[Show abstract][Hide abstract] ABSTRACT: The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.
Full-text · Article · Oct 2015 · Glycoconjugate Journal
[Show abstract][Hide abstract] ABSTRACT: β-Site amyloid precursor protein cleaving enzyme-1 (BACE1) is a protease essential for amyloid-β (Aβ) production in Alzheimer's disease (AD). BACE1 protein is known to be upregulated by oxidative stress-inducing stimuli but the mechanism for this upregulation remains to be clarified. We have recently found that BACE1 is modified with bisecting GlcNAc by N -acetylglucosaminyltransferase-III (GnT-III, encoded by Mgat3 gene) and that GnT-III deficiency reduces Aβ plaque formation in the brain by accelerating lysosomal degradation of BACE1. Therefore, we hypothesized that bisecting GlcNAc would stabilize BACE1 protein upon oxidative stress. In this study, we first show that Aβ deposition in the mouse brain induces oxidative stress concomitant with an increase in levels of BACE1 and bisecting GlcNAc. Furthermore, pro-oxidant treatment induces expression of BACE1 protein in wild-type mouse embryonic fibroblasts (MEFs), whereas it reduces BACE1 protein in GnT-III ( Mgat3 ) KO MEFs by accelerating lysosomal degradation of BACE1. We purified BACE1 from Neuro2A cells and performed LC-ESI-MS analysis for BACE1-derived glycopeptides and mapped bisecting GlcNAc-modified sites on BACE1. Point mutations at two N -glycosylation sites (N153 and N223) abolish the bisecting GlcNAc modification on BACE1. These mutations almost cancelled the enhanced BACE1 degradation seen in Mgat3(-/-) MEFs, indicating that bisecting GlcNAc on BACE1 indeed regulates its degradation. Finally, we show that traumatic brain injury-induced BACE1 upregulation is significantly suppressed in Mgat3(-/-) brain. These results highlight the role of bisecting GlcNAc in oxidative stress-induced BACE1 expression and offer a novel glycan-targeted strategy for suppressing Aβ generation.
No preview · Article · Oct 2015 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: In order to verify the protein enriched from pooled human sera to be a lung-specific protein surfactant protein-D (SP-D), we performed peptide mass fingerprinting (PMF)-based protein identification. MASCOT search results of the obtained PMF unequivocally demonstrated that it is identical to human SP-D. Meanwhile, we performed MALDI-QIT-TOF mass spectrometry-based N-glycomic analysis of the recombinant human SP-D produced in murine myeloma cells. The obtained mass spectra of N-glycans from the recombinant SP-D demonstrated that the recombinant protein is almost exclusively modified with core-fucosylated N-glycans. .
[Show abstract][Hide abstract] ABSTRACT: Biological significance:
It has been proposed that serum SP-D concentrations are predictive of COPD pathogenesis, but distinguishing between COPD patients and healthy individuals to establish a clear cut-off value is difficult because smoking status highly affects circulating SP-D levels. Herein, we focused on N-glycosylation in SP-D and examined whether or not N-glycosylation patterns in SP-D are associated with the pathogenesis of COPD. We performed an N-glycomic analysis of human serum SP-D and the results show that a core-fucose is present in its N-glycan. We also found that the N-glycosylation in serum SP-D was indeed altered in COPD, that is, fucosylation levels including core-fucosylation are significantly increased in COPD patients compared with non-COPD smokers. The severity of emphysema was positively associated with fucosylation levels in serum SP-D in smokers. Our findings shed new light on the discovery and/or development of a useful biomarker based on glycosylation changes for diagnosing COPD.
No preview · Article · Jul 2015 · Journal of proteomics
[Show abstract][Hide abstract] ABSTRACT: E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.Oncogene advance online publication, 20 July 2015; doi:10.1038/onc.2015.225.
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand-protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest that 18 is a broad-spectrum MMP inhibitor.
No preview · Article · May 2015 · Bioscience Biotechnology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: While many examples have been reported that glycoclusters interact with target lectins more strongly than single molecules of glycans, through multivalency effects, literature examples to support lectin interactions/modulations on cell surface and in live animals is quite rare. Our N-glycoclusters, which were efficiently prepared by immobilizing 16 molecules of the asparagine-linked glycans (N-glycans) onto a lysine-based dendron template through histidine-mediated Huisgen cycloaddition, were shown to efficiently detect platelet endothelial cell adhesion molecule (PECAM) on human umbilical vein endothelial cells (HUVEC) as a α(2-6)-sialylated oligosaccharides recognizing lectin. Furthermore, the identity of the N-glycans on our N-glycoclusters allowed control over organ-selective accumulation and serum clearance properties when intravenously injected into mice.
No preview · Article · May 2015 · Glycoconjugate Journal
[Show abstract][Hide abstract] ABSTRACT: O-GlcNAcylation is a reversible post-translational modification. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. More recent evidence indicates that regulation of O-GlcNAcylation is important for inflammatory diseases and tumorigenesis. In this study, we revealed that O-GlcNAcylation was increased in the colonic tissues of dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models. Moreover, the O-GlcNAcylation level was elevated in human CAC tissues compared with matched normal counterparts. To investigate the functional role of O-GlcNAcylation in colitis, we used OGA heterozygote mice, which have an increased level of O-GlcNAcylation. OGA+/- mice have higher susceptibility to DSS-induced colitis than OGA+/+ mice. OGA +/- mice exhibited a higher incidence of colon tumors than OGA+/+ mice. In molecular studies, elevated O-GlcNAc levels were shown to enhance the activation of NF-κB signaling through increasing the binding of RelA/p65 to its target promoters. We also found that Thr-322 and Thr352 in the p65-O-GlcNAcylation sites are critical for p65 promoter binding. These results suggest that the elevated O-GlcNAcylation level in colonic tissues contributes to the development of colitis and CAC by disrupting regulation of NF-κB-dependent transcriptional activity.
