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Klarić TS, Lauc G. The dynamic brain N-glycome. Glycoconj J 2022; 39:443-471. [PMID: 35334027 DOI: 10.1007/s10719-022-10055-x] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2021] [Revised: 02/27/2022] [Accepted: 03/09/2022] [Indexed: 01/17/2023]
Abstract
The attachment of carbohydrates to other macromolecules, such as proteins or lipids, is an important regulatory mechanism termed glycosylation. One subtype of protein glycosylation is asparagine-linked glycosylation (N-glycosylation) which plays a key role in the development and normal functioning of the vertebrate brain. To better understand the role of N-glycans in neurobiology, it's imperative we analyse not only the functional roles of individual structures, but also the collective impact of large-scale changes in the brain N-glycome. The systematic study of the brain N-glycome is still in its infancy and data are relatively scarce. Nevertheless, the prevailing view has been that the neuroglycome is inherently restricted with limited capacity for variation. The development of improved methods for N-glycomics analysis of brain tissue has facilitated comprehensive characterisation of the complete brain N-glycome under various experimental conditions on a larger scale. Consequently, accumulating data suggest that it's more dynamic than previously recognised and that, within a general framework, it has a given capacity to change in response to both intrinsic and extrinsic stimuli. Here, we provide an overview of the many factors that can alter the brain N-glycome, including neurodevelopment, ageing, diet, stress, neuroinflammation, injury, and disease. Given this emerging evidence, we propose that the neuroglycome has a hitherto underappreciated plasticity and we discuss the therapeutic implications of this regarding the possible reversal of pathological changes via interventions. We also briefly review the merits and limitations of N-glycomics as an analytical method before reflecting on some of the outstanding questions in the field.
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Affiliation(s)
| | - Gordan Lauc
- Genos Glycoscience Research Laboratory, Zagreb, Croatia.,Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
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Quantitative structural analysis of glycans expressed within tumors derived from pancreatic cancer patient-derived xenograft mouse models. Biochem Biophys Res Commun 2020; 534:310-316. [PMID: 33288196 DOI: 10.1016/j.bbrc.2020.11.087] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Accepted: 11/20/2020] [Indexed: 12/17/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an intractable malignancy for which novel therapeutic targets are in high demand. To uncover glycans expressed within PDAC, we previously performed glycome profiling of PDAC cell lines using lectin microarray and found that the lectin rBC2LCN with specificity to a Fucα1-2Galβ1-3 motif exhibited strong binding to a PDAC cell line (Capan-1) and to all tumor tissues derived from 69 pancreatic cancer patients. Nevertheless, no information was available as to whether glycans containing the Fucα1-2Galβ1-3 motif are expressed within PDAC. Here we used HPLC combined with MALDI-TOFMS to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from two types of patient-derived PDAC xenograft mouse models, PC3 (well-differentiated) and PC42 (poorly-differentiated). A higher percentage of highly branched and sialylated complex-type N-glycans was detected in PC42 relative to PC3. The percentage of core 1 O-glycans was higher in PC42 relative to PC3, whereas that of core 3 O-glycans was higher in PC3. Cancer-related glycan epitopes such as Lewis A and Lewis Y were detected in core 3 O-glycans of both PC3 and PC42. H-type3 containing the Fucα1-2Galβ1-3 motif was detected in Core 2 O-glycans in both models, explaining the molecular mechanism of the binding of rBC2LCN to PDAC.
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Higashi M, Yoshimura T, Usui N, Kano Y, Deguchi A, Tanabe K, Uchimura Y, Kuriyama S, Suzuki Y, Masaki T, Ikenaka K. A Potential Serum N-glycan Biomarker for Hepatitis C Virus-Related Early-Stage Hepatocellular Carcinoma with Liver Cirrhosis. Int J Mol Sci 2020; 21:ijms21238913. [PMID: 33255418 PMCID: PMC7727814 DOI: 10.3390/ijms21238913] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 11/20/2020] [Accepted: 11/21/2020] [Indexed: 02/07/2023] Open
Abstract
Detection of early-stage hepatocellular carcinoma (HCC) is beneficial for prolonging patient survival. However, the serum markers currently used show limited ability to identify early-stage HCC. In this study, we explored human serum N-glycans as sensitive markers to diagnose HCC in patients with cirrhosis. Using a simplified fluorescence-labeled N-glycan preparation method, we examined non-sialylated and sialylated N-glycan profiles from 71 healthy controls and 111 patients with hepatitis and/or liver cirrhosis (LC) with or without HCC. We found that the level of serum N-glycan A2G1(6)FB, a biantennary N-glycan containing core fucose and bisecting GlcNAc residues, was significantly higher in hepatitis C virus (HCV)-infected cirrhotic patients with HCC than in those without HCC. In addition, A2G1(6)FB was detectable in HCV-infected patients with early-stage HCC and could be a more accurate marker than alpha-fetoprotein (AFP) or protein induced by vitamin K absence or antagonists-II (PIVKA-II). Moreover, there was no apparent correlation between the levels of A2G1(6)FB and those of AFP or PIVKA-II. Thus, simultaneous use of A2G1(6)FB and traditional biomarkers could improve the accuracy of HCC diagnosis in HCV-infected patients with LC, suggesting that A2G1(6)FB may be a reliable biomarker for early-stage HCC patients.
