Altering the Modular Architecture of Galectins Affects its Binding with Synthetic α-Dystroglycan O-Mannosylated Core M1 Glycoconjugates In situ.

The 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. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.

Clinical utility of a serum glycome analysis in patients with colorectal cancer.

BACKGROUND 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. The levels of glycans in the serum were examined with a comprehensive, quantitative, high-throughput unique glycome analysis, and their diagnostic and prognostic abilities were evaluated. RESULTS: Among 34 stably detected glycans, nine were differentially expressed between CRC and CRA. Serum levels of hybrid type glycans were increased in patients with CRC compared with those with CRA (P < 0.001), and both hybrid-type and multi-antennary glycans were significantly increased in advanced cancer cases. The glycan, m/z 1914, showed the highest diagnostic value among the decreased glycans, whereas m/z 1708 showed the highest among the increased glycans. The glycan ratio m/z 1708/1914 showed a higher area under the receiver operating characteristic curve (0.889) than any other single glycan or conventional tumor marker, such as carcinoembryonic antigen (0.766, P = 0.040) and carbohydrate antigen 19-9 (0.615, P < 0.001). High m/z 1708/1914 was also correlated with an advanced cancer stage and short overall survival. CONCLUSION: Serum glycans, especially the m/z 1708/1914 ratio, were useful for the diagnosis, staging, and prognosis prediction of CRC.

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody 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. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Effect of Site-Specific O-Glycosylation on the Structural Behavior of NOTCH1 Receptor Extracellular EGF-like Domains 11 and 10.

Human 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. Our strategy enabled us to synthesize four EGF11 and five EGF10 modules. The specific O-glycosylation states affected in vitro folding of EGF10 more than EGF11, while calcium ion had a larger effect on EGF11 folding. Comprehensive NMR studies shed light on the new type "sugar bridges" crosslinking Thr-O-GlcNAc in the consensus sequence C5-X-X-G-X-(T/S)-G-X-X-C6 and an amino acid in the hinge region between the domains, 445Thr-O-GlcNAc-IIe451 in domain 11 and 405Thr-O-GlcNAc-Gln411 in domain 10, respectively.

Serum N-glycan profiling can predict biopsy-proven graft rejection after living kidney transplantation.

BACKGROUND: 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 developed an integrated score to predict graft rejection based on a combination of age, gender, immunological risk factors, and serum N-glycan levels at post-KT day D1 and D7. Rejection-free survival rates stratified by the sum of integrated scores (D1 + D7) were evaluated using Kaplan-Meier curves. RESULTS: Of 174, 52 showed graft rejection (Rejection-pos. group) and 122 recipients did not show graft rejection (Rejection-neg. group). The integrated scores were significantly higher in the Rejection-pos. group than those of the Rejection-neg. group. Area-under-curve (AUC) value of integrated scores at post-KT D1, and D7 were 0.84 and 0.84, respectively. The sum of integrated scores (D1 + D7) ≥ 0.50 identified graft rejection with 81% sensitivity and 80% specificity; with an AUC value of 0.87. Recipients with higher sum of integrated scores (D1 + D7 ≥ 0.5) had significantly shorter rejection-free survival than those with lower scores. CONCLUSION: Evaluation of serum N-glycosylation profiles can identify recipients who are prone to rejection.

Exploring serum and immunoglobulin G N-glycome as diagnostic biomarkers for early detection of breast cancer in Ethiopian women.

BACKGROUND: 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. METHODS: We employed a comprehensive glycomics approach by integrating glycoblotting-based glycan purification with MALDI-TOF/MS based quantitative analysis. Sera of BC patients belonging to stages I-IV and normal controls (NC) were collected from Ethiopian women during 2015-2016. IgG was purified by affinity chromatography using protein G spin plate and further subjected to glycoblotting for glycan release. Mass spectral data were further processed and evaluated rigorously, using various bioinformatics and statistical tools. RESULTS: Out of 35 N-glycans that were significantly up-regulated in the sera of all BC patients compared to the NC, 17 complex type N-glycans showed profound expression abundance and diagnostic potential (AUC = 0.8-1) for the early stage (I and II) BC patients. Most of these glycans were core-fucosylated, multiply branched and sialylated structures, whose abundance has been strongly associated with greater invasive and metastatic potential of cancer. N-glycans quantified form IgG confirmed their abundance in BC patients, of which two core-fucosylated and agalactosylated glycans (m/z 1591, 1794) could specifically distinguish (AUC = 0.944 and 0.921, p ≤ 0.001) stage II patients from NC. Abundance of such structural features in IgG is associated with a decrease in its immunosuppressive potential towards tumor cells, which in part may correlate with the aggressive nature of BC commonly noticed in black population. CONCLUSIONS: Our comprehensive study has addressed for the first time both whole serum and IgG N-glycosylation signatures of native black women suffering from BC and revealed novel glyco-biomarkers with marked overexpression and distinguishing ability at early stage patients. Further studies on direct identification of the intact glycoproteins using a glycoprteomics approach will provide a deeper understanding of specific biomarkers towards their clinical utility.

