Publications by authors named "Hisashi Narimatsu"

243 Publications

O-glycosylated HBsAg peptide can induce specific antibody neutralizing HBV infection.

Biochim Biophys Acta Gen Subj 2021 Sep 25;1866(1):130020. Epub 2021 Sep 25.

Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan. Electronic address:

Background: Hepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg.

Methods: HBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined.

Results: Mass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(β1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro.

Conclusions: Glycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection.

General Significance: The detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.
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http://dx.doi.org/10.1016/j.bbagen.2021.130020DOI Listing
September 2021

Association between the expression of core 3 synthase and survival outcomes of patients with cholangiocarcinoma.

Oncol Lett 2021 Nov 3;22(5):760. Epub 2021 Sep 3.

Graduate School of Comprehensive Human Sciences, Major in Medical Sciences, Clinical Sciences Program, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan.

Cholangiocarcinoma (CCA) is a highly aggressive and metastatic type of malignant carcinoma that is associated with high mortality rates and is difficult to detect at early stages. Core 3 structure is a mucin-type -glycans synthesized by β1,3--acetylglucosaminyltransferase 6 (core 3 synthase), which plays an important role in the digestive system, in particular gastrointestinal goblet cells. It has been reported that core 3 synthase-expressing cells show lower migratory and invasive rates, and lower metastatic activity. A immunohistochemical study also showed that this enzyme was expressed in normal epithelial cells of the colon, but completely disappeared in colorectal cancer cells. The present study aimed to identify biomarkers that could be used to predict the prognosis of patients with CCA. Pathological specimens of 185 CCA tissues were immunohistochemically stained with two antibodies, G8-144 and MECA-79, which recognize core 3 synthase and 6-sulfated -acetyllactosamine on the extended core-1 -glycans, respectively. The association between G8-144 or MECA-79 positivity and patient prognosis was statistically analyzed. Positive expression of G8-144 was associated with improved prognosis in patients with distal CCA (dCCA). Patients with dCCA positive for G8-144 showed lower mortality rates than those with negative expression. However, the positive expression of MECA-79 was associated with CCA progression and metastasis, indicating that it is a poor prognostic marker for CCA. In conclusion, as both antibodies resulted in mirror-image staining, the involvement of G8-144 and MECA-79 in -glycan synthesis could be considered as potential favorable and unfavorable biomarkers, respectively, for CCA prognosis.
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http://dx.doi.org/10.3892/ol.2021.13021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8436361PMC
November 2021

N-glycan structures of Wisteria floribunda agglutinin-positive Mac2 binding protein in the serum of patients with liver fibrosis†.

Glycobiology 2021 Jun 29. Epub 2021 Jun 29.

Glycoscience & Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science & Technology, Tsukuba, Ibaraki 305-8568, Japan.

The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: 1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), 2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), 3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), 4) recombinant M2BP produced by HEK293 cells (rM2BP), and 5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that, reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc, and in part of recombinant, depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.
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http://dx.doi.org/10.1093/glycob/cwab060DOI Listing
June 2021

Polypeptide N-acetylgalactosaminyltransferase 18 retains in endoplasmic reticulum depending on its luminal regions interacting with ER resident UGGT1, PLOD3 and LPCAT1.

Glycobiology 2021 Sep;31(8):947-958

Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.

Mucin-type O-glycosylation is initiated by the polypeptide: N-acetylgalactosaminyltransferase (ppGalNAc-T) family of enzymes, which consists of 20 members in humans. Among them, unlike other ppGalNAc-Ts located in Golgi apparatus, ppGalNAc-T18 distributes primarily in the endoplasmic reticulum (ER) and non-catalytically regulates ER homeostasis and O-glycosylation. Here, we report the mechanism for ppGalNAc-T18 ER localization and the function of each structural domain of ppGalNAc-T18. By using ppGalNAc-T18 truncation mutants, we revealed that the luminal stem region and catalytic domain of ppGalNAc-T18 are essential for ER localization, whereas the lectin domain and N-glycosylation of ppGalNAc-T18 are not required. In the absence of the luminal region (i.e., stem region, catalytic and lectin domains), the conserved Golgi retention motif RKTK within the cytoplasmic tail combined with the transmembrane domain ensure ER export and Golgi retention, as observed for other Golgi resident ppGalNAc-Ts. Results from coimmunoprecipitation assays showed that the luminal region interacts with ER resident proteins UGGT1, PLOD3 and LPCAT1. Furthermore, flow cytometry analysis showed that the entire luminal region is required for the non-catalytic O-GalNAc glycosylation activity of ppGalNAc-T18. The findings reveal a novel subcellular localization mechanism of ppGalNAc-Ts and provide a foundation to further characterize the function of ppGalNAc-T18 in the ER.
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http://dx.doi.org/10.1093/glycob/cwab031DOI Listing
September 2021

Clinicopathological significance of core 3 O-glycan synthetic enzyme, β1,3-N-acetylglucosaminyltransferase 6 in pancreatic ductal adenocarcinoma.