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Affiliation(s)
- Mikito Higashi
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan; (M.H.); (Y.K.)
- Mitsubishi Chemical Group Science and Technology Research Center, Yokohama, Kanagawa 227-8502, Japan;
| | - Takeshi Yoshimura
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan; (M.H.); (Y.K.)
- Department of Physiological Sciences, School of Life Sciences, SOKENDAI (The Graduate University for Advanced Studies), Hayama, Kanagawa 240-0193, Japan
- Department of Child Development and Molecular Brain Science, United Graduate School of Child Development, Osaka University, Suita, Osaka 565-0871, Japan
- Correspondence:
| | - Noriyoshi Usui
- Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan;
- Addiction Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center, Osaka 541-8567, Japan
| | - Yuichiro Kano
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan; (M.H.); (Y.K.)
| | - Akihiro Deguchi
- Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa 761-0793, Japan; (A.D.); (T.M.)
| | - Kazuhiro Tanabe
- Mitsubishi Chemical Group Science and Technology Research Center, Yokohama, Kanagawa 227-8502, Japan;
| | - Youichi Uchimura
- Mitsubishi Chemical Group Science and Technology Research Center, Yokohama, Kanagawa 227-8502, Japan;
| | - Shigeki Kuriyama
- Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa 761-0793, Japan; (A.D.); (T.M.)
| | - Yasuyuki Suzuki
- Department of Gastroenterological Surgery, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa 761-0793, Japan;
| | - Tsutomu Masaki
- Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa 761-0793, Japan; (A.D.); (T.M.)
| | - Kazuhiro Ikenaka
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan; (M.H.); (Y.K.)
- Department of Physiological Sciences, School of Life Sciences, SOKENDAI (The Graduate University for Advanced Studies), Hayama, Kanagawa 240-0193, Japan
- Correspondence:
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Takashima Y, Yoshimura T, Kano Y, Hayano A, Hondoh H, Ikenaka K, Yamanaka R. Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells. BMC Cancer 2019; 19:910. [PMID: 31510952 PMCID: PMC6739943 DOI: 10.1186/s12885-019-6129-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Accepted: 09/03/2019] [Indexed: 01/19/2023] Open
Abstract
Background Oligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell–cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma. Methods Here, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens. Results We first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated. Discussion This is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma, Conclusion These results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.
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Affiliation(s)
- Yasuo Takashima
- Laboratory of Molecular Target Therapy for Cancer, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Takeshi Yoshimura
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan.,Present Address: Department of Child Development and Molecular Brain Science, United Graduate School of Child Development, Osaka University, Suita, Osaka, 565-0871, Japan
| | - Yuichiro Kano
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan
| | - Azusa Hayano
- Laboratory of Molecular Target Therapy for Cancer, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan
| | - Hiroaki Hondoh
- Department of Neurosurgery, Toyama Prefectural Central Hospital, Toyama, 930-8550, Japan
| | - Kazuhiro Ikenaka
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan
| | - Ryuya Yamanaka
- Laboratory of Molecular Target Therapy for Cancer, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan.