[Show abstract][Hide abstract] ABSTRACT: Core fucosylation is an important post-translational modification, which is catalyzed by α1,6-fucosyltransferase (Fut8). Increased expression of Fut8 has been shown in diverse carcinomas including hepatocarcinoma. In this study, we investigated the role of Fut8 expression in liver regeneration by using the 70% partial hepatectomy (PH) model, and found that Fut8 is also critical for the regeneration of liver. Interestingly, we show that the Fut8 activities were significantly increased in the beginning of PH (~4d), but returned to the basal level in the late stage of PH. Lacking Fut8 led to delayed liver recovery in mice. This retardation mainly resulted from suppressed hepatocyte proliferation, as supported not only by a decreased phosphorylation level of epidermal growth factor (EGF) receptor and hepatocyte growth factor (HGF) receptor in the liver of Fut8(-/-) mice in vivo, but by the reduced response to exogenous EGF and HGF of the primary hepatocytes isolated from the Fut8(-/-) mice. Furthermore, an administration of L-fucose, which can increase GDP-fucose synthesis through a salvage pathway, significantly rescued the delayed liver regeneration of Fut8(+/-) mice. Overall, our study provides the first direct evidence for the involvement of Fut8 in liver regeneration.
Full-text · Article · Feb 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Objecitive: Fucosyltransferase 8 (FUT8), the only enzyme responsible for the core α1,6-fucosylation of asparagine-linked oligosaccharides of glycoproteins, is a vital enzyme in cancer development and progression. We examined FUT8 expression in non-small cell lung cancers (NSCLCs) to analyze its clinical significance. We also examined the expression of guanosine diphosphate-mannose-4,6-dehydratase (GMD), which is imperative for the synthesis of fucosylated oligosaccharides.
Using immunohistochemistry, we evaluated the expression of FUT8 and GMD in relation to patient survival and prognosis in potentially curatively resected NSCLCs.
High expression of FUT8 was found in 67 of 129 NSCLCs (51.9%) and was significantly found in non-squamous cell carcinomas (p = 0.008). High expression of FUT8 was associated with poor survival (p = 0.03) and was also a significant and independent unfavorable prognostic factor in patients with potentially curatively resected NSCLCs (p = 0.047). High expression of GMD was significantly associated with high FUT8 expression (p = 0.04).
High expression of FUT8 is associated with an unfavorable clinical outcome in patients with potentially curatively resected NSCLCs, suggesting that FUT8 can be a prognostic factor. The analysis of FUT8 expression and its core fucosylated products may provide new insights for the therapeutic targets of NSCLCs.
[Show abstract][Hide abstract] ABSTRACT: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is not fully reversible. It is a highly complex disease in which both environmental exposures and host susceptibility factors should be taken into account in its management. Although cigarette smoke is the major risk factor, quitting smoking does not fully repress the airway inflammation and restore the lost lung function.
Our group has been interested in changes of glycosylation that are related to the onset, biomarker discovery, and therapeutics of various diseases including cancer, neurological disease, and lifestyle-related diseases such as diabetes and COPD. Changes in glycan structures are currently being used as cancer biomarkers, prognosis, and treatment of the disease. Cell membrane receptors are mostly glycosylated proteins, and changes in the glycosylation of them have been shown to lead to dysfunctions in downstream signal pathways.
In COPD, alterations in the structures of glycans lead to dysfunction of receptor molecules, such as the TGF-beta receptor related to the upregulation of protease, or result in alterations in mucus viscosity and impaired mucociliary transport. Although the mechanism responsible for this is still uncertain, it appears that environmental factors such as exposure to cigarette smoke, pollutants, and bacteria/virus infections can alter the activities of some glycosyltransferases, and eventually this may affect the synthesis of glycans. Here, we show some glycan structures and relationships with the pathogenesis of COPD. Our recent findings regarding the role of glycan changes in COPD may open a new avenue toward the development of glycotherapeutics for COPD.
[Show abstract][Hide abstract] ABSTRACT: Cellular glycosylation plays an important role in many biological processes, as well as in disease states such as diabetes and cancer. Nucleotide sugar s are donor substrates of glycosyltransferases and their availability and localization regulate glycosylation status. The nucleotide sugar level is controlled by cellular metabolic states. To investigate the fate of nucleotide sugars in glycosylation, two methods for monitoring nucleotide sugar metabolism, namely, ion-pair reversed-phase HPLC and LC-MS, have been previously described (Nakajima et al., Glycobiology 20(7):865–871, 2010; Mol Cell Proteomics (9):2468–2480, 2013). Using the HPLC method, cellular levels of eight unique nucleotide sugars were simultaneously determined. Using the LC-MS method, which is based on mass isotopomer analysis of nucleotide sugars metabolically labeled with 13C6-glucose, the metabolic pathways governing UDP-GlcNAc synthesis and utilization were characterized. Here, these strategies are reviewed and the biochemical significance of nucleotide sugars in cellular glycosylation is discussed.