PLoS One 2020 30;15(11):e0242851. Epub 2020 Nov 30.

Division of Molecular Pathology, National Cancer Center Research Institute, Tokyo, Japan.

Mucin-type O-glycans are involved in cancer initiation and progression, although details of their biological and clinicopathological roles remain unclear. The aim of this study was to investigate the clinicopathological significance of β1,3-N-acetylglucosaminyltransferase 6 (β3Gn-T6), an essential enzyme for the synthesis of core 3 O-glycan and several other O-glycans in pancreatic ductal adenocarcinoma (PDAC). We performed immunohistochemical and lectin-histochemical analyses to detect the expression of β3Gn-T6 and several O-glycans in 156 cases of PDAC with pancreatic intraepithelial neoplasias (PanINs) and corresponding normal tissue samples. The T antigen, Tn antigen, sialyl Lewis X (sLeX) antigen, and sLeX on core 2 O-glycan were more highly expressed in PDAC cells than in normal pancreatic duct epithelial cells (NPDEs). Conversely, the expression of 6-sulfo N-acetyllactosamine on extended core 1 O-glycan was found in NPDEs and was low in PDAC cells. These glycan expression levels were not associated with patient outcomes. β3Gn-T6 was expressed in ~20% of PDAC cases and 30-40% of PanINs but not in NPDEs. Higher expression of β3Gn-T6 was found in PDAC cells in more differentiated adenocarcinoma cases showing significantly longer disease-free survival in both univariate and multivariate analyses. In addition, the expression of β3Gn-T6 in PDAC cells and PanINs significantly correlated with the expression of MUC5AC in these cells, suggesting that β3Gn-T6 expression is related to cellular differentiation status of the gastric foveolar phenotype. Thus, it is likely that β3Gn-T6 expression in PDAC cells is a favorable prognostic factor in PDAC patients, and that the expression of β3Gn-T6 correlates with the gastric foveolar phenotype in pancreatic carcinogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242851PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703919PMC
January 2021

Glycogene Expression Profiling of Hepatic Cells by RNA-Seq Analysis for Glyco-Biomarker Identification.

Front Oncol 2020 28;10:1224. Epub 2020 Jul 28.

Molecular and Cellular Glycoproteomics Research Group, Department of Life Science and Biotechnology, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.

Glycans are primarily generated by "glycogenes," which consist of more than 200 genes for glycosynthesis, including sugar-nucleotide synthases, sugar-nucleotide transporters, and glycosyltransferases. Measuring the expression level of glycogenes is one of the approaches to analyze the glycomes of particular biological and clinical samples. To develop an effective strategy for identifying the glycosylated biomarkers, we performed transcriptome analyses using quantitative real-time polymerase chain reaction (qRT-PCR) arrays and RNA sequencing (RNA-Seq). First, we measured and analyzed the transcriptome from the primary culture of human liver cells and hepatocarcinoma cells using RNA-Seq. This analysis revealed similar but distinctive expression profiles of glycogenes among hepatic cells as indicated by the qRT-PCR arrays, which determined a copy number of 186 glycogenes. Both data sets indicated that altered expression of glycosyltransferases affect the glycosylation of particular glycoproteins, which is consistent with the mass analysis data. Moreover, RNA-Seq analysis can uncover mutations in glycogenes and search differently expressed genes out of more than 50,000 distinct human gene transcripts including candidate biomarkers that were previously reported for hepatocarcinoma cells. Identification of candidate glyco-biomarkers from the expression profile of the glycogenes and proteins from liver cancer tissues available from public database emphasized the possibility that even though the expression level of biomarkers might not be altered, the expression of the glycogenes modifying biomarkers, generating glyco-biomarkers, might be different. Pathway analysis revealed that ~20% of the glycogenes exhibited different expression levels in normal and cancer cells. Thus, transcriptome analyses using both qRT-PCR array and RNA-Seq in combination with glycome and glycoproteome analyses can be advantageous to identify "glyco-biomarkers" by reinforcing information at the expression levels of both glycogenes and proteins.
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http://dx.doi.org/10.3389/fonc.2020.01224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402167PMC
July 2020

Screening siRNAs against host glycosylation pathways to develop novel antiviral agents against hepatitis B virus.