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Structural and quantitative evidence of α2-6-sialylated N-glycans as markers of the differentiation potential of human mesenchymal stem cells. Glycoconj J 2016; 34:797-806. [PMID: 27314244 PMCID: PMC5711977 DOI: 10.1007/s10719-016-9699-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Revised: 06/01/2016] [Accepted: 06/02/2016] [Indexed: 02/02/2023]
Abstract
Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such, they hold promise for use in stem cell-based therapies. However, no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously, we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that α2–6-sialylation is a marker of the differentiation potential of these cells. Nevertheless, no information was available about the structural details of these of α2–6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs), human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of α2–6-sialylated N-glycans was detected in early passage cells (24–28 % of sialylated N-glycans) compared with late passage cells (13–15 %). A major α2–6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes, Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast, no significant differences were observed between early and late passage hMSCs with respect to α2–6-sialylated O-glycan percentages. These results demonstrate that levels of α2–6-sialylated N-glycans, but not O-glycans, could be used as markers of the differential potential of hMSCs.
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Torii T, Yoshimura T, Narumi M, Hitoshi S, Takaki Y, Tsuji S, Ikenaka K. Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex. Glycoconj J 2014; 31:671-83. [PMID: 25417067 PMCID: PMC4245497 DOI: 10.1007/s10719-014-9566-2] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2014] [Revised: 10/03/2014] [Accepted: 10/23/2014] [Indexed: 11/25/2022]
Abstract
Oligosaccharides of glycoproteins expressed on the cell surface play important roles in cell-cell interactions, particularly sialylated N-glycans having a negative charge, which interact with sialic acid-binding immunoglobulin-like lectins (siglecs). The entire structure of sialylated N-glycans expressed in the mouse brain, particularly the linkage type of sialic acid residues attached to the backbone N-glycans, has not yet been elucidated. An improved method to analyze pyridylaminated sugar chains using high performance liquid chromatography (HPLC) was developed to determine the entire structure of sialylated N-linked sugar chains expressed in the adult and developing mouse cerebral cortices. Three classes of sialylated sugar chains were prevalent: 1) N-glycans containing α(2-3)-sialyl linkages on a type 2 antennary (Galβ(1-4)GlcNAc), 2) sialylated N-glycans with α(2-6)-sialyl linkages on a type 2 antennary, and 3) a branched sialylated N-glycan with a [Galβ(1-3){NeuAcα(2-6)}GlcNAc-] structure, which was absent at embryonic day 12 but then increased during development. This branched type sialylated N-glycan structure comprised approximately 2 % of the total N-glycans in the adult brain. Some N-glycans (containing type 2 antennary) were found to change their type of sialic acid linkage from α(2-6)-Gal to α(2-3)-Gal. Thus, the linkages and expression levels of sialylated N-glycans change dramatically during brain development.
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Affiliation(s)
- Tomohiro Torii
- Department of Physiological Sciences, School of Life Sciences, The Graduate University for Advanced Studies (SOKENDAI), Shonan Village, Hayama, Kanagawa, 240-0193, Japan
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Onsirisakul N, Nakakita SI, Boonarkart C, Kongchanagul A, Suptawiwat O, Puthavathana P, Chaichuen K, Kittiniyom K, Suzuki Y, Auewarakul P. Substrate specificity of avian influenza H5N1 neuraminidase. World J Virol 2014; 3:30-36. [PMID: 25396120 PMCID: PMC4229813 DOI: 10.5501/wjv.v3.i4.30] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/19/2014] [Revised: 09/03/2014] [Accepted: 10/16/2014] [Indexed: 02/05/2023] Open
Abstract
AIM: To characterise neuraminidase (NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus.
METHODS: Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography (HPLC) analysis of the enzymatic product.
RESULTS: The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked (P < 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin (HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid.
CONCLUSION: H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.
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Yoshimura T, Yamada G, Narumi M, Koike T, Ishii A, Sela I, Mitrani-Rosenbaum S, Ikenaka K. Detection of N-glycans on small amounts of glycoproteins in tissue samples and sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 2012; 423:253-60. [PMID: 22369894 DOI: 10.1016/j.ab.2012.01.023] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2011] [Revised: 01/21/2012] [Accepted: 01/23/2012] [Indexed: 11/30/2022]
Abstract
N-linked glycans harbored on glycoproteins profoundly affect the character of proteins by altering their structure or capacity to bind to other molecules. Specific knowledge of the role of N-glycans in these changes is limited due to difficulties in identifying precise carbohydrate structures on a given glycoprotein, which arises from the large amounts of glycoprotein required for N-glycan structural determination. Here, we refined a simple method to purify and detect trace amounts of N-glycans. During the N-glycan purification step, most contaminants were removed by two kinds of columns: a graphite carbon column and a cellulose column. N-Glycans were identified with a three-dimensional high-performance liquid chromatography (HPLC) system. Using our method, a global analysis of N-glycans from human muscle biopsy samples and mouse brain sections was possible. By combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with our method, we refined analytical procedures for N-glycans from SDS-PAGE gels using hydrazinolysis to achieve a high N-glycan recovery rate. N-Glycans on as little as 1 μg of the target protein transferrin or immunoglobulin G (IgG) were easily detected. These methods allowed us to efficiently determine glycoprotein N-glycans at picomole (pmol) levels.
