Profuse color-evolution based aptasensor for mucin 1 detection utilizing urease-mediated color mixing of the mixed pH indicator.

Mucin 1 is a significant tumor marker, and developing portable and cost-effective methods for its detection is crucial, especially in resource-limited areas. Herein, we developed an innovative approach for mucin 1 detection using a visible multicolor aptasensor. Urease-encapsulated DNA microspheres were used to mediate multicolor change facilitated by the color mixing of the mixed pH indicator, a mixed methyl red and bromocresol green solution. Distinct color changes were exhibited in response to varying mucin 1 concentrations. Notably, the color mixing of the mixed pH indicator was used to display various hues of colors, broadening the range of color variation. And color tonality is much easier to differentiate than color intensity, improving the resolution with naked-eyes. Besides, the variation of color from red to green (a pair of complementary colors) enhanced the color contrast, heightening sensitivity for visual detection. Importantly, the proposed method was successfully applied to detect mucin 1 in real samples, demonstrating a clear differentiation of colors between the samples of healthy individuals and breast cancer patients. The use of a mixed pH indicator as a multichromatic substrate offers the merits of low cost, fast response to pH variation, and plentiful color-evolution. And the incorporation of calcium carbonate microspheres to encapsulate urease ensures stable urease activity and avoids the need for extra urease decoration. The color-mixing dependent strategy opens a new way for multicolor detection of MUC1, characterized by vivid color changes.

Multifunctional Lipidated Protein Carrier with a Built-In Adjuvant as a Universal Vaccine Platform Potently Elevates Immunogenicity of Weak Antigens.

Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated protein as a carrier, in which the TLR1/2 agonist Pam3CSK4 was conjugated to the N-terminus of MUC1-loaded carrier protein BSA through pyridoxal 5'-phosphate-mediated transamination reaction. The resulting Pam3CSK4-BSA-MUC1 conjugate was subsequently incorporated into liposomes, which biomimics the membrane structure of tumor cells. The results indicated that this lipidated protein carrier significantly enhanced antigen uptake by APCs and obviously augmented the retention of the vaccine at the injection site. Compared with the BSA-MUC1 and BSA-MUC1 + Pam3CSK4 groups, Pam3CSK4-BSA-MUC1 evoked 22- and 11-fold increases in MUC1-specific IgG titers. Importantly, Pam3CSK4-BSA-MUC1 elicited robust cellular immunity and significantly inhibited tumor growth. This is the first time that lipidated protein was constructed to enhance antigen immunogenicity, and this universal carrier platform exhibits promise for utilization in various vaccines, holding the potential for further clinical application.

Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing.

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry-based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostic, targeting, and treatment strategies.

Direct observation of glycans bonded to proteins and lipids at the single-molecule level.

Proteins and lipids decorated with glycans are found throughout biological entities, playing roles in biological functions and dysfunctions. Current analytical strategies for these glycan-decorated biomolecules, termed glycoconjugates, rely on ensemble-averaged methods that do not provide a full view of positions and structures of glycans attached at individual sites in a given molecule, especially for glycoproteins. We show single-molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy. Intact glycoconjugate ions from electrospray are soft-landed on a surface for their direct single-molecule imaging. The submolecular imaging resolution corroborated by quantum mechanical modeling unveils whole structures and attachment sites of glycans in glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins densely decorated with glycans.

MUC1 Glycopeptide Vaccine Modified with a GalNAc Glycocluster Targets the Macrophage Galactose C-type Lectin on Dendritic Cells to Elicit an Improved Humoral Response.

Mucin expression and glycosylation patterns on cancer cells differ markedly from healthy cells. Mucin 1 (MUC1) is overexpressed in several solid tumors and presents high levels of aberrant, truncated O-glycans (e.g., Tn antigen). Dendritic cells (DCs) express lectins that bind to these tumor-associated carbohydrate antigens (TACAs) to modulate immune responses. Selectively targeting these receptors with synthetic TACAs is a promising strategy to develop anticancer vaccines and to overcome TACA tolerance. In this work, we prepared, via a solid phase peptide synthesis approach, a modular tripartite vaccine candidate, incorporating a high-affinity glycocluster based on a tetraphenylethylene scaffold, to target the macrophage galactose-type lectin (MGL) on antigen presenting cells. MGL is a C-type lectin receptor that binds Tn antigens and can route them to human leukocyte antigen class II or I, making it an attractive target for anticancer vaccines. Conjugation of the glycocluster to a library of MUC1 glycopeptides bearing the Tn antigen is shown to promote uptake and recognition of the TACA by DCs via MGL. In vivo testing revealed that immunization with the newly designed vaccine construct bearing the GalNAc glycocluster induced a higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone. Additionally, the antibodies obtained bind a library of tumor-associated saccharide structures on MUC1 and MUC1-positive breast cancer cells. Conjugation of a high-affinity ligand for MGL to tumor-associated MUC1 glycopeptide antigens has a synergistic impact on antibody production.