Hepatol Res 2020 Oct 24;50(10):1128-1140. Epub 2020 Aug 24.

Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.

Aim: Hepatitis B virus (HBV) relies on glycosylation for crucial functions, such as entry into host cells, proteolytic processing and protein trafficking. The aim of this study was to identify candidate molecules for the development of novel antiviral agents against HBV using an siRNA screening system targeting the host glycosylation pathway.

Methods: HepG2.2.15.7 cells that consistently produce HBV were employed for our in vitro study. We investigated the effects of siRNAs that target 88 different host glycogenes on hepatitis B surface antigen (HBsAg) and HBV DNA secretion using the siRNA screening system.

Results: We identified four glycogenes that reduced HBsAg and/or HBV DNA secretion; however, the observed results for two of them may be due to siRNA off-target effects. Knocking down ST8SIA3, a member of the sialyltransferase family, significantly reduced both HBsAg and HBV DNA secretion. Knocking down GALNT7, which transfers N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus, also significantly reduced both HBsAg and HBV DNA levels.

Conclusions: These results showed that knocking down the ST8SIA3 and GALNT7 glycogenes inhibited HBsAg and HBV DNA secretion in HepG2.2.15.7 cells, indicating that the host glycosylation pathway is important for the HBV life cycle and could be a potential target for the development of novel anti-HBV agents.
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http://dx.doi.org/10.1111/hepr.13552DOI Listing
October 2020

Multi-serum glycobiomarkers improves the diagnosis and prognostic prediction of cholangiocarcinoma.

Clin Chim Acta 2020 Nov 11;510:142-149. Epub 2020 Jul 11.

Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8565, Japan. Electronic address:

Background: Aberrant glycosylation has been reported to play important roles in progression of cholangiocarcinoma (CCA) and hence the aberrant expressed glycans are beneficial markers for diagnosis and prognostic prediction of CCA.

Methods: Five CCA-associated glycobiomarkers-carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen-S27 (CA-S27), CCA-associated carbohydrate antigen (CCA-CA), WFA-positive MUC1 (WFA-MUC1), and WFA-positive M2BP (WFA-M2BP), in the sera from CCA patients (N = 138) were determined in comparison with non-CCA control subjects (N = 246).

Results: Receiver operating characteristic analysis suggested the significance of each glycobiomarker in discriminating CCA from non-CCA with area under curve of 0.580-0.777. High levels of CA19-9, CCA-CA, CA-S27, or WFA-MUC1 were associated with poor prognosis and poor survival of CCA patients. Combination of these glycobiomarkers and graded as a GlycoBiomarker (GB)-score could increase the power of the tests in diagnosis than an individual marker with 81% of sensitivity, specificity and accuracy.

Conclusions: According to the GB-score, these glycobiomarkers not only increased diagnostic power but also discriminated survival of patients indicating the diagnostic and prognostic values of GB-score.
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http://dx.doi.org/10.1016/j.cca.2020.07.017DOI Listing
November 2020

O-linked N-acetylgalactosamine modification is present on the tumor suppressor p53.

Biochim Biophys Acta Gen Subj 2020 08 14;1864(8):129635. Epub 2020 May 14.

Key Laboratory of Systems Biomedicine (Ministry of Education) and Collaborative Innovation Center of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China; SCSB (China)-AIST (Japan) Joint Medical Glycomics Laboratory, Shanghai, China. Electronic address:

Background: Mucin-type O-glycosylation (referred to as O-GalNAc glycosylation) is the most abundant O-glycosylation on membrane and secretory proteins. Recently several evidences suggest that nuclear or cytoplasmic proteins might also have O-GalNAc glycosylation. However, what nucleocytoplasmic proteins are O-GalNAc glycosylated and what the biological function of this modification in cells are still poorly understood. Previously, we reported the tumor suppressor p53 could be O-GalNAc glycosylated in vitro. To investigate the existence and function of O-GalNAc glycosylation on nucleocytoplasmic proteins in cell, p53 as a representative nucleocytoplasmic protein was studied.

Methods: Using lectin blotting with GalNAc specific lectins, enzymatic treatments with O-GlcNAcase, core 1 β1, 3-galactosyltransferase and O-glycosidase, and metabolic labeling with un-O-acetylated GalNAz in UDP-Gal/UDP-GalNAc 4-epimerase (GALE) knockout cells, we validated the O-GalNAc glycosylation on p53. Using mass spectrometry analysis and site-directed mutagenesis, we identified the glycosylated sites and studied the functions of O-GalNAc glycosylation on p53.