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Affiliation(s)
- Takeshi Yoshimura
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan
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Miyazaki T, Matsumoto Y, Matsuda K, Kurakata Y, Matsuo I, Ito Y, Nishikawa A, Tonozuka T. Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642. Glycoconj J 2011; 28:563-71. [PMID: 22020441 DOI: 10.1007/s10719-011-9356-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2011] [Revised: 10/06/2011] [Accepted: 10/06/2011] [Indexed: 01/12/2023]
Abstract
A gene for processing α-glucosidase I from a filamentous fungus, Aspergillus brasiliensis (formerly called Aspergillus niger) ATCC 9642 was cloned and fused to a glutathione S-transferase tag. The active construct with the highest production level was a truncation mutant deleting the first 16 residues of the hydrophobic N-terminal domain. This fusion enzyme hydrolyzed pyridylaminated (PA-) oligosaccharides Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA and the products were identified as Glc(2)Man(9)GlcNAc(2)-PA and Glc(2)Man(4)-PA, respectively. Saturation curves were obtained for both Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA, and the K (m) values for both substrates were estimated in the micromolar range. When 1 μM Glc(3)Man(4)-PA was used as a substrate, the inhibitors kojibiose and 1-deoxynojirimycin had similar effects on the enzyme; at 20 μM concentration, both inhibitors reduced activity by 50%.
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Affiliation(s)
- Takatsugu Miyazaki
- Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
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Toda T, Nakamura M, Yamada M, Nishine T, Torii T, Ikenaka K, Hashimoto R, Mori M. Glycoproteomic analysis of abnormal N-glycosylation on the kappa chain of cryocrystalglobulin in a patient of multiple myeloma. ACTA ACUST UNITED AC 2009. [DOI: 10.2198/jelectroph.53.1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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Korekane H, Shida K, Murata K, Ohue M, Sasaki Y, Imaoka S, Miyamoto Y. Evaluation of laser microdissection as a tool in cancer glycomic studies. Biochem Biophys Res Commun 2007; 352:579-86. [PMID: 17150194 DOI: 10.1016/j.bbrc.2006.10.191] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2006] [Accepted: 10/30/2006] [Indexed: 11/24/2022]
Abstract
Laser microdissection (LMD) is a recent development that enables the isolation of specific cell populations from tissue sections. This study focuses on the potential of LMD as a tool in cancer glycomics using colon cancer as a model. LMD was performed on hematoxylin and eosin stained frozen tissue sections. Tumor cells and normal epithelial cells were selectively microdissected. N-Glycans from the LMD- and the bulk tissue-derived samples were liberated by hydrazinolysis and then labeled with 2-aminopyridine. After sialidase digestion, the resulting asialo-N-glycans were analyzed by normal and reversed phase HPLC combined with mass spectrometry. Comparison of the various N-glycan profiles with the aid of LMD identified seven characteristic N-glycans with significantly different expression profiles between normal and cancerous cells that could not be detected by conventional analysis. Thus, LMD is a potent and useful tool for analyzing variations in the expression of N-glycans by overcoming the problem of tissue sample heterogeneity.
