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Gao T, Lott AA, Huang F, Rohokale R, Li Q, Olivos HJ, Chen S, Guo Z. Structural characterization and analysis of different epimers of neutral glycosphingolipid LcGg4 by ion mobility spectrometry-mass spectrometry. Analyst 2022; 147:3101-3108. [PMID: 35695136 DOI: 10.1039/d2an00224h] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
LcGg4, a neutral glycosphingolipid (GSL) and cancer antigen, its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, and three lipid forms of GalNAc-LcGg4 were studied by mass spectrometry (MS). It was found that different forms of GalNAc-LcGg4 carrying homologous (d16:1/18:0) and (d18:1/18:0) lipids were easily separated and identified using liquid chromatography (LC)-MS. In addition, like gangliosides, homologous lipid forms of GalNAc-LcGg4 showed the same fragmentation pattern, except for a uniform shift of their glycolipid product ions by a certain m/z number determined by the varied lipid structure. It was also disclosed that LcGg4 and its epimers GalNAc-LcGg4 and GlcNAc-LcGg4, which are different only in the C4-configuration of their non-reducing end sugar residues, gave the same MS/MS product ions in similar relative intensities, as well as the same LC retention time, suggesting the challenge to differentiate epimeric GSLs by LC-MS. However, ion mobility spectrometry (IMS)-MS was able to efficiently separate and distinguish these epimers. This study has demonstrated the promise of IMS-MS for isomeric GSL characterization and the IMS-MS and LC-MS/MS combination for natural GSL analysis.
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Affiliation(s)
- Tianqi Gao
- Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
| | - Aneirin A Lott
- Department of Biology, Genetics Institute, University of Florida, Gainesville, FL 32611, USA.,Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32610, USA
| | - Fanran Huang
- Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
| | - Rajendra Rohokale
- Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
| | - Qingjiang Li
- Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
| | - Hernando J Olivos
- Waters Corporation, 5 Technology Drive, Building B, Milford, MA 01757, USA
| | - Sixue Chen
- Department of Biology, Genetics Institute, University of Florida, Gainesville, FL 32611, USA.,Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32610, USA
| | - Zhongwu Guo
- Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.
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Ye H, Wang X, Wang L, Chu X, Hu X, Sun L, Jiang M, Wang H, Wang Z, Zhao H, Yang X, Wang J. Full high-throughput sequencing analysis of differences in expression profiles of long noncoding RNAs and their mechanisms of action in systemic lupus erythematosus. Arthritis Res Ther 2019; 21:70. [PMID: 30836987 PMCID: PMC6402184 DOI: 10.1186/s13075-019-1853-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2018] [Accepted: 02/24/2019] [Indexed: 11/17/2022] Open
Abstract
Background The specific function of long noncoding RNAs (lncRNAs) in systemic lupus erythematosus (SLE) and the mechanism of their involvement in related pathological changes remain to be elucidated, so, in this study, we analyzed the differences in the expression profiles of lncRNAs and their mechanisms of action in SLE using full high-throughput sequencing, bioinformatics, etc. methods. Methods We used high-throughput sequencing to detect differences in the expression profiles of lncRNAs, miRNAs, and mRNAs in PBMCs from patients with SLE at the genome-wide level. Next, we predicted target genes of 30 lincRNAs (long intergenic noncoding RNAs) by constructing a coexpression network of differential lincRNAs and mRNAs and identified the role of lincRNAs. Then, we analyzed the coexpression network of 23 optimized lincRNAs and their corresponding 353 miRNAs, evaluated the cis- and trans-effects of these lincRNAs, and performed GO and KEGG analyses of target genes. We also selected 8 lincRNAs and 2 newly discovered lncRNAs for q-PCR validation and lncRNA–miRNA–mRNA analysis. Finally, we also analyzed respectively the relation between lncRNAs and gender bias in SLE patients using RT-qPCR, the relation between Systemic Lupus Erythematosus Disease Activity Index score and the “IFN signature” using ELISA, and the relation between the differential expression of lncRNAs and a change in the number of a cell type of PBMCs in SLE patients using RT-qPCR. Results The profiles of 1087 lncRNAs, 102 miRNAs, and 4101 mRNAs in PBMCs significantly differed between patients with SLE and healthy controls. The coexpression network analysis showed that the network contained 23 lincRNAs and 353 mRNAs. The evaluation of the cis- and trans-effects showed that the 23 lincRNAs acted on 704 target genes. GO and KEGG analyses of the target genes predicted the biological functions of the 23 lincRNAs. q-PCR validation showed 7 lincRNAs and 2 novel lncRNAs were identical to the sequencing results. The ceRNA network contained 7 validated lincRNAs, 15 miRNAs, and 155 mRNAs. In addition, the differential expression of lncRNAs may be gender dependent in SLE patients, SLE patients also exhibit a robust “IFN signature,” and PBMCs exhibiting differential expression of lncRNAs may be due to a change in the number of a cell type. Conclusion This work determined specific lncRNAs that play important biological functions in the pathogenesis of lupus and provided a new direction for diagnosis and treatment of disease. Electronic supplementary material The online version of this article (10.1186/s13075-019-1853-7) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Hui Ye
- Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China.