Results: The p53 was O-GalNAc glycosylated in cells. Ser121 residue was one of the glycosylated sites on p53. The O-GalNAc glycosylation at Ser121 was associated with the stability and activity of p53.

Conclusions: These results revealed that the O-GalNAc glycosylation was a novel modification on p53.

General Significance: Our study provided a pilot evidence that the O-GalNAc glycosylation existed on nucleocytoplasmic protein.
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http://dx.doi.org/10.1016/j.bbagen.2020.129635DOI Listing
August 2020

Comparative Glycomic Analysis of Exosome Subpopulations Derived from Pancreatic Cancer Cell Lines.

J Proteome Res 2020 06 6;19(6):2516-2524. Epub 2020 May 6.

Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8565, Japan.

Extracellular vesicles such as exosomes are generally covered with an array of glycans, which are controlled by the host-cell glyco-synthetic machinery, similar to secreted and membrane glycoproteins. Several exosome subpopulations classified by their tetraspanin expression have been investigated in the context of diseases. However, a comparative analysis of their glycomics has never been attempted. Herein, we report a method for the comparative glycomic analysis of exosome subpopulations among pancreatic cancer cell lines. Glycomic profiles were obtained for extracellular vesicles, secreted glycoproteins, and membrane glycoproteins from eight cell lines. Statistical analyses revealed high populations of PHA-L-binding proteins in the vesicles. The surfaces of extracellular vesicles were labeled with Cy3 and captured by magnetic beads with antibodies against tetraspanins (CD9, CD63, and CD81). The coprecipitated vesicles were lysed and subjected to a lectin microarray analysis. A hierarchical clustering analysis using 19 glycomic profiles confirmed that most subpopulations, except CD81-positive exosomes, could be distinguished according to the host-cell species. Principal component analysis and subsequent lectin-affinity capturing of intact exosomes highlighted that CD81-positive exosomes preferentially expressed not PHA-L- but LEL-binding proteins on their surfaces. These data suggested that exosomal glycomics depended on the host-cell type and subpopulation.
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http://dx.doi.org/10.1021/acs.jproteome.0c00200DOI Listing
June 2020

Mice lacking core 1-derived O-glycan in podocytes develop transient proteinuria, resulting in focal segmental glomerulosclerosis.

Biochem Biophys Res Commun 2020 03 20;523(4):1007-1013. Epub 2020 Jan 20.

Laboratory Animal Resource Center in Transborder Medical Research Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Japan. Electronic address:

The glomerular filtration barrier is composed of podocytes, glomerular basement membrane, and endothelial cells. Disruption of these structures causes several glomerular injuries, such as focal segmental glomerulosclerosis (FSGS). The surface of podocyte apical membranes is coated by negatively charged sialic acids on core 1-derived mucin-type O-glycans. Here, we aimed to investigate the physiological role of core 1-derived O-glycans in the podocytes using adult mice lacking podocyte-specific core 1-derived O-glycans (iPod-Cos). iPod-Cos mice exhibited early and transient proteinuria with foot process effacements and developed typical FSGS-like disease symptoms. To identify the key molecules responsible for the FSGS-like phenotype, we focused on podocalyxin and podoplanin, which possess mucin-type O-glycans. Expression and localization of podocalyxin did not change in iPod-Cos glomeruli. Besides, western blot analysis revealed significantly lower levels of intact podocalyxin in isolated glomeruli of iPod-Cos mice, and high levels of processed forms in iPod-Cos glomeruli, as compared to that in control glomeruli. Conversely, podoplanin mRNA, and protein levels were lower in iPod-Cos mice than in control mice. These results demonstrated that core 1-derived O-glycan on podocytes is required for normal glomerular filtration and may contribute to the stable expression of podocalyxin and podoplanin.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.033DOI Listing
March 2020

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Hum Mol Genet 2019 12;28(24):4053-4066

Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the β3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose β1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.
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http://dx.doi.org/10.1093/hmg/ddz225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6991180PMC
December 2019

Fucosyltransferase 2 induces lung epithelial fucosylation and exacerbates house dust mite-induced airway inflammation.

J Allergy Clin Immunol 2019 09 21;144(3):698-709.e9. Epub 2019 May 21.

Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan. Electronic address:

Background: One of the pathognomonic features of asthma is epithelial hyperproduction of mucus, which is composed of a series of glycoproteins; however, it remains unclear how glycosylation is induced in lung epithelial cells from asthmatic patients and how glycan residues play a role in the pathogenesis of asthma.

Objective: The objective of this study was to explore comprehensive epithelial glycosylation status induced by allergic inflammation and reveal its possible role in the pathogenesis of asthma.

