Pancreatic cancer is highly metastatic and has poor prognosis, mainly due to delayed detection, often after metastasis has occurred. A novel method to enable early detection and disease intervention is strongly needed. Here we unveil for the first time that pancreatic cancer cells (PANC-1) and secreted exosomes express MUC1 bearing cancer-relevant dynamic epitopes recognized specifically by an anti-MUC1 antibody (SN-131), which binds specifically core 1 but not core 2 type -glycans found in normal cells.
View Article and Find Full Text PDFThe multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively.
View Article and Find Full Text PDFThis study presents "mouse tissue glycome atlas" representing the profiles of major N-glycans of mouse glycoproteins that may define their essential functions in the surface glycocalyx of mouse organs/tissues and serum-derived extracellular vesicles (exosomes). Cell surface glycocalyx composed of a variety of N-glycans attached covalently to the membrane proteins, notably characteristic "N-glycosylation patterns" of the glycocalyx, plays a critical role for the regulation of cell differentiation, cell adhesion, homeostatic immune response, and biodistribution of secreted exosomes. Given that the integrity of cell surface glycocalyx correlates significantly with maintenance of the cellular morphology and homeostatic immune functions, dynamic alterations of N-glycosylation patterns in the normal glycocalyx caused by cellular abnormalities may serve as highly sensitive and promising biomarkers.
View Article and Find Full Text PDFDystroglycan (DG), which constitutes a part of the dystrophin-glycoprotein complex, connects the extracellular matrix to the cytoskeleton. The matriglycans presented by the extracellular α-DG serve as a contact point with extracellular matrix proteins (ECM) containing laminin G-like domains, providing cellular stability. However, it remains unknown whether core M1 (GlcNAcβ1-2Man) structures can serve as ligands among the various O-Mannosylated glycans.
View Article and Find Full Text PDFClusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg with a biantennary disialyl -glycan (A2G2S2 or FA2G2S2) at Asn374 differed significantly prior to and after curative nephrectomy for clear cell renal cell carcinoma (RCC) patients motivated us to verify the feasibility of this glycopeptide as a novel biomarker of RCC. To determine the precise -glycan structure attached to Asn374, whether A2G2S2 is composed of the Neu5Acα2,3Gal or/and the Neu5Acα2,6Gal moiety, we synthesized key glycopeptides having one of the two putative isomers.
View Article and Find Full Text PDFBackground And Aim: Serum glycans are known to be good markers for the early diagnosis and prognostic prediction in many cancers. The aims of this study were to reveal the serum glycan changes comprehensively during the process of carcinogenesis from colorectal adenoma (CRA) to colorectal cancer (CRC) and to evaluate the usefulness of the glycan profiles as clinical markers for CRC.
Methods: Serum samples were obtained from 80 histologically proven CRC and 36 CRA cases.
Despite emerging importance of tumor cells-derived exosomes in cancer metastasis, the heterogeneity of exosome populations has largely hampered systemic characterization of their molecular composition, biogenesis, and functions. This study communicates a novel method for predicting and targeting pre-metastatic sites based on an exosome model "fluorescent cancer glyconanosomes" displaying N-glycans of cultured tumor cells. Glycoblotting by antiadhesive quantum dots provides a nice tool to shed light on the pivotal functions of the glycocalyx reconstructed from four cancer cell types without bias due to other compositions of exosomes.
View Article and Find Full Text PDFThe N‑glycoforms of glycoproteins modify protein function and control a number of biological pathways. The aim of the present study was to investigate the correlation between alterations in N‑glycans and cancer aggressiveness in terms of cancer cell invasion ability. The expression of urokinase‑type plasminogen activator (uPA) and N‑acetylglucosaminyltransferase V (GnT‑V) in liver cancer cell lines was analyzed by western blotting.
View Article and Find Full Text PDFIn many human carcinomas, mucin-1 (MUC1) is overexpressed and aberrantly glycosylated, resulting in the exposure of previously hidden antigens. This generates new patient antibody profiles that can be used in cancer diagnosis. In the present study, we focused on the MUC1-associated Tn antigen (α--GalNAc-Ser/Thr) and substituted the GalNAc monosaccharide by a glycomimic to identify MUC1-based glycopeptides with increased antigenicity.
View Article and Find Full Text PDFHuman NOTCH1 receptor contains 36 epidermal growth factor (EGF)-like repeating domains, in which O-glycosylation status of EGF12 domain regulates the interaction with Notch ligands. Our interest is focused on the effect of specific O-glycosylation states on the structural behavior of EGF11 and EGF10, because they appeared to affect molecular mechanism in receptor-ligand interactions by inducing some conformational alterations in these domains and/or the regions connecting two domains. To understand the structural impact of various O-glycosylation patterns on the pivotal EGF-like repeats 10, 11, and 12, we performed chemical synthesis and NMR studies of site-specifically O-glycosylated EGF11 and EGF10.
View Article and Find Full Text PDFAberrantly truncated immature -glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature -glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis.
View Article and Find Full Text PDFThe Tn antigen (GalNAc-α-1--Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography.
View Article and Find Full Text PDFAutoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs.
View Article and Find Full Text PDFBackground: To evaluate whether serum N-glycan profile can be used as a diagnostic marker of graft rejection after living-donor kidney transplants (KT).