Chemoenzymatic Synthesis of Glycopeptides to Explore the Role of Mucin†1 Glycosylation in Cell Adhesion.

Post-translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O-glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell-adhesion experiments with MCF-7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Helical self-assembly of a mucin segment suggests an evolutionary origin for von Willebrand factor tubules.

The glycoprotein von Willebrand factor (VWF) contributes to hemostasis by stanching injuries in blood vessel walls. A distinctive feature of VWF is its assembly into long, helical tubules in endothelial cells prior to secretion. When VWF is released into the bloodstream, these tubules unfurl to release linear polymers that bind subendothelial collagen at wound sites, recruit platelets, and initiate the clotting cascade. VWF evolved from gel-forming mucins, the polymeric glycoproteins that coat and protect exposed epithelia. Despite the divergent function of VWF in blood vessel repair, sequence conservation and shared domain organization imply that VWF retained key aspects of the mucin bioassembly mechanism. Here, we show using cryo-electron microscopy that the ability to form tubules, a property hitherto thought to have arisen as a VWF adaptation to the vasculature, is a feature of the amino-terminal region of mucin. This segment of the human intestinal gel-forming mucin (MUC2) was found to self-assemble into tubules with a striking resemblance to those of VWF itself. To facilitate a comparison, we determined the residue-resolution structure of tubules formed by the homologous segment of VWF. The structures of the MUC2 and VWF tubules revealed the flexible joints and the intermolecular interactions required for tubule formation. Steric constraints in full-length MUC2 suggest that linear filaments, a previously observed supramolecular assembly form, are more likely than tubules to be the physiological mucin storage intermediate. Nevertheless, MUC2 tubules indicate a possible evolutionary origin for VWF tubules and elucidate design principles present in mucins and VWF.

Design of a Novel Recombinant Multi-Epitope Vaccine against Triple-Negative Breast Cancer

Background: Triple-negative breast cancer (TNBC) is determined by the absence of ERBB2, estrogen and progesterone receptors' expression. Cancer vaccines, as the novel immunotherapy strategies, have emerged as promising tools for treating the advanced stage of TNBC. The aim of this study was to evaluate Carcinoembryonic antigen (CEA), Metadherin (MTDH), and Mucin 1 (MUC-1) proteins as vaccine candidates against TNBC. Methods: In this research, a novel vaccine was designed against TNBC by using different immunoinformatics and bioinformatics approaches. Effective immunodominant epitopes were chosen from three antigenic proteins, namely CEA, MTDH, and MUC-1. Recombinant TLR4 agonists were utilized as an adjuvant to stimulate immune responses. Following the selection of antigens and adjuvants, appropriate linkers were chosen to generate the final recombinant protein. To achieve an excellent 3D model, the best predicted 3D model was required to be refined and validated. To demonstrate whether the vaccine/TLR4 complex is stable or not, we performed docking analysis and dynamic molecular simulation. Result: Immunoinformatics and bioinformatics evaluations of the designed construct demonstrated that this vaccine candidate could effectively be used as a therapeutic armament against TNBC. Conclusion: Bioinformatics studies revealed that the designed vaccine has an acceptable quality. Investigating the effectiveness of this vaccine can be confirmed by supplementary in vitro and in vivo studies.

Nanoparticle conjugated with aptamer anti-MUC1/Y for inflammatory arthritis.

Aptamers may form well-defined three-dimensional structures binding with high affinity and stability to a specific receptor. The aptamer anti-MUC1 isoform Y is one the most used due the affinity to MUC1, which is overexpressed in several types of cancer and inflammation process. In this study we have developed, characterized, in vitro as in vivo evaluated a nanoaptamer (anti-MUC1/Y) as a nanoagent for rheumatoid arthritis treatment. The results showed that a nanoaptamer with a size range of 241 nm was produced. The entrapment efficacy was 90% with a biodistribution showing a high hepatic uptake (>98%). The results in vivo showed a potent effect in arthritis experimental model, especially in low doses. The results corroborate the applicability of this nanosystem for RA treatment.

Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101.

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.

A novel humanized MUC1 antibody-drug conjugate for the treatment of trastuzumab-resistant breast cancer.