Methods: We evaluated the glycosylation status of lung epithelium using a lectin microarray. We next searched for molecular mechanisms underlying epithelial glycosylation. We also examined whether epithelial glycosylation is involved in induction of allergic inflammation.

Results: On allergen inhalation, lung epithelial cells were heavily α(1,2)fucosylated by fucosyltransferase 2 (Fut2), which was induced by the IL-13-signal transducer and activator of transcription 6 pathway. Importantly, Fut2-deficient (Fut2) mice, which lacked lung epithelial fucosylation, showed significantly attenuated eosinophilic inflammation and airway hyperresponsiveness in house dust mite (HDM)-induced asthma models. Proteome analyses and immunostaining of the HDM-challenged lung identified that complement C3 was accumulated in fucosylated areas. Indeed, Fut2 mice showed significantly reduced levels of C3a and impaired accumulation of C3a receptor-expressing monocyte-derived dendritic cells in the lung on HDM challenge.

Conclusion: Fut2 induces epithelial fucosylation and exacerbates airway inflammation in asthmatic patients in part through C3a production and monocyte-derived dendritic cell accumulation in the lung.
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http://dx.doi.org/10.1016/j.jaci.2019.05.010DOI Listing
September 2019

Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2.

J Biol Chem 2019 05 21;294(18):7433-7444. Epub 2019 Mar 21.

From the Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan and

The type-I LacdiNAc (LDN; GalNAcβ1-3GlcNAc) has rarely been observed in mammalian cells except in the -glycan of α-dystroglycan, in contrast to type-II LDN structures (GalNAcβ1-4GlcNAc) in - and -glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a β1,3--acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified. In this study, using HeLa cells, we demonstrate that inhibition of Golgi-resident glycosyltransferase increases the abundance of B3GALNT2-synthesized type-I LDN structures, recognized by agglutinin (WFA). Using isotope-coded glycosylation site-specific tagging (IGOT)-LC/MS analysis of Lec8 Chinese hamster cells lacking galactosylation and of cells transfected with the gene, we identified the glycoproteins that carry B3GALNT2-generated type-I LDN in their -glycans. Our results further revealed that LDN presence on low-density lipoprotein receptor-related protein 1 and nicastrin depends on B3GALNT2, indicating the occurrence of type-I LDN in mammalian cells. Our analysis also uncovered that most of the identified glycoproteins localize to intracellular organelles, particularly to the endoplasmic reticulum. Whereas B4GALNT3 and B4GALNT4 synthesized LDN on extracellular glycoproteins, B3GALNT2 primarily transferred LDN to intracellular glycoproteins, thereby clearly delineating proteins that carry type-I or type-II LDNs. Taken together, our results indicate the presence of mammalian glycoproteins carrying type-I LDN on -glycans and suggest that type-I and type-II LDNs have different roles .
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http://dx.doi.org/10.1074/jbc.RA118.006892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509504PMC
May 2019

Polypeptide N-acetylgalactosaminyltransferase 18 non-catalytically regulates the ER homeostasis and O-glycosylation.

Biochim Biophys Acta Gen Subj 2019 05 21;1863(5):870-882. Epub 2019 Feb 21.

Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China; SCSB (China)-AIST (Japan) Joint Medical Glycomics Laboratory, Shanghai, China. Electronic address:

Mucin-type O-glycosylation plays important roles in various biological processes. It is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Unlike most ppGalNAc-Ts localized to the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on the endoplasmic reticulum (ER) and exhibits no ppGalNAc-T catalytic activity in vitro. Herein, we found that ppGalNAc-T18 silencing in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cell, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found that ppGalNAc-T18 exerts its functions in O-glycosylation and ER stress via a non-catalytic mechanism. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner.
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http://dx.doi.org/10.1016/j.bbagen.2019.01.009DOI Listing
May 2019

Mutation of gene identified in patients diagnosed with asthenozoospermia.

Hum Fertil (Camb) 2020 Dec 10;23(4):226-233. Epub 2019 Jan 10.

Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

Asthenozoospermia is commonly observed in infertile men. However, very few causative gene mutations have been identified because an efficient detection method has not been established. We previously identified a patient with asthenozoospermia carrying a heterozygous point deletion in by detecting an abnormal reduction in the abundance of GALNTL5 and other marker proteins. To identify other mutations in , we screened sperm samples from 208 infertile men mainly diagnosed with asthenozoospermia using the same method, and conducted next-generation sequencing. Consequently, another case of mutation was detected only in sperm at a low frequency but not in the somatic blood cells of a patient diagnosed with asthenozoospermia. In this patient, sperm motility improved and the mutation disappeared at 2 years after the first observation. In this man, carrying a heterozygotic deficiency of , the swim-up method was useful to concentrate the spermatozoa without mutation. Intracytoplasmic sperm injection of the selected motile spermatozoa into oocytes of the patient's partner resulted in successful conception, and a female child was safely delivered. These results suggest the feasibility of our approach for the screening and treatment of asthenozoospermia associated with mutation.
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http://dx.doi.org/10.1080/14647273.2018.1562239DOI Listing
December 2020

Current Technologies for Complex Glycoproteomics and Their Applications to Biology/Disease-Driven Glycoproteomics.

J Proteome Res 2018 12 25;17(12):4097-4112. Epub 2018 Oct 25.

Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education) , Shanghai Jiao Tong University , 800 Dong Chuan Road , Minhang , Shanghai 200240 , P.R. China.

Glycoproteomics is an important recent advance in the field of glycoscience. In glycomics, glycan structures are comprehensively analyzed after glycans are released from glycoproteins. However, a major limitation of glycomics is the lack of insight into glycoprotein functions. The Biology/Disease-driven Human Proteome Project has a particular focus on biological and medical applications. Glycoproteomics technologies aimed at obtaining a comprehensive understanding of intact glycoproteins, i.e., the kind of glycan structures that are attached to particular amino acids and proteins, have been developed. This Review focuses on the recent progress of the technologies and their applications. First, the methods for large-scale identification of both N- and O-glycosylated proteins are summarized. Next, the progress of analytical methods for intact glycopeptides is outlined. MS/MS-based methods were developed for improving the sensitivity and speed of the mass spectrometer, in parallel with the software for complex spectrum assignment. In addition, a unique approach to identify intact glycopeptides using MS1-based accurate masses is introduced. Finally, as an advance of glycomics, two approaches to provide the spatial distribution of glycans in cells are described, i.e., MS imaging and lectin microarray. These methods allow rapid glycomic profiling of different types of biological samples and thus facilitate glycoproteomics.
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http://dx.doi.org/10.1021/acs.jproteome.8b00515DOI Listing
December 2018

Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

Sci Rep 2018 09 24;8(1):14251. Epub 2018 Sep 24.

Kanagawa Cancer Center Research Institute, Yokohama, Japan.

The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser and Ser. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.
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http://dx.doi.org/10.1038/s41598-018-32534-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155162PMC
September 2018

Highly Sensitive Glycan Profiling of Hepatitis B Viral Particles and a Simple Method for Dane Particle Enrichment.

Anal Chem 2018 09 20;90(17):10196-10203. Epub 2018 Aug 20.

Research Center for Medical Glycoscience (RCMG) , National Institute of Advanced Industrial Science and Technology , AIST Tsukuba Central 2, 1-1-1, Umezono , Tsukuba , Ibaraki 305-8568 , Japan.

Hepatitis B virus (HBV) is a double-stranded DNA virus composed of three types of viral particles. The virions are called Dane particles and the others are noninfectious subviral particles (SVPs). In blood, SVPs are detected in abundance, about 1000-10000 fold higher than Dane particles. Dane particles are hazardous because of their strong infectivity, unlike SVPs. Dane particles are covered with an envelope of glycoprotein called HBV surface antigen (HBsAg). HBsAg glycosylation is involved in viral particle formation and secretion. In this study, we established a novel and highly sensitive method for viral glycan profiling of HBsAg using small aliquots of patient serum. Our lectin microarray system could sensitively profile the glycans exposed on HBV while retaining the intact viral particle structure under nonreducing conditions. Several typical lectins were chosen from the lectin microarray results. Specifically, jacalin, which recognizes O-glycan, showed specific and strong reactivity to the M-HBsAg required for Dane particle secretion. Employing the lectin-fractionation method using jacalin, HBV particles were fractionated into jacalin-bound and unbound fractions from patient serum. We measured HBsAg titer and viral DNA load in each fraction using clinical tests. Interestingly, the jacalin-bound fraction contained a major fraction of the HBV viral DNA load. Thus, in this study we have presented a glycan profiling method for HBsAg on the intact HBV particle and an easy and simple method to enrich Dane particles from patient serum by jacalin fractionation.
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http://dx.doi.org/10.1021/acs.analchem.8b01030DOI Listing
September 2018

Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE.

J Am Soc Mass Spectrom 2018 06 19;29(6):1138-1152. Epub 2018 Apr 19.

Glycoscience & Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science & Technology, Tsukuba, Ibaraki, 305-8568, Japan.