Methods: We retrospectively examined 174 KT recipients at five medical centers. N-Glycan levels were analyzed in postoperative serum samples using glycoblotting combined with mass spectrometry.
We investigated the diagnostic and prognostic potential of serum N-glycan profiling for castration-resistant prostate cancer (CRPC). We retrospectively investigated serum N-glycan structural analysis by glycoblotting for 287 patients with benign prostatic hyperplasia (BPH), 289 patients with newly diagnosed prostate cancer (PC), 57 patients with PC treated with androgen-deprivation therapy without disease progression (PC-ADT), and 60 patients with CRPC. N-Glycan profiling was compared between the non-CRPC (BPH, newly diagnosed PC and PC-ADT) and CRPC patients.
View Article and Find Full Text PDFPandemic influenza, triggered by the mutation of a highly pathogenic avian influenza virus (IFV), has caused considerable damage to public health. In order to identify such pandemic IFVs, antibodies that specifically recognize viral surface proteins have been widely used. However, since the analysis of a newly discovered virus is time consuming, this delays the availability of suitable detection antibodies, making this approach unsuitable for the early identification of pandemic IFVs.
View Article and Find Full Text PDFActivated endocytosis of extracellular macromolecules and their intracellular trafficking to lysosomes is an essential metabolic mechanism in cancer cells during their rapid proliferation. Cancer cells reuse a vast amount of N-acetylglucosamine (GlcNAc) supplied from the GlcNAc salvage pathway for the accelerated synthesis of a pivotal uridine diphosphate (UDP)-GlcNAc. A method to inactivate key glycosidases in lysosomes could critically contribute to the development of potent anticancer therapy.
View Article and Find Full Text PDFBackground: Alterations in protein glycosylation patterns have potentially been targeted for biomarker discovery in a wide range of diseases including cancer. Although there have been improvements in patient diagnosis and survival for breast cancer (BC), there is no clinically validated serum biomarker for its early diagnosis. Here, we profiled whole serum and purified Immunoglobulin G (IgG) fraction N-glycome towards identification of non-invasive glycan markers of BC.
View Article and Find Full Text PDFStructural and functional effects of core M1 type glycan modification catalyzed by protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) were investigated using a core M1 glycoform focused library of an α-dystroglycan fragment, TRGAIIQTPTLGPIQPTRV. Evanescent-field fluorescence-assisted microarray system illuminated the specific binding pattern of plant lectins that can discriminate the glycan structure of core M1 glycan of the library. The comparative NMR analysis of synthetic glycopeptide having different length of the O-mannosylated glycans revealed a conformational change of the peptide backbone along with core M1 disaccharide formation.
View Article and Find Full Text PDFBackground: Most glycomics studies have focused on understanding disease mechanisms and proposing serum markers for various diseases, yet the influence of ethnic variation on the identified glyco-biomarker remains poorly addressed. This study aimed to investigate the inter-ethnic serum N-glycan variation among US origin control, Japanese, Indian, and Ethiopian healthy volunteers.
Methods: Human serum from 54 healthy subjects of various ethnicity and 11 Japanese hepatocellular carcinoma (HCC) patients were included in the study.
The glycan part of glycoproteins is known to be involved in the structure and modulatory functions of glycoproteins, serving as ligands for cell-to-cell interactions, and as specific ligands for cell-to-microbe interactions. It is believed that intraspecies and interspecies variations in glycosylation exist. As an approach to better understand glycan diversity, egg whites (EW) from four different quail species are studied by the well-established glycoblotting procedure, a glycan enrichment and analysis method.
View Article and Find Full Text PDFGlycans, including glycosphingolipids, are broadly expressed in plasma membranes and play important roles in cell-cell interactions. Recently, it has been revealed that glycans participate in the regulation of malignant phenotypes of cancer cells, e.g.
View Article and Find Full Text PDFGlycans attached to the IgG Fc domain affect strongly biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of therapeutic antibodies. However, molecular mechanism in the glycoform-dependent functional modulation of the IgGs remains elusive. The present study communicates that selected reaction monitoring (SRM)-based assay of tryptic IgG Fc glycopeptides is a promising approach for the characterization of antibodies when combined with structure-defined synthetic Fc peptides having a focused -glycoform as a calibration standard.
View Article and Find Full Text PDFPrevious studies on the large-scale glycomics of more than 3500 human serum samples revealed that the serum glycoproteins of cancer patients often have more dominant and specific glycoforms, namely, branched tri- and tetra-antennary N-glycans, most cancer patient groups than normal control groups. We herein established an efficient synthetic protocol of glycopeptides having highly complicated N-glycan structures that may be generated by direct tryptic digestion of serum glycoproteins. A preliminary selected reaction monitoring (SRM) assay using the synthetic model glycopeptide , Ser-Val-Gln-Glu-Ile-Gln-Ala-Thr-Phe-Phe-Tyr-Phe-Thr-Pro-Asn-Lys-Thr-Glu-Asp-Thr-Ile-Phe-Leu-Arg having an asialo tri-antennary N-glycan at the Asn54 residue as a designated calibration standard allowed for the rapid and absolute quantitation of the tryptic fragment derived from the serum α1-acid glycoprotein carrying a focused N-glycoform of cancer patients and healthy controls in a range between 200 and 1600 fmole μL without any enrichment process for the target glycoprotein.
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