The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

GalNAc-glycopeptides derived from mucin MUC1 23 are an important class of tumor-associated antigens. α-O-24 glycosylation forces the peptide to adopt an extended 25 conformation in solution, which is far from the structure 26 observed in complexes with a model anti-MUC1 antibody. 27 Herein, we propose a new strategy for designing potent antigen 28 mimics based on modulating peptide/carbohydrate interactions 29 by means of O → S/Se replacement at the glycosidic linkage. 30 These minimal chemical modifications bring about two key 31 structural changes to the glycopeptide. They increase the 32 carbohydrate−peptide distance and change the orientation and 33 dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody 34 binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative 35 to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative 36 bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice 37 without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer 38 cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the 39 mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of 40 tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer 41 diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it 42 may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors. ■ INTRODUCTION44 MUC1 mucin is an O-glycoprotein overexpressed in many 45 tumor tissues. 1−4 Although in healthy cells the backbone of 46 this protein is decorated with complex glycans, in cancer cells J. Am. Chem. Soc. XXXX, XXX, XXX−XXX clr00 | ACSJCA | JCA11.1.4300/W Library-x64 | research.3f (R4.1.i3 HF01:4938 | 2.1) 2018/08/24 11:08:00 | PROD-WS-120 | rq_962204 | 2/12/2019 21:32:37 | 10 | JCA-DEFAULT 47 this backbone carries rather simple and truncated oligosac-48 charides. Consequently, different tumor-associated carbohy-49 drate antigens, such as the Tn determinant (α-O-GalNAc-Ser/ 50 Thr), 5 are presented to the immune system and can be 51 identified by anti-MUC1 antibodies. Peptide fragment Ala-Pro-52 Asp-Thr-Arg-Pro, which includes the immunodominant 53 PDTRP region of MUC1 tandem repeats, 6 constitutes the 54 minimum epitope recognized by these antibodies. 7 Partially 55 glycosylated MUC1 derivatives have been used to prepare 56 immunogenic formulations for the development of therapeutic 57 cancer vaccines. 8−12 Similarly, unnatural glycopeptides that 58 mimic tumor-associated MUC1 can find application as...