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Affiliation(s)
- Hiroaki Korekane
- Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-2 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan
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Ishii A, Ikeda T, Hitoshi S, Fujimoto I, Torii T, Sakuma K, Nakakita SI, Hase S, Ikenaka K. Developmental changes in the expression of glycogenes and the content of N-glycans in the mouse cerebral cortex. Glycobiology 2006; 17:261-76. [PMID: 17172259 DOI: 10.1093/glycob/cwl076] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Biosynthesis of N-glycans varies significantly among tissues and is strictly regulated spatially and temporally within the tissue. The strict molecular mechanisms that are responsible for control of N-glycan synthesis remain largely unknown. We developed complementary deoxyribonucleic acid (cDNA) macroarray system and analyzed gene expression levels of more than 140 glycosyltransferases and glycosidases in the cerebral cortex from developing and adult mice. We also analyzed the relative amounts of major N-glycans present in the cerebral cortex and examined how the synthesis of N-glycans might be regulated through the expression of these genes. We demonstrated that the content of N-linked oligosaccharides dramatically changed during the course of brain development. Some of these changes could not be explained by alterations in the expression of the corresponding genes. For example, the amount of core fucosylated sugar chains in the early embryonic brain and the expression level of fucosyltransferase VIII, the only gene known to be responsible for core fucosylation, did not change proportionately. This result suggests that post-transcriptional regulation of this gene plays an important role in regulating its enzymatic activity. On the other hand, the amount of beta1,3-galactose residue-containing sugar chains increased postnatally following an increase in the level of beta1,3-galactosyltransferase messenger ribonucleic acid (mRNA). Furthermore, the amount of sugar chains with an outer fucose residue, containing LewisX-BA-2, correlated well with the expression of fusocyltransferase IX mRNA. These findings add to our understanding of the molecular mechanisms responsible for the regulation of N-glycan biosynthesis in the cerebral cortex.
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Affiliation(s)
- Akihiro Ishii
- Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan
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Sakuma K, Fujimoto I, Hitoshi S, Tanaka F, Ikeda T, Tanabe K, Toyokuni S, Wada H, Mio T, Mishima M, Ikenaka K. An N-glycan structure correlates with pulmonary metastatic ability of cancer cells. Biochem Biophys Res Commun 2006; 340:829-35. [PMID: 16380076 DOI: 10.1016/j.bbrc.2005.12.072] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2005] [Accepted: 12/12/2005] [Indexed: 11/23/2022]
Abstract
N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.
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Affiliation(s)
- Keiichiro Sakuma
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Japan.
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Tanabe K, Ikenaka K. In-column removal of hydrazine and N-acetylation of oligosaccharides released by hydrazionolysis. Anal Biochem 2006; 348:324-6. [PMID: 16325143 DOI: 10.1016/j.ab.2005.10.035] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2005] [Revised: 10/14/2005] [Accepted: 10/18/2005] [Indexed: 10/25/2022]
Affiliation(s)
- Kazuhiro Tanabe
- Analytical Services Division, Mitsubishi Chemical Science and Technology Research Center, Yokohama 227-8502, Japan.
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15
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Shoji H, Ikenaka K, Nakakita SI, Hayama K, Hirabayashi J, Arata Y, Kasai KI, Nishi N, Nakamura T. Xenopus galectin-VIIa binds N-glycans of members of the cortical granule lectin family (xCGL and xCGL2). Glycobiology 2005; 15:709-20. [PMID: 15761024 DOI: 10.1093/glycob/cwi051] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.
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Affiliation(s)
- Hiroki Shoji
- Department of Endocrinology, Kagawa University, 1750-1 Ikenobe, Kita-gun, Kagawa 761-0793, Japan
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16
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Menon KN, Ikeda T, Fujimoto I, Narimatsu H, Nakakita SI, Hase S, Ikenaka K. Changes inN-linked sugar chain patterns induced by moderate-to-high expression of the galactosyltransferase I gene in a brain-derived cell line, CG4. J Neurosci Res 2005; 80:29-36. [PMID: 15723386 DOI: 10.1002/jnr.20416] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Oligosaccharides with biantennae and bisecting N-acetyl glucosamine (GlcNAc) residues attached to the mannose in the beta1-4 trimannosyl core (BA2) are enriched in the brain and considered brain-type sugar chains. We investigated the significance of the interplay between galactosyltransferase I (GalTase I) and BA2 formation in a brain-derived cell line, CG4. Increased GalTase expression in different glial- and neuronal-derived cell lines was accompanied by decreased or undetectable levels of BA2, depending on the level of GalTase expression. Forceful expression of GalTase I in CG4 cells expressing high levels of BA2 and low GalTase activity significantly reduced BA2 levels. In addition, a sixfold increase in an abnormal sugar chain A1(6)G1Fo and a moderate increase in A2G2Fo(6)F were evident. The increased levels of A1(6)G1Fo indicate a diversion or abrogation of the N-linked sugar chain biosynthetic pathway from normal. The accumulation of A1(6)G1Fo and increased A2G2Fo(6)F levels were accompanied by decreased levels of the high mannose-type sugar chains, M5A, M6B, M8A, and M9A. Increased GalTase I expression also led to stunted growth and abnormal morphology of CG4 cells, with increased mortality. Even moderate overexpression of GalTase I thus disrupts the normal biosynthetic pathway of N-linked sugar chains, and high overexpression is fatal to CG4 cells.