Synthetic glycopeptides reveal specific binding pattern and conformational change at O-mannosylated position of α-dystroglycan by POMGnT1 catalyzed GlcNAc modification.

Structural and functional effects of core M1 type glycan modification catalyzed by protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) were investigated using a core M1 glycoform focused library of an α-dystroglycan fragment, 372TRGAIIQTPTLGPIQPTRV390. 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. No long-range NOE signals of glycan-amino acid nor inter amino acid indicate the conformational change is induced by steric hindrance of core M1, the sole 1,2-O-modified form among protein binding sugar residue found in mammals.

Antiadhesive Nanosomes Facilitate Targeting of the Lysosomal GlcNAc Salvage Pathway through Derailed Cancer Endocytosis.

Activated 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. Here we demonstrate that "nanosomes" made of core metals covered by an antiadhesive mixed self-assembled monolayer allow for avoiding nonspecific surface protein corona and targeted molecular delivery through activated endocytosis. Nanosomes carrying suicide substrates showed that lysosomal glycosidases such as ß-hexosaminidase and ß-galactosidase in cancer cells are promising targets for novel anticancer therapeutic nanomedicine that induce apoptotic cell death through lysosomal membrane permeabilization. The advantage of this method is evident because multivalent surface loading by antiadhesive nanosomes makes it possible to highlight "weak interactions" such as carbohydrate-lectin interactions independent of surface protein corona.

Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

Glycans, 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. growth and invasion. However, their roles in irradiation-tolerant cancer cells have not yet been elucidated. In this study, we show that specific glycosphingolipids are highly expressed in invasive, irradiation-tolerant lung cancer cells. Particularly, the glycosphingolipid GM2 contributes to the development of an invasive phenotype in these lung cancer cells. Our results suggest that glycosphingolipids, including GM2, are implicated in the regulation of invasiveness in irradiation-tolerant lung cancer cells and may therefore serve as potential therapeutic targets for lung cancers following radiotherapy.Key words: glycosphingolipids, GM2, invasion, lung cancer cells, radiotherapy.

Chemical Synthesis Demonstrates That Dynamic O-Glycosylation Regulates the Folding and Functional Conformation of a Pivotal EGF12 Domain of the Human NOTCH1 Receptor.

The interaction of the human NOTCH1 receptor and its ligands is a crucial step in initiating the intracellular signal transductions, in which O-glycosylation of the extracellular EGF-like domain strongly affects multiple aspects of cell differentiation, development, and cancer biology. However, consequences of biosynthetic O-glycosylation processes in the endoplasmic reticulum (ER) and Golgi on the folding of EGF domains remain unclear. Synthetic human NOTCH1 EGF12 modules allow for new insight into the crucial roles of O-glycosylation in the folding and conformation of this pivotal domain. Here, we show for the first time that predominant O-glucosylation at Ser458 facilitates proper folding of the EGF12 domain in the presence of calcium ion, while the nonglycosylated linear EGF12 peptide affords large amounts of misfolded products (>50%) during in vitro oxidative folding. Strikingly, O-fucosylation at Thr466 prior to O-glucosylation at Ser458 totally impedes folding of EGF12 independent of calcium ion, whereas modification of the Fucα1→ moiety with ß-linked GlcNAc dramatically enhances folding efficiency. In addition, we elicit that extension of the Glcß1→ moiety with xyloses is a negative-regulation mechanism in the folding of EGF12 when synthesis of a trisaccharide (Xylα1→3Xylα1→3Glcß1→) dominates over the posttranslational modification at Thr466. Comprehensive nuclear magnetic resonance studies of correctly folded EGF12 modules demonstrate that noncovalently bonded bridges between sugars and peptide moieties, namely sugar bridges, contribute independently to the stabilization of the antiparallel ß-sheet in the ligand-binding region. Our results provide evidence that the dynamic O-glycosylation status of the EGF12 domain elaborated in the ER and Golgi strongly affects folding and trafficking of the human NOTCH1 receptor.

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery.

BACKGROUND: Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers. METHODS: We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). RESULTS: The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. CONCLUSION: Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. GENERAL SIGNIFICANCE: The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal control sera can facilitate the discovery research of highly sensitive and reliable serum biomarkers for an early diagnosis of AD. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

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.

An Efficient Glycoblotting-Based Analysis of Oxidized Lipids in Liposomes and a Lipoprotein.

Although widely occurring lipid oxidation, which is triggered by reactive oxygen species (ROS), produces a variety of oxidized lipids, practical methods to efficiently analyze oxidized lipids remain elusive. Herein, it is shown that the glycoblotting platform can be used to analyze oxidized lipids. Analysis is based on the principle that lipid aldehydes, one of the oxidized lipid species, can be captured selectively, enriched, and detected. Moreover, 3-methyl-1-p-tolyltriazene (MTT) methylates phosphoric and carboxylic acids, and this MTT-mediated methylation is, in combination with conventional tandem mass spectrometry (MS/MS) analysis, an effective method for the structural analysis of oxidized lipids. By using three classes of standards, liposomes, and a lipoprotein, it is demonstrated that glycoblotting represents a powerful approach for focused lipidomics, even in complex macromolecules.