Mucin 1 (MUC1) has been regarded as an ideal target for cancer treatment, since it is overexpressed in a variety of different cancers including the majority of breast cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1. In this study, we generated a humanized MUC1 (HzMUC1) antibody from our previously developed MUC1 mouse monoclonal antibody that only recognizes MUC1 on the surface of tumor cells. Furthermore, an antibody-drug conjugate (ADC) was generated by conjugating HzMUC1 with monomethyl auristatin (MMAE), and the efficacy of HzMUC1-MMAE on the MUC1-positive HER2+ breast cancer in vitro and in 'Xenograft' model was tested. Results from western blot analysis and immunoprecipitation revealed that the HzMUC1 antibody did not recognize cell-free MUC1-N in sera from breast cancer patients. Confocal microscopy analysis showed that HzMUC1 antibody bound to MUC1 on the surface of breast cancer cells. Results from mapping experiments suggested that HzMUC1 may recognize an epitope present in the interaction region between MUC1-N and MUC1-C. Results from colony formation assay and flow cytometry demonstrated that HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in trastuzumab-resistant HER2-positive breast cancer cells. Meanwhile, HzMUC1-MMAE significantly reduced the growth of HCC1954 xenograft tumors by inhibiting cell proliferation and enhancing cell death. In conclusion, our results indicate that HzMUC1-ADC is a novel therapeutic drug that can overcome trastuzumab resistance of breast cancer. HzMUC1-ADC should also be an effective therapeutic drug for the treatment of different MUC1-positive cancers in clinic.

MUC1-C Contributes to the Maintenance of Human Embryonic Stem Cells and Promotes Somatic Cell Reprogramming.

Mucin 1 (MUC1) is a transmembrane glycoprotein overexpressed in several cancer cells in which it regulates cell surface properties, tumor invasion, and cell death. Recently, we reported that MUC1-C, the C-terminal subunit of MUC1, is involved in the growth of mouse embryonic stem (ES) cells. However, the functional significance of MUC1-C in human ES cells remains unclear. In this study, we investigated the expression and function of MUC1-C in human ES cells. Based on reverse transcription-polymerase chain reaction, western blotting, and confocal microscopy following immunostaining, undifferentiated human ES cells expressed MUC1-C and the expression level decreased during differentiation. Inhibition of MUC1-C, by the peptide inhibitor GO201 that targets the cytoplasmic domain of MUC1-C (MUC1-CD), reduced cell proliferation and OCT4 protein expression, and promoted cell death. Moreover, the inhibition of MUC1-C increased the intracellular reactive oxygen species (ROS) levels and downregulated expression of glycolysis-related enzymes. These findings indicate that expression and function of MUC1-C are required for stem cell properties involved in cell proliferation, maintenance of pluripotency and optimal ROS levels, and a high glycolytic flux in human ES cells. In addition, forced overexpression of MUC1-CD increased the efficiency of reprogramming from fibroblast cells to induced pluripotent stem cells, suggesting that MUC1-C expression can contribute to the reprogramming process.

MUC1: Structure, Function, and Clinic Application in Epithelial Cancers.

The transmembrane glycoprotein mucin 1 (MUC1) is a mucin family member that has different functions in normal and cancer cells. Owing to its structural and biochemical properties, MUC1 can act as a lubricant, moisturizer, and physical barrier in normal cells. However, in cancer cells, MUC1 often undergoes aberrant glycosylation and overexpression. It is involved in cancer invasion, metastasis, angiogenesis, and apoptosis by virtue of its participation in intracellular signaling processes and the regulation of related biomolecules. This review introduces the biological structure and different roles of MUC1 in normal and cancer cells and the regulatory mechanisms governing these roles. It also evaluates current research progress and the clinical applications of MUC1 in cancer therapy based on its characteristics.

Dual-potential electrochemiluminescence of single luminophore for detection of biomarker based on black phosphorus quantum dots as co-reactant.

Simultaneous cathodic and anodic electrochemiluminescence (ECL) emissions of needle-like nanostructures of Ru(bpy)32+ (RuNDs) as the only luminophore are reported based on different co-reactants. Cathodic ECL was attained from RuNDs/K2S2O8 system, while anodic ECL was achieved from RuNDs/black phosphorus quantum dots (BPQDs) system. Ferrocene attached to the hairpin DNA could quench the cathodic and anodic ECL simultaneously. Subsequently, the ECL signals recovered in the presence of tumor marker mucin 1 (MUC1), which made it possible to quantitatively detect MUC1. The variation of ECL signal was related linearly to the concentrations of MUC1 in the range 20 pg mL-1 to 10 ng mL-1, and the detection limits were calculated to 2.5 pg mL-1 (anodic system, 3σ) and 6.2 pg mL-1 (cathodic system, 3σ), respectively. The recoveries were 97.0%, 105%, and 95.2% obtained from three human serum samples, and the relative standard deviation (RSD) is 5.3%. As a proof of concept, this work realized simultaneous ECL emission of  a single luminophore, which initiates a new thought in biomarker ECL detection beyond the traditional ones. Simultaneous cathodic and anodic ECL emissions of RuNDs were reported based on different co-reactants. Ferrocene could quench the ECL emission in the cathode and the anode simultaneously. Thus, an aptasensor was constructed based on the variation of ECL intensity. As a proof of concept, this work realized simultaneous ECL emission of a single luminophore, which initiates a new thought in biomarker ECL detection beyond the traditional ones by avoiding the false positive signals.