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Affiliation(s)
- Krishnakumar N Menon
- Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi, Japan
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17
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Hase S. Chapter 28 Pre- and post-column detection-oriented derivatization techniques in HPLC of carbohydrates. JOURNAL OF CHROMATOGRAPHY LIBRARY 2002. [DOI: 10.1016/s0301-4770(02)80053-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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18
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Yoshida S, Miyazaki M, Zhang QZ, Sakai K, Fujimoto I, Ikenaka K, Ikemoto A, Watanabe S, Okuyama H. Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task. J Neurosci Res 2001; 63:185-95. [PMID: 11169628 DOI: 10.1002/1097-4547(20010115)63:2<185::aid-jnr1010>3.0.co;2-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
We have analyzed oligosaccharide chains in brain microsomes of rats fed an n-3 polyunsaturated fatty acid-deficient (safflower oil group; S group) or -rich (perilla oil group; P group) diet before and after brightness-discrimination learning tasks. The amount of concanavalin A-binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex-type oligosaccharides became dominant after the learning task and especially a tetra-antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S-group than the P group. When polysialylated glycans were analyzed on serotonin-conjugated HPLC column, the glycans in the S-group microsomes before the learning task contained larger amount of higher affinity-polysialylated glycans to serotonin column than those in the P-group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P-group. Removal of mannoside from microsomes by alpha-mannosidase-treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1-anilinonaphthalene-8-sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho-glycans were changed by dietary n-3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin.
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Affiliation(s)
- S Yoshida
- Research Laboratory Center, Oita Medical University, Hasama-cho, Japan.
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19
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Manzi AE, Norgard-Sumnicht K, Argade S, Marth JD, van Halbeek H, Varki A. Exploring the glycan repertoire of genetically modified mice by isolation and profiling of the major glycan classes and nano-NMR analysis of glycan mixtures. Glycobiology 2000; 10:669-89. [PMID: 10910972 DOI: 10.1093/glycob/10.7.669] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
The production of mice with genetic alterations in glycosyltransferases has highlighted the need to isolate and study complex mixtures of the major classes of oligosaccharides (glycans) from intact tissues. We have found that nano-NMR spectroscopy of whole mixtures of N- and O-glycans can complement HPLC profiling methods for elucidating structural details. Working toward obtaining such glycan mixtures from mouse tissues, we decided to develop an approach to isolate not only N- and O-glycans, but also to separate out glycosphingolipids, glycosaminoglycans and glycosylphosphatidylinositol anchors. We describe here a comprehensive Glycan Isolation Protocol that is based primarily upon the physicochemical characteristics of the molecules, and requires only commonly available reagents and equipment. Using radiolabeled internal tracers, we show that recovery of each major class of glycans is as good or better than with conventional approaches for isolating individual classes, and that cross-contamination is minimal. The recovered glycans are of sufficient purity to provide a "glycoprofile" of a cell type or tissue. We applied this approach to compare the N- and O-glycans from wild type mouse tissues with those from mice genetically deficient in glycosyltransferases. N- and O-glycan mixtures from organs of mice deficient in ST6Gal-I (CMP-Sia:Galbeta1-4GlcNAc alpha2-6 sialyltransferase) were studied by the nano-NMR spectroscopy approach, showing no detectable alpha2-6-linked sialic acids. Thus, ST6Gal-I is likely responsible for generating most or all of these residues in normal mice. Similar studies indicate that this linkage is very rare in ganglioside glycans, even in wild-type tissues. In mice deficient in GalNAcT-8 (UDP-GalNAc:polypeptide O-Ser/Thr GalNAc transferase 8), HPLC profiling indicates that O-glycans persist in the thymus in large amounts, without a major change in overall profile, suggesting that other enzymes can synthesize the GalNAc-O-Ser/Thr linkage in this tissue. These results demonstrate the applicability of nano-NMR spectroscopy to complex glycan mixtures, as well as the versatility of the Glycan Isolation Protocol, which makes possible the concurrent examination of multiple glycan classes from intact vertebrate tissues.
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Affiliation(s)
- A E Manzi
- Departments of Medicine and Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla 92093-0687, USA
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