Serum Aberrant N-Glycan Profile as a Marker Associated with Early Antibody-Mediated Rejection in Patients Receiving a Living Donor Kidney Transplant.

We determined if the serum N-glycan profile can be used as a diagnostic marker of antibody-mediated rejection (ABMR) in living donor kidney transplant (LKTx) recipients. Glycoblotting, combined with mass spectrometry, was used to retrospectively examine N-glycan levels in the postoperative sera of 197 LKTx recipients of whom 16 recipients had ABMR with or without T-cell-mediated rejection (TCMR), 40 recipients had TCMR, and 141 recipients had no adverse events. Multivariate discriminant analysis for prediction of ABMR was performed by inputting an ABMR event as an explanatory variable and sex, age, and serum N-glycan level as objective variables. The N-glycan score was calculated by multiplying the level of candidate objective variables by objective function values. The ABMR predictive performance of the N-glycan score was assessed by receiver operator characteristic curve and Kaplan-Meier curve analyses. The N-glycan score discriminated ABMR with 81.25% sensitivity, 87.85% specificity, and an area under the curve (AUC) of 0.892 that was far superior to that of preformed donor-specific antibody status (AUC, 0.761). Recipients with N-glycan-positive scores >0.8770 had significantly shorter ABMR survival than that of recipients with N-glycan-negative scores. Although the limitations of our study includ its small sample size and retrospective nature, the serum N-glycan score may contribute to prediction of ABMR.

Aberrant N-Glycosylation Profile of Serum Immunoglobulins is a Diagnostic Biomarker of Urothelial Carcinomas.

The aim of this study to determine whether the aberrant N-glycosylated serum immunoglobulins (Igs) can be applied as a diagnostic marker of urothelial carcinoma (UC). Between 2009 and 2016, we randomly obtained serum available from 237 UC and also 96 prostate cancer as other cancer controls from our serum bank and also obtained-from 339 healthy volunteers (HV)-controls obtained from community-dwelling volunteers in Iwaki Health Promotion Project. A total of 32 types of N-glycan levels on Igs were determined by high-throughput N-glycomics and analyzed by multivariable discriminant analysis. We found five UC-associated aberrant N-glycans changes on Igs and also found that asialo-bisecting GlcNAc type N-glycan on Igs were significantly accumulated in UC patients. The diagnostic N-glycan Score (dNGScore) established by combination of five N-glycans on Igs discriminated UC patients from HV and prostate cancer (PC) patients with 92.8% sensitivity and 97.2% specificity. The area under the curve (AUC) for of the dNGScore was 0.969 for UC detection that was much superior to that of urine cytology (AUC, 0.707) and hematuria (AUC, 0.892). Furthermore, dNGScore can detect hematuria and urine cytology negative patients. The dNGscore based on aberrant N-glycosylation signatures of Igs were found to be promising diagnostic biomarkers of UCs.

Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O-Glycans in the Ligand-Binding Region.

The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a ß-D-glucopyranose-initiated glycan (Xylα1 → 3Xylα1 → 3Glcß1 →) at Ser458 and α-L-fucopyranose-initiated glycan (Neu5Acα2 → 3Galß1 → 4GlcNAcß1 → 3Fucα1 →) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel ß-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.

A comprehensive glycome profiling of Huntington's disease transgenic mice.

BACKGROUND: Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers. METHODS: Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice. RESULTS: We estimated the structure and expression levels of 87 and 58N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice. CONCLUSION: Total glycome expression levels are significantly different between HD transgenic and control group mice. GENERAL SIGNIFICANCE: Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era.

Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases.

Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macromolecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice.

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6 Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

Alteration of serum N-glycan profile in patients with autoimmune pancreatitis.

OBJECTIVES: The aims of this study were to determine the change in whole-serum N-glycan profile in autoimmune pancreatitis (AIP) patients and to investigate its clinical utility. METHODS: We collected serum from 21 AIP patients before any treatment, and from 60 healthy volunteers (HLTs). Serum glycan profile was measured by comprehensive and quantitative high-throughput glycome analysis. RESULTS: Of the 53 glycans detected, 14 were differentially expressed in AIP patients. Pathway analysis demonstrated that agalactosyl and monogalactosyl bi-antennary glycans were elevated in AIP patients. Among the 14 glycans, #3410, #3510, and #4510 showed high area under receiver operating characteristic (AUROC) values (0.955, 0.964, and 0.968 respectively) for the diagnosis of AIP. These three glycans were mainly bound to immunoglobulin G; however, their serum levels were significantly higher, even in AIP patients who showed lower serum IgG4 levels, than in HLTs. CONCLUSIONS: We demonstrated, for the first time, whole-serum glycan profiles of AIP patients and showed that the levels of glycans #3410, #3510, and #4510 were increased in AIP patients. These glycans might be valuable biomarkers of AIP.