccines based on carbohydrate epitopes are currently being evaluated as potential immunotherapeutics in the treatment of cancer. In an effort to study the role that the amino acid moiety (L-serine and/or L-threonine residues) plays on the global shape of the resulting glycopeptides and on the dynamics of the carbohydrate moiety, diverse glycopeptides based on the Tn antigen have been synthesized and studied in aqueous solution by combining NMR spectroscopic experiments and molecular dynamics simulations. Our results demonstrate that although the effect of the clustering of Tn on the peptide backbone is not remarkable, it substantially modifies the dynamics, and thus, the presentation features of the carbohydrate moiety. In fact, the selected sequence has a crucial influence on both the orientation and flexibility of the sugar region. Thus, although a serine-threonine pair shows a well-defined spatial disposition of the Tn epitopes, its analogue sequence threonine-serine allows a certain degree of mobility that could favor the interaction with a diversity of receptors without a major energy penalty. These features can be explained by attending to the different conformational behavior of the glycosidic linkage of threonine-containing glycopeptides when compared with those of the serine analogues. On this basis, and taking into account that these carbohydrates interact with components of the immune system, these findings could have implications for further design of new cancer vaccines.

Principles of mucin structure: implications for the rational design of cancer vaccines derived from MUC1-glycopeptides

“…A hydrogen bond between the NH proton of the glycosylated Thr and the carbonyl group of the sugar, along with a steric effect provoked by this residue, could be responsible for the conformational change in the peptide backbone. A similar deduction was reached for clusters of the Tn 29,30 or STn antigens. 31 Strikingly, the study conducted by Campbell's group on glycopeptides derived from the sequence GVT 3 S 4 APDTRPAPGSTA, with singly or doubly Tn-glycosylated versions at T3 or/and S4, points out that the hydrogen bond interaction should be stronger for the aGaNAc-Thr pair than for the a-GalNAc-Ser set, as suggested by the temperature coefficient data.…”

“…Corzana and co-workers have conducted conformational analyses of short glycopeptides derived from MUC1. 30,[36][37][38] As a result of the extra flexibility displayed by the peptide motif in this kind of compounds, the NOE-derived distances and coupling constants are used with MD-tar. In stark contrast to classical constraints, this technique provides a distribution of low-energy conformers that can quantitatively reproduce the NMR spectroscopic data.…”

“…37 Besides, as reported by Campbell et al, 27 different conformational behaviour between the two Tn antigens (a-O-GalNAc-Ser/Thr) was observed in solution for various investigated glycopeptides (see Section 2.3). 15,30 We conclude this section by highlighting that a-O-glycosylation forces the peptide backbone into a stable conformation, which minimises the steric effects by further modification, and serves as the preferred peptide conformation of the epitopes recognised by some mAbs. 22 It seems that the sugar elongation from the first GalNAc residue does not influence peptide conformation.…”

Microarray Analysis of Antibodies Induced with Synthetic Antitumor Vaccines: Specificity against Diverse Mucin Core Structures

“…If the clustered T N ‐antigen GS*T*A epitope was present, then additional glycosylation with various single core glycans in PDT*R did not undermine the antibody affinity (peptides a – d , Figure ). Clustered S*T* glycosylation has been shown by NMR structural studies to fix the spatial disposition and flexibility of the carbohydrate moiety on the peptide backbone . The high antibody specificity may be a direct consequence of the hapten clustering.…”

Peptide-tethered monodentate and chelating histidylidene metal complexes: synthesis and application in catalytic hydrosilylation

“…Cleavage of the Boc protecting group at the N-terminus was effected with methanolic HCl thus providing the dihydrochloride salt H-His(Me)-OMe•2HCl 2. Deprotection with TFA was equally successful, 14 however the obtained trifluoroacetate salt was significantly more hygroscopic than the chloride salt and hence less easy to handle. The C-protected methyl-histidine 2 was subsequently used for amino acid coupling reactions.…”