Jun-ichi Furukawa

Comprehensive Approach to Structural and Functional Glycomics Based on Chemoselective Glycoblotting and Sequential Tag Conversion

Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.

High Throughput Quantitative Glycomics and Glycoform-focused Proteomics of Murine Dermis and Epidermis

Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L and γ-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e.g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high mannose glycans.

Quantitative Glycomics of Human Whole Serum Glycoproteins Based on the Standardized Protocol for Liberating N-Glycans

Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis.

Decreased Amyloid-β Pathologies by Intracerebral Loading of Glycosphingolipid-enriched Exosomes in Alzheimer Model Mice

Background: Exosome, a type of extracellular vesicles, can associate with Aβ in vitro. Results: Intracerebrally injected exosomes trapped Aβ on surface glycosphingolipids and transported it into microglia in AD mouse brains, resulting in reductions in Aβ pathology. Conclusion: Exogenous exosomes act as potent scavengers for Aβ in mouse brains. Significance: The findings provide a novel therapeutic approach for AD. Elevated levels of amyloid-β peptide (Aβ) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aβ can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aβ metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aβ and with the associated Aβ were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-β precursor protein transgenic mice resulted in marked reductions in Aβ levels, amyloid depositions, and Aβ-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aβ binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aβ by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aβ clearance in the central nervous system. Improving Aβ clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.

BlotGlycoABC™, an Integrated Glycoblotting Technique for Rapid and Large Scale Clinical Glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an “all-in-one” protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC™ bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.

Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.

Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

A potential function for neuronal exosomes: Sequestering intracerebral amyloid‐β peptide

Elevated amyloid‐β peptide (Aβ) in brain contributes to Alzheimers disease (AD) pathogenesis. We demonstrated the presence of exosome‐associated Aβ in the cerebrospinal fluid (CSF) of cynomolgus monkeys and APP transgenic mice. The levels of exosome‐associated Aβ notably decreased in the CSF of aging animals. We also determined that neuronal exosomes, but not glial exosomes, had abundant glycosphingolipids and could capture Aβ. Infusion of neuronal exosomes into brains of APP transgenic mice decreased Aβ and amyloid depositions, similarly to what reported previously on neuroblastoma‐derived exosomes. These findings highlight the role of neuronal exosomes in Aβ clearance, and suggest that their downregulation might relate to Aβ accumulation and, ultimately, the development of AD pathology.

Identification of novel serum biomarkers of hepatocellular carcinoma using glycomic analysis.

The altered N‐glycosylation of glycoproteins has been suggested to play an important role in the behavior of malignant cells. Using glycomics technology, we attempted to determine the specific and detailed N‐glycan profile for hepatocellular carcinoma (HCC) and investigate the prognostic capabilities. From 1999 to 2011, 369 patients underwent primary curative hepatectomy in our facility and were followed up for a median of 60.7 months. As normal controls, 26 living Japanese related liver transplantation donors were selected not infected by hepatitis B and C virus. Their mean age was 40.0 and 15 (57.7%) were male. We used a glycoblotting method to purify N‐glycans from preoperative blood samples from this cohort (10 μL serum) which were then identified and quantified using mass spectrometry (MS). Correlations between the N‐glycan levels and the clinicopathologic characteristics and outcomes for these patients were evaluated. Our analysis of the relative areas of all the sugar peaks identified by MS, totaling 67 N‐glycans, revealed that a proportion had higher relative areas in the HCC cases compared with the normal controls. Fourteen of these molecules had an area under the curve of greater than 0.80. Analysis of the correlation between these 14 N‐glycans and surgical outcomes by univariate and multivariate analysis identified G2890 (m/z value, 2890.052) as a significant recurrence factor and G3560 (m/z value, 3560.295) as a significant prognostic factor. G2890 and G3560 were found to be strongly correlated with tumor number, size, and vascular invasion. Conclusion: Quantitative glycoblotting based on whole serum N‐glycan profiling is an effective approach to screening for new biomarkers. The G2890 and G3560 N‐glycans determined by tumor glycomics appear to be promising biomarkers for malignant behavior in HCCs. (HEPATOLOGY 2013;)

Crucial Role of Aspartic Acid at Position 265 in the CH2 Domain for Murine IgG2a and IgG2b Fc-Associated Effector Functions

Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (FcγRIIB and FcγRIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other FcγR (FcγRI and FcγRIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of FcγR and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.