Fayna Garcia-Martin

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor-Associated Mucin Glycopeptides by the Human Macrophage Galactose-Type Lectin

The human macrophage galactose-type lectin (MGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc-α-1-O-Ser/Thr) in mucins. NMR and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca(2+) . NMR data were complemented with molecular dynamics simulations and Corcema-ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn-presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH-π contacts involving exclusively the NHAc moiety.

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

BACKGROUND Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. METHODS A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. RESULTS Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats. CONCLUSION We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. GENERAL SIGNIFICANCE The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.

The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR.

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.

Effects of the multiple O-glycosylation states on antibody recognition of the immunodominant motif in MUC1 extracellular tandem repeats

Antibodies that react with human epithelial cell membrane MUC1 glycoprotein with aberrant glycoforms are promising diagnostic and therapeutic reagents in various cancers and interstitial lung diseases. However, the precise epitopes for anti-MUC1 antibodies have remained unclear. Although the MUC1 extracellular domain has multiple O-glycosylation sites within the tandem repeats, there have been few systematic approaches to determine the effects of the multiple O-glycosylation states, in the context of the disease-relevant epitope regions, on antibody recognition. In this study, we established a comprehensive approach for the characterization of anti-MUC1 antibodies by combining microarray-based epitope profiling and NMR-based conformational analysis of synthetic MUC1 glycopeptides. Epitope mapping analysis using a microarray that displayed 23 synthetic MUC1 glycopeptides revealed that anti-KL6/MUC1 monoclonal antibody (anti-KL6 mAb) has absolute binding specificity with an essential epitope, Pro-Asp-Thr[Neu5Acα(2→3)Galβ(1→3)GalNAcα1→]-Arg-Pro-Ala-Pro, in an ultimately glycoform-specific manner when compared with the other well-studied anti-MUC1 mAbs DF3 and SM3, which are directed against the same Pro-Asp-Thr-Arg (PDTR) motif in the tandem repeats. Multiple O-glycosylations at the neighbouring Ser/Thr residues did not disturb this specific recognition by anti-KL6 mAb, even when modified by sterically hindered core 2-type pentasaccharide moieties (SC2). To our surprise, both DF3 and SM3 exhibited a drastic decrease in binding ability with putative MUC1 fragments with an immunodominant PDTR motif when other glycosylation sites were occupied by Tn antigen (GalNAcα1→) or T antigen [Galβ(1→3)GalNAcα1→]. However, modification at the two adjacent Ser residues by O-glycans that contained ST antigen [Neu5Acα(2→3)Galβ(1→3)GalNAcα1→] resulted, exceptionally, in a substantial enhancement of the affinity of DF3 for the PDTR region. These results demonstrated for the first time that the O-glycosylation states around the immunodominant PDTR motif strongly influence the binding potency and profile of DF3 and SM3. NMR studies of the synthetic MUC1 fragments discovered the molecular mechanisms by which multiple O-glycosylations at the adjacent Ser/Thr residues induce significant conformational alterations in the PDTR motif in a glycoform-dependent manner. Anti-KL6 mAb was proved to be the only anti-MUC1 mAb that can recognise a unique glycopeptidic neo-epitope generated via site-specific posttranslational modification by ST antigen independently from O-glycosylation states at the adjacent Ser/Thr residues within the MUC1 tandem repeats.

H-bonding promotion of peptide solubility and cyclization by fluorinated alcohols

A promotion effect for peptide cyclization by strong H-bonding of fluorinated alcohols was revealed via a synthetic study of a cyclic AFGP skeleton. Combination of fluorinated alcohol-DCM solvent system and DIC-additive system afforded the cyclic hexapeptide skeleton in more than 80% yield. The ratio of intra- vs. inter-peptide condensation depended upon the H-bonding donor strength. This effect was quenched by H-bond acceptor solvents.

Synthesis of oligonucleotide derivatives using ChemMatrix supports

The synthesis of oligonucleotides on poly(ethylene glycol)‐based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide–peptide conjugates and for the ligation of oligonucleotides using Cu+‐catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.

Design and synthesis of FAJANU: a de novo C(2) symmetric cyclopeptide family.

A novel cyclic peptide has been designed from several potent marine cytotoxic peptides, including IB-01212, luzopeptin, triostin, and thiocoraline. The FAJANU scaffold maintains C 2 symmetry, cyclic structure, and the construction of aromatic and aliphatic character at the N- and C-terminal extremes. A first six-member family was previously synthesized and evaluated biologically. Several analogues presented greater activity than IB-01212. Furthermore, on the basis of the most active candidate, we have performed a more exhaustive synthetic and structural analysis: (i) structure-activity relationship provided clues about the key elements in the framework, (ii) NMR assignment confirmed C 2 symmetry, and (iii) confocal images revealed its penetration and cellular localization.

Glycopeptides as Targets for Dendritic Cells: Exploring MUC1 Glycopeptides Binding Profile toward Macrophage Galactose-Type Lectin (MGL) Orthologs

The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL.

Synthetic Mucin-Like Glycopeptides as Versatile Tools to Measure Effects of Glycan Structure/Density/Position on the Interaction with Adhesion/Growth-Regulatory Galectins in Arrays.

Functional pairing of cellular glycoconjugates with tissue lectins is a highly selective process, whose determinative factors have not yet been fully delineated. Glycan structure and modes of presentation, that is, its position and density, can contribute to binding, as different members of a lectin family can regulate degrees of responsiveness to these factors. Using a peptide repeat sequence motif of the glycoprotein mucin-1, the principle of introducing synthetic (glyco)peptides with distinct variations in these three parameters to an array-based screening of tissue lectins is illustrated. Interaction profiles of seven adhesion/growth-regulatory galectins cover the range from intense signals with core 2 pentasaccharides and core 1 binding for galectins-3 and -5 to a lack of binding for galectin-1 and also the galectin-related protein, which was included as a negative control. Remarkably, the two tandem-repeat-type galectins-4 and -8 were distinguished by core 1 sialylation, as the two separated domains were. These results encourage further synthetic elaboration of the glycopeptide library and testing of the network of natural galectins and rationally engineered variants of the lectins.

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.