Synthesis and Structural Model of an α(2,6)‐Sialyl‐T Glycosylated MUC1 Eicosapeptide under Physiological Conditions

Abstract

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, a 20-residue peptide (GSTAPPAHGVTSAPDTRPAP) representing the full length tandem repeat sequence of the human mucin MUC1 and its analogue glycosylated with the (2,6)-sialyl-T antigen on Thr11, were prepared and investigated by NMR and molecular modeling. The peptides contain both the GVTSAP sequence, which is an effective substrate for GalNAc transferases, and the PDTRP fragment, a known epitope recognized by several anti-MUC1 monoclonal antibodies. It has been shown that glycosylation of threonine in the GVTSAP sequence is a prerequisite for subsequent glycosylation of the serine at GVTSAP. Furthermore, carbohydrates serve as additional epitopes for MUC1 antibodies. Investigation of the solution structure of the sialyl-T glycoeicosapeptide in a H(2)O/D(2)O mixture (9:1) under physiological conditions (25 degrees C and pH 6.5) revealed that the attachment of the saccharide side-chain affects the conformational equilibrium of the peptide backbone near the glycosylated Thr11 residue. For the GVTSA region, an extended, rod-like secondary structure was found by restrained molecular dynamics simulation. The APDTR region formed a turn structure which is more flexibly organized. Taken together, the joined sequence GVTSAPDTR represents the largest structural model of MUC1 derived glycopeptides analyzed so far.

Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine

“…Conformational studies by NMR complemented by light scattering measurements have indicated that deglycosylation of MUC1 results in a less extended and more globular structure (50). Similar studies using MUC1-related O-glycopeptides have shown that the carbohydrate moieties exert conformational effects (10)(11)(12), which may provide a rationale for differences in immune responses. Also, the use of glycosylated 1 led to the efficient activation of CTLs, which were able to recognize glycosylated and unglycosylated structures, with the former ones being preferred.…”

“…Furthermore, modification of the peptides with αGalNAc (Tn-antigen) led to stronger antibody binding. It has been proposed that the improved binding is due to saccharide induced conformational change of the peptide backbone (10)(11)(12).…”

“…In general, these strategies have failed to elicit effective immune responses to MUC1-expressing cancer cells, probably due to the conformational disparities between nonglycosylated vaccine sequences and tumor-expressed, aberrantly glycosylated MUC1 (10)(11)(12). The immunogenicity of carbohydrate epitopes (Tn-or sialosyl-Tn) conjugated to an antigenically irrelevant carrier protein has been examined in mice, however, these constructs elicited only modest IgM and IgG antibody responses (28)(29)(30)(31).…”

Vollsynthetische Vakzinen aus tumorassoziierten MUC1‐Glycopeptiden und einem Lipopeptid‐Liganden des Toll‐like Rezeptors 2

“…Boons et al [5] berichteten kürzlich über eine Vakzine, in der ein Glycoundecapeptid mit monosaccharidischer T N -Antigen-Seitenkette aus dem Tandem-Repeat von MUC1 direkt mit einem T-Zellepitop aus Poliovirus [6] und dem oben genannte TLR2-Agonisten verknüpft ist und durch die in Mäusen selektive Antiseren induziert wurden. In der Synthese dieser Vakzine wurde die selektive Entfernung der O-Acetylgruppen aus dem N-Acetylgalactosaminteil durch Umesterung mit Hydrazin in Methanol [7] Um den Einfluss des an basischen Gruppen reichen Lipopeptids auf die Konformation des MUC1-Glycopeptids [8] zu minimieren, wurde ein Oligoethylenglycol-Spacer zwischen TLR2-Ligand und B-Zellepitop eingebaut. Trägt die aktivierte Carbonsäureeinheit des Lipopeptids nur säurela-bile Schutzgruppen und ist das Glycopeptid im Kohlenhydrat bereits deblockiert, dann werden beim abschließenden Deblockieren die Palmitoylester nicht angegriffen, sodass eine reine, synthetische Vakzine erhalten wird.…”

Synthetic Vaccines of Tumor‐Associated Glycopeptide Antigens by Immune‐Compatible Thioether Linkage to Bovine Serum Albumin

“…[23] Therefore, the development of antitumor vaccines requires nonimmunogenic linkers. As the specificity of the glycopeptide antigens obviously depends upon their conformation, [24] such linker structures should contain as few hydrogen-bond donors and acceptors as possible but should still promote water solubility. Alkyl thioethers are expected to fulfill these requirements.…”