Classification, structural biology, and applications of mucin domain-targeting proteases.

Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high density of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus production and breakdown thus determines the composition of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Additionally, immunoglobulin A (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiology.

Synthesis and immunological evaluation of the unnatural ß-linked mucin-1 Thomsen-Friedenreich conjugate.

MUC1 glycopeptides are attractive antigens for anti-cancer vaccine development. One potential drawback in using the native MUC1 glycopeptide for vaccine design is the instability of the O-glycosyl linkage between the glycan and the peptide backbone to glycosidase. To overcome this challenge, a MUC1 glycopeptide mimic has been synthesized with the galactose-galactosamine disaccharide linked with threonine (Thomsen-Friedenreich or Tf antigen) through an unnatural ß-glycosyl bond. The resulting MUC1-ß-Tf had a much-enhanced stability toward a glycosidase capable of cleaving the glycan from the corresponding MUC1 glycopeptide with the natural α-Tf linkage. The MUC1-ß-Tf was subsequently conjugated with a powerful carrier bacteriophage Qß. The conjugate induced high levels of IgG antibodies in clinically relevant human MUC1 transgenic mice, which cross-recognized not only the natural MUC1-α-Tf glycopeptide but also MUC1 expressing tumor cells, supporting the notion that a simple switch of the stereochemistry of the glycan/peptide linkage can be a strategy for anti-cancer vaccine epitope design for glycopeptides.

Exploring Molecular Contacts of MUC1 at CIN85 Binding Interface to Address Future Drug Design Efforts.

The modulation of protein-protein interactions (PPIs) by small molecules represents a valuable strategy for pharmacological intervention in several human diseases. In this context, computer-aided drug discovery techniques offer useful resources to predict the network of interactions governing the recognition process between protein partners, thus furnishing relevant information for the design of novel PPI modulators. In this work, we focused our attention on the MUC1-CIN85 complex as a crucial PPI controlling cancer progression and metastasis. MUC1 is a transmembrane glycoprotein whose extracellular domain contains a variable number of tandem repeats (VNTRs) regions that are highly glycosylated in normal cells and under-glycosylated in cancer. The hypo-glycosylation fosters the exposure of the backbone to new interactions with other proteins, such as CIN85, that alter the intracellular signalling in tumour cells. Herein, different computational approaches were combined to investigate the molecular recognition pattern of MUC1-CIN85 PPI thus unveiling new structural information useful for the design of MUC1-CIN85 PPI inhibitors as potential anti-metastatic agents.

Highly glycosylated MUC1 mediates high affinity L-selectin binding at the human endometrial surface.

BACKGROUND: Sialyl-Lewis X/L-selectin high affinity binding interactions between transmembrane O-glycosylated mucins proteins and the embryo have been implicated in implantation processes within the human reproductive system. However, the adhesive properties of these mucins at the endometrial cell surface are difficult to resolve due to known discrepancies between in vivo models and the human reproductive system and a lack of sensitivity in current in vitro models. To overcome these limitations, an in vitro model of the human endometrial epithelial was interrogated with single molecule force spectroscopy (SMFS) to delineate the molecular configurations of mucin proteins that mediate the high affinity L-selectin binding required for human embryo implantation. RESULTS: This study reveals that MUC1 contributes to both the intrinsic and extrinsic adhesive properties of the HEC-1 cellular surface. High expression of MUC1 on the cell surface led to a significantly increased intrinsic adhesion force (148 pN vs. 271 pN, p < 0.001), whereas this adhesion force was significantly reduced (271 pN vs. 118 pN, p < 0.001) following siRNA mediated MUC1 ablation. Whilst high expression of MUC1 displaying elevated glycosylation led to strong extrinsic (> 400 pN) L-selectin binding at the cell surface, low expression of MUC1 with reduced glycosylation resulted in significantly less (≤200 pN) binding events. CONCLUSIONS: An optimal level of MUC1 together with highly glycosylated decoration of the protein is critical for high affinity L-selectin binding. This study demonstrates that MUC1 contributes to cellular adhesive properties which may function to facilitate trophoblast binding to the endometrial cell surface through the L-selectin/sialyl-Lewis x adhesion system subsequent to implantation.

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin.

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.