| | - Xue Wang
- Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Lei Wang
- School of the Second Clinical Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Xiaoying Chu
- Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Xuanxuan Hu
- Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Li Sun
- The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Minghua Jiang
- The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Hong Wang
- The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Zihan Wang
- School of Stomatology, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Han Zhao
- School of the Second Clinical Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China
| | - Xinyu Yang
- Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China.
| | - Jianguang Wang
- Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, 325035, Zhejiang, China.
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Analysis of Differentially Expressed Genes in Necrotic Enteritis-infected Fayoumi Chickens using RNA Sequencing. J Poult Sci 2017; 54:121-133. [PMID: 32908417 PMCID: PMC7477130 DOI: 10.2141/jpsa.0160053] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
We identified and evaluated differentially expressed genes (DEGs) by RNA-Sequencing (RNA-Seq) in the intestinal mucosa of two Fayoumi chicken lines, M5.1 and M15.2, that are affected by necrotic enteritis (NE); these chicken lines share the same genetic background but have different major histocompatibility complexes. RNA-Seq generated over 49 and 40 million reads for lines M5.1 and M15.2, respectively. The alignment of these sequences with the Gallus gallus genome database revealed the expression of more than 14,500 genes in two lines, among which 581, 1270, and 1140 DEGs were detected when lines M15.2 and M5.1 were compared with the control and compared between each other. The analysis of all DEGs using the gene ontology database revealed annotations for 111 biological processes, 32 cellular components, and 17 molecular functions, and KEEG pathway mapping indicated that the DEGs were primarily involved in immunity, responses to various stimuli, and signal transduction. In addition, we analyzed 183 innate immune genes that were differentially expressed in NE-induced chicken lines, including 46 CD molecular genes, 89 immune-related genes, and 13 β-defensin genes with 3 lineage-specific duplications. Taken together, the transcriptional profiles showed that line M5.1 was more resistant to NE than line M15.2 and that differential gene expression patterns were associated with host genetic differences in resistance to NE. qRT-PCR and RNA-Seq analyses showed that all the genes examined had similar responses to NE (correlation coefficient R=0.84 to 0.88, p<0.01) in both lines. To the best of our knowledge, this is the first study that describes NE-induced DEGs using RNA-seq in two lines with different levels of susceptibility to NE. These results will lead to increased insights on NE disease resistance mechanisms and the role of host genes in the control of the host immune response.
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Abstract
Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Galα4Galβ4Glcβ1Cer) and isoglobotriaosylceramide (Galα3Galβ4Glcβ1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6. Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9. Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.