To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1938-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004004PMC
June 2018

GGDonto ontology as a knowledge-base for genetic diseases and disorders of glycan metabolism and their causative genes.

J Biomed Semantics 2018 04 18;9(1):14. Epub 2018 Apr 18.

Glycoscience and Glycotechnology Research Group, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

Background: Inherited mutations in glyco-related genes can affect the biosynthesis and degradation of glycans and result in severe genetic diseases and disorders. The Glyco-Disease Genes Database (GDGDB), which provides information about these diseases and disorders as well as their causative genes, has been developed by the Research Center for Medical Glycoscience (RCMG) and released in April 2010. GDGDB currently provides information on about 80 genetic diseases and disorders caused by single-gene mutations in glyco-related genes. Many biomedical resources provide information about genetic disorders and genes involved in their pathogenesis, but resources focused on genetic disorders known to be related to glycan metabolism are lacking. With the aim of providing more comprehensive knowledge on genetic diseases and disorders of glycan biosynthesis and degradation, we enriched the content of the GDGDB database and improved the methods for data representation.

Results: We developed the Genetic Glyco-Diseases Ontology (GGDonto) and a RDF/SPARQL-based user interface using Semantic Web technologies. In particular, we represented the GGDonto content using Semantic Web languages, such as RDF, RDFS, SKOS, and OWL, and created an interactive user interface based on SPARQL queries. This user interface provides features to browse the hierarchy of the ontology, view detailed information on diseases and related genes, and find relevant background information. Moreover, it provides the ability to filter and search information by faceted and keyword searches.

Conclusions: Focused on the molecular etiology, pathogenesis, and clinical manifestations of genetic diseases and disorders of glycan metabolism and developed as a knowledge-base for this scientific field, GGDonto provides comprehensive information on various topics, including links to aid the integration with other scientific resources. The availability and accessibility of this knowledge will help users better understand how genetic defects impact the metabolism of glycans as well as how this impaired metabolism affects various biological functions and human health. In this way, GGDonto will be useful in fields related to glycoscience, including cell biology, biotechnology, and biomedical, and pharmaceutical research.
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http://dx.doi.org/10.1186/s13326-018-0182-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905134PMC
April 2018

Mac-2 binding protein glycan isomer (M2BPGi) is a new serum biomarker for assessing liver fibrosis: more than a biomarker of liver fibrosis.

J Gastroenterol 2018 Jul 9;53(7):819-826. Epub 2018 Jan 9.

Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba, Japan.

Assessing liver fibrosis is important for predicting the efficacy of antiviral therapy and patient prognosis. Liver biopsy is the gold standard for diagnosing liver fibrosis, despite its invasiveness and problematic diagnostic accuracy. Although noninvasive techniques to assess liver fibrosis are becoming important, reliable serum surrogate markers are not available. A glycoproteomics study aimed at identifying such markers discovered Mac 2-Binding Protein Gylcan Isomer (M2BPGi), which is a reliable marker for assessing liver fibrosis in patients with viral hepatitis and other fibrotic liver diseases such as primary biliary cholangitis, biliary atresia, autoimmune hepatitis, and nonalcoholic fatty liver disease. M2BPGi predicts the development of hepatocellular carcinoma (HCC) in patients infected with hepatitis B and C as well as the prognosis of liver cirrhosis in those with HCC after therapy. The unique features of M2BPGi are as follows: (1) cut-off values differ for the same stages of fibrosis according to the cause of fibrosis; and (2) M2BPGi levels rapidly decrease after patients achieve a sustained antiviral response to hepatitis C virus. These observations cannot be explained if M2BPGi levels reflect the amount of fibrotic tissue. Hepatic stellate cells (HSCs) secrete M2BPGi, which may serve as a messenger between HSCs and Kupffer cells via Mac-2 (galectin 3) that is expressed in Kupffer cells during fibrosis progression. Here we show that M2BPGi is a surrogate marker for assessing HSC activation. These findings may reveal the roles of HSCs in extrahepatic fibrotic disease progression.
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http://dx.doi.org/10.1007/s00535-017-1425-zDOI Listing
July 2018

Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2.

PLoS One 2017 29;12(12):e0190333. Epub 2017 Dec 29.

Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190333PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747463PMC
February 2018

Wisteria floribunda agglutinin positive glycobiomarkers: a unique lectin as a serum biomarker probe in various diseases.

Expert Rev Proteomics 2018 02 21;15(2):183-190. Epub 2017 Dec 21.

a Research Center for Medical Glycoscience , National Institute of Advanced Industrial Science and Technology , Tsukuba , Japan.