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Zhou Z, Zhang C, Xia C, Chen W, Zhu H, Shang P, Ma F, Wang PG, Zhang J, Xu W, Tian Z. Enhanced Antitumor Effects by Chemical Modified IGb3 Analogues. Mol Cancer Ther 2011; 10:1375-84. [PMID: 21653685 DOI: 10.1158/1535-7163.mct-11-0030] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
MESH Headings
- Animals
- Antigens, CD1d/chemistry
- Antigens, CD1d/metabolism
- Antineoplastic Agents/chemistry
- Antineoplastic Agents/pharmacology
- Binding Sites
- Dendritic Cells/drug effects
- Dendritic Cells/immunology
- Globosides/chemistry
- Globosides/pharmacology
- Killer Cells, Natural/drug effects
- Killer Cells, Natural/immunology
- Melanoma, Experimental/immunology
- Melanoma, Experimental/metabolism
- Melanoma, Experimental/pathology
- Mice
- Mice, Inbred C57BL
- Models, Molecular
- Neoplasm Metastasis
- Protein Binding
- Receptors, Antigen, T-Cell, alpha-beta/chemistry
- Receptors, Antigen, T-Cell, alpha-beta/metabolism
- STAT1 Transcription Factor/metabolism
- T-Box Domain Proteins/metabolism
- Trihexosylceramides/chemistry
- Trihexosylceramides/pharmacology
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Affiliation(s)
- Zhixia Zhou
- Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
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Chuang K, Elford EL, Tseng J, Leung B, Harris HW. An expanding role for apolipoprotein E in sepsis and inflammation. Am J Surg 2010; 200:391-7. [PMID: 20409531 PMCID: PMC2909338 DOI: 10.1016/j.amjsurg.2009.10.017] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2009] [Revised: 10/19/2009] [Accepted: 10/27/2009] [Indexed: 11/28/2022]
Abstract
BACKGROUND Apolipoprotein E (apoE), a component of plasma lipoproteins, plays an important, but poorly defined role in sepsis. We have shown that injecting apoE increases septic mortality in a rat model of gram-negative bacterial sepsis, with concomitant hepatic natural killer T (NKT) cell proliferation and activation. The presumed mechanism for this apoE-mediated mortality is that apoE can bind and traffic antigens, presumed to include lipopolysaccharide (LPS), and promote activation of dendritic cells (DC) with subsequent NKT activation and cytokine release. Thus, we sought to prove that LPS was the antigen responsible for the increased NKT activation enhanced by the presence of apoE. METHODS We isolated murine marrow-derived DCs, pulsed them with lipid antigen (LPS, and positive controls alpha-galactosylceramide [alpha-GalCer] and isoglobotrihexosylceramide 3 [iGb3]) with or without apoE, and then cocultured the DCs with hybridoma NKTs. NKT activation was measured by interleukin-2 (IL-2) supernatant levels using enzyme-linked immunosorbent assay (ELISA). RESULTS LPS at different concentrations was a weak stimulus for NKT activation regardless of apoE presence. When apoE was present, iGb3, an endogenous ligand analog, elicited more than a 2-fold increase in IL-2 response when compared with iGb3 alone (P < .05). CONCLUSIONS These results indicate an endogenous ligand, not LPS, may be responsible for NKT activation. A molecular remnant similar to iGb3 could act as a damage-associated molecular pattern and play a prominent role in animal models of sepsis.
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Affiliation(s)
- Kelley Chuang
- Department of Surgery, University of California, San Francisco, East Bay, Oakland, USA
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Dias BR, Rodrigues EG, Nimrichter L, Nakayasu ES, Almeida IC, Travassos LR. Identification of iGb3 and iGb4 in melanoma B16F10-Nex2 cells and the iNKT cell-mediated antitumor effect of dendritic cells primed with iGb3. Mol Cancer 2009; 8:116. [PMID: 19968878 PMCID: PMC2795753 DOI: 10.1186/1476-4598-8-116] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2009] [Accepted: 12/07/2009] [Indexed: 01/23/2023] Open
Abstract
Background CD1d-restricted iNKT cells are protective against murine melanoma B16F10-Nex2 growing subcutaneously in syngeneic C57Bl/6 mice as inferred from the fast tumor development in CD1d-KO in comparison with wild type animals. CD1d glycoproteins are related to the class I MHC molecules, and are involved in the presentation, particularly by dentritic cells (DC), of lipid antigens to iNKT cells. In the present work we attempted to identify the endogenous lipid mediator expressed in melanoma cells inducing such immunesurveillance response and study the possibility of protecting animals challenged with tumor cells with lipid-primed DC. Results Crude cytosolic and membrane fractions from in vivo growing melanoma contained iNKT-stimulating substances. Lipids were then extracted from these cells and one of the fractions (i.e. F3A) was shown to prime bone marrow-derived dendritic cells (BMDC) to stimulate iNKT murine hybridoma (DN32D3) cells to produce IL-2. The active fraction was analyzed by electrospray ionization-mass spectrometry (ESI-LIT-MS) and both iGb3 and iGb4 were identified along with GM3. When iGb3 was incubated with BMDC and tested with DN32D3 cells, IL-2 was equally produced indicating iNKT cell activation. GM3 consistently inhibited this response. To assess the antitumor response-induced by iGb3, a cytotoxicity assay in vitro was used with [3H]-thymidine labeled B16F10-Nex2 cells. At target/effector (iGb3-activated iNKT) cell ratio of 100-1-100-4 tumor cell lysis was shown. The antitumor activity in vivo was tested in mice challenged i.v. with B16F10-Nex2 cells and treated with iGb3- or α-galactosylceramide-primed DCs. A 4-fold lower tumor load in the lungs was observed with either treatment. Conclusion Our results show the expression of globo and isoglobohexosylceramides in murine melanoma B16F10-Nex2. The expression of iGb3 and its precursor, iGb4, on tumor cells may prime an effective iNKT cell-dependent antitumor response, modulated negatively by GM3 which is also produced in these cells. iGb3-primed BMDC exerted a significant iNKT cell-mediated anti-tumor activity in mice challenged with melanoma cells.
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Affiliation(s)
- Bianca R Dias
- Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil.
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Ben Ya’acov A, Lalazar G, Livovsky DM, Kanovich D, Axelrod E, Preston S, Schwarzmann G, Ilan Y. Decreased STAT-1 phosphorylation by a thio analogue of beta-d-glucosylceramide is associated with altered NKT lymphocyte polarization. Mol Immunol 2009; 47:526-33. [DOI: 10.1016/j.molimm.2009.07.030] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2009] [Accepted: 07/28/2009] [Indexed: 10/20/2022]
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Glucocerebroside: an evolutionary advantage for patients with Gaucher disease and a new immunomodulatory agent. Immunol Cell Biol 2009; 87:514-24. [PMID: 19529001 DOI: 10.1038/icb.2009.42] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Gaucher disease (GD) is caused by the reduced activity of a lysosomal enzyme, glucocerebrosidase, leading to the accumulation of glucocerebroside (GC). The relatively high prevalence of this disease within an ethnic group is believed to reflect a selective advantage. Treatment with enzyme replacement therapy (ERT) is safe and effective in ameliorating the primary symptoms of the disease, yet there have been reports that some patients on ERT have developed type 2 diabetes or metabolic syndrome, malignancies and central nervous system disorders. A series of animal studies suggest that these complications may be related to the reduction of GC levels by the enzyme administered. GC has been shown to have an immunomodulatory effect through the promotion of dendritic cells, natural killer T cells, and regulatory T cells. The break down of GC to ceramide can underline part of these findings. Clinical trials suggested a beneficial effect of GC in type 2 diabetes or nonalcoholic steatohepatitis. This review of the data from animal models and humans proposes that the increased level of GC may provide an evolutionary advantage for patients with GD. Indirectly, these data support treating symptomatic patients with mild/moderate GD with low-dose ERT and re-evaluating the use of ERT in asymptomatic patients.
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Li Y, Thapa P, Hawke D, Kondo Y, Furukawa K, Furukawa K, Hsu FF, Adlercreutz D, Weadge J, Palcic MM, Wang PG, Levery SB, Zhou D. Immunologic glycosphingolipidomics and NKT cell development in mouse thymus. J Proteome Res 2009; 8:2740-51. [PMID: 19284783 PMCID: PMC2720133 DOI: 10.1021/pr801040h] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting molecule. Genetic evidence suggested that beta-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. N-butyldeoxygalactonojirimycin (NB-DGJ), a drug that specifically inhibits the glucosylceramide synthase, inhibits the endogenous ligands for NKT cells. Furthermore, we and others have found a beta-linked glycosphingolipid, isoglobotriaosylceramide (iGb3), is a stimulatory NKT ligand. The iGb3 synthase knockout mice have a normal NKT development and function, indicating that other ligands exist and remain to be identified. In this study, we have performed a glycosphingolipidomics study of mouse thymus, and studied mice mutants which are deficient in beta-hexosaminidase b or alpha-galactosidase A, two glycosidases that are up- and downstream agents of iGb3 turnover, respectively. Our mass spectrometry methods generated a first database for glycosphingolipids expressed in mouse thymus, which are specifically regulated by rate-limiting glycosidases. Among the identified thymic glycosphingolipids, only iGb3 is a stimulatory ligand for NKT cells, suggesting that large-scale fractionation, enrichment and characterization of minor species of glycosphingolipids are necessary for identifying additional ligands for NKT cells. Our results also provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycosphingolipids.