Introduction: Serum proteins are generally glycosylated and solubilized, and are thus present as glycoproteins. The glycan structure of glycoproteins reflects cell differentiation status; glycan structures generated by diseased cells are distinguishable from those produced by healthy cells. Proteins may therefore serve as markers of tissues that secrete them. Several strategies for the identification of novel serum biomarkers using a combination of glycoscience-based technologies have been recently proposed. The selection of lectins for use as probes for identification of altered glycan structures represents a critical step. Areas covered: This review describes the identification of Wisteria floribunda agglutinin (WFA) as a probe that recognizes the altered glycan structure of glycoproteins secreted by diseased cells. WFA may be employed as a probe for several diseases, e.g., liver fibrosis, liver cirrhosis, prostate cancer, ovarian cancer, and IgA nephropathy. The advantage of employing WFA as a serum biomarker probe is that only very small amounts of WFA-positive glycoproteins are present in serum; therefore, WFA background in serum is very low. Expert commentary: Based on the findings to date, several WFA-positive serum biomarkers may be measured without pre-purification of target glycoproteins, indicating their utility as serum biomarkers in patients with various diseases.
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http://dx.doi.org/10.1080/14789450.2018.1419066DOI Listing
February 2018

Incomplete clearance of apoptotic cells by core 1-derived O-glycan-deficient resident peritoneal macrophages.

Biochem Biophys Res Commun 2018 01 13;495(2):2017-2023. Epub 2017 Dec 13.

Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Japan; Laboratory Animal Resource Center, Japan; Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, Japan. Electronic address:

The core 1 β1,3-galactosyltransferase-specific molecular chaperon (Cosmc) is essential for the synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of core 1-derived O-glycans in macrophages, we exploited the LysM-Cre transgene to generate a conditional Cosmc mutant allele (conditional Cosmc knockout; cKO) in myeloid cells. cKO mice developed normally with no gross phenotypic abnormalities or abnormal peripheral blood counts. Resident peritoneal macrophages (rpMacs) of cKO mice exhibited impaired engulfment of apoptotic cells but showed normal macrophage differentiation and counts. T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) is a phosphatidylserine (PS) receptor expressed on rpMacs and possesses a heavily O-glycosylated domain. Tim4 tethers apoptotic cells through PS binding. Expression of the Tim4 transcript was unchanged in cKO rpMacs, whereas flow cytometric, Western and dot blot analyses revealed that Tim4 protein expression in cKO rpMacs was significantly lower than that in wild-type (WT) rpMacs. Moreover, the expression levels of other efferocytosis-related molecules, Mertk, Itgav and Itgb3, were normal in rpMacs. In addition, hypoglycosylated Tim4-FLAG fusion protein sufficiently recognized PS. These results demonstrated that core 1-derived O-glycan is required for Tim4-dependent normal efferocytosis and may contribute to the stable expression of the Tim4 glycoprotein.
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http://dx.doi.org/10.1016/j.bbrc.2017.12.066DOI Listing
January 2018

Glycobiomarker, Fucosylated Short-Form Secretogranin III Levels Are Increased in Serum of Patients with Small Cell Lung Carcinoma.

J Proteome Res 2017 12 12;16(12):4495-4505. Epub 2017 Oct 12.

Department of Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba , 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

Secretogranin III (SgIII) is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins. Granins are expressed in endocrine and neuroendocrine cells and subsequently processed into bioactive hormones. Although granin-derived peptide expression is correlated with neuroendocrine carcinomas, little is known about SgIII. We previously identified SgIII by a comparative glycoproteomics approach for elucidation of glycobiomarker candidates in lung carcinoma. Here, we examined the expression, secretion, and glycosylation of SgIII to identify novel biomarkers of small cell lung carcinoma (SCLC). In comparative immunohistochemical analysis and secretion profiling, SgIII was observed in all types of lung cancer. However, low-molecular-weight SgIII (short-form SgIII) was specifically found in SCLC culture medium. Glycoproteomics analysis showed that a fucosylated glycan was attached to the first of three potential N-glycosylation sites and an unfucosylated glycan was detected on the second site; however, the third site was not glycosylated. Next, we performed lectin capture with a fucose-binding lectin and detected short-form SgIII specifically in the sera of patients with SCLC. The results suggested an association between the fucosylated glycoform of short-form SgIII and SCLC. Thus, fucosylated short-form SgIII may be a valuable biomarker for SCLC and could be used to monitor development of the disease. All MS data are available via ProteomeXchange and jPOST with identifiers PXD007626 and JPST000313, respectively.
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http://dx.doi.org/10.1021/acs.jproteome.7b00484DOI Listing
December 2017
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