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Affiliation(s)
- Yunsen Li
- Department of Melanoma Medical Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77054, USA
| | - Prakash Thapa
- Department of Melanoma Medical Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77054, USA
| | - David Hawke
- Mass Spectrometry Core Facility, Department of Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77054, USA
| | - Yuji Kondo
- Department of Biochemistry II, Nagoya University School of Medicine, Tsurumai, Showa-ku, Nagoya 466-0065, Japan
| | - Keiko Furukawa
- Department of Biochemistry II, Nagoya University School of Medicine, Tsurumai, Showa-ku, Nagoya 466-0065, Japan
| | - Koichi Furukawa
- Department of Biochemistry II, Nagoya University School of Medicine, Tsurumai, Showa-ku, Nagoya 466-0065, Japan
| | - Fong-Fu Hsu
- Mass Spectrometry Resource, Division of Endocrinology, Diabetes, Metabolism, and Lipid Research, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA
| | | | - Joel Weadge
- Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Denmark
| | - Monica M Palcic
- Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Denmark
| | - Peng G. Wang
- Department of Biochemistry, The Ohio State University, Columbus, OH 43210
| | - Steven B Levery
- Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark
| | - Dapeng Zhou
- Department of Melanoma Medical Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77054, USA
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Lalazar G, Ben Ya'acov A, Livovsky DM, El Haj M, Pappo O, Preston S, Zolotarov L, Ilan Y. Beta-glycoglycosphingolipid-induced alterations of the STAT signaling pathways are dependent on CD1d and the lipid raft protein flotillin-2. THE AMERICAN JOURNAL OF PATHOLOGY 2009; 174:1390-9. [PMID: 19246642 DOI: 10.2353/ajpath.2009.080841] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Beta-glucosylceramide has been shown to affect natural killer T cell function in models of inflammation. We, therefore, investigated the effects of different beta-glycosphingolipids, including beta-glucosylceramide, on STAT (signal transducers and activators of transcription) signaling pathways and determined whether these effects were mediated by lipid raft microdomains and/or CD1d molecules. The effects of alpha- and beta-structured ligands on the lipid raft protein flotillin-2 were studied in both natural killer T hybridoma cells and leptin-deficient mice. To determine whether CD1d was involved in the effects of the beta-glycosphingolipids, an anti-CD1d blocking antibody was used in a cell proliferation assay system. The downstream effects on the protein phosphorylation levels of STAT1, STAT3, and STAT6 were examined in both immune-mediated hepatitis and hepatoma models. The effects of beta-glycosphingolipids on the STAT signaling pathways were found to be dependent on CD1d. Lipid rafts were affected by both the dose and ratio of the beta-glycosphingolipids and the acyl chain length, and these effects were followed by downstream effects on STAT proteins. Our results show that beta-glycosphingolipids have beneficial effects in natural killer T cell-dependent immune-mediated metabolic and malignant animal models in vivo.
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Affiliation(s)
- Gadi Lalazar
- Liver Unit, Department of Medicine, Hebrew University-Hadassah Medical Center, P.O.B 12000, Jerusalem, Israel, IL-91120
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Lalazar G, Ben Ya'acov A, Eliakim-Raz N, Livovsky DM, Pappo O, Preston S, Zolotarov L, Ilan Y. β-Glycosphingolipids-mediated lipid raft alteration is associated with redistribution of NKT cells and increased intrahepatic CD8+ T lymphocyte trapping. J Lipid Res 2008; 49:1884-93. [DOI: 10.1194/jlr.m800113-jlr200] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
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Blanchard B, Nurisso A, Hollville E, Tétaud C, Wiels J, Pokorná M, Wimmerová M, Varrot A, Imberty A. Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I. J Mol Biol 2008; 383:837-53. [PMID: 18762193 DOI: 10.1016/j.jmb.2008.08.028] [Citation(s) in RCA: 115] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2008] [Revised: 08/11/2008] [Accepted: 08/13/2008] [Indexed: 11/16/2022]
Abstract
The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.
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NKT lymphocyte polarization determined by microenvironment signaling: a role for CD8+ lymphocytes and beta-glycosphingolipids. J Autoimmun 2008; 31:188-95. [PMID: 18710796 DOI: 10.1016/j.jaut.2008.07.003] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2008] [Revised: 07/09/2008] [Accepted: 07/09/2008] [Indexed: 01/15/2023]
Abstract
Natural killer T-cell (NKT) regulatory lymphocytes have been shown to behave differently in various immune settings. The aim of the present study was to determine the effect of microenvironmental signaling on NKT polarization and the process of active CD8 and NKT intrahepatic lymphocyte sequestration. In an in vitro assay, double negative (DN) NKT hybridoma cells were incubated with Hep3B hepatoma cells. This caused a significant increase in the secretion of alpha-fetoprotein (AFP) from Hep3B cells. When NKT cells were exposed to beta-glucoslyceramide (beta-GC) prior to incubation, Hep3B cells exhibited increased proliferation, increased IFN secretion, and reduced AFP secretion. In vivo, the adoptive transfer of naïve DN NKT cells into athymic nude-nu mice transplanted with human Hep3B hepatocellular carcinoma (HCC) caused accelerated tumor growth. This effect was inhibited by prior ex vivo exposure of DN NKT lymphocytes to beta-GC. To assess the effect of the immunological environment on NKT cells, immune mediated hepatitis and colitis were induced simultaneously in mice. Induction of TNBS colitis prior to administration of concanavalin A (Con A) hepatitis resulted in an aggravation of the liver damage caused by Con A hepatitis alone. This effect was associated with reduced intrahepatic CD8+ T cell trapping and an increase in intrahepatic NKT cells. The presence of different ligands altered host microenvironment signaling and influenced the fate and polarization of NKT cells and the sequestration of active intrahepatic lymphocytes. These data support the notion that NKT regulatory lymphocytes have an inherent plasticity that may be important for their regulatory function.
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Abstract
beta-Glycosphingolipids have emerged as a family of potential ligands for natural killer T (NKT)-regulatory lymphocytes. This subset of regulatory lymphocytes has been implicated in the regulation of autoimmune processes. The major histocompatibility complex (MHC) Class I-like CD1d glycoprotein is a member of the CD1 family of antigen-presenting molecules and is responsible for selection of NKT cells. beta-Glycolipids have been shown to alter immune responses in the opposing settings of autoimmune diseases or cancer. In this review, we discuss the potential use of beta-glycoshpingolipids for NKT-based immunotherapy.
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Affiliation(s)
- Tomer Adar
- Liver Unit, Hebrew University-Hadassah Medical Center, Jerusalem, Israel
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16
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Mizrahi M, Lalazar G, Ben Ya’acov A, Livovsky DM, Horowitz Y, Zolotarov L, Adler R, Shouval D, Ilan Y. β-Glycoglycosphingolipid-induced augmentation of the anti-HBV immune response is associated with altered CD8 and NKT lymphocyte distribution: A novel adjuvant for HBV vaccination. Vaccine 2008; 26:2589-95. [PMID: 18423947 DOI: 10.1016/j.vaccine.2008.03.026] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2007] [Revised: 02/26/2008] [Accepted: 03/12/2008] [Indexed: 01/12/2023]
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17
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Shang P, Zhang C, Xia C, Chen W, Han Q, Wang PG, Zhang J, Tian Z. Chemical modification of iGb3 increases IFN-gamma production by hepatic NKT cells. Int Immunopharmacol 2008; 8:645-53. [PMID: 18387506 DOI: 10.1016/j.intimp.2008.01.003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2007] [Revised: 01/04/2008] [Accepted: 01/04/2008] [Indexed: 10/22/2022]
Abstract
Isoglobotrihexosylceramide (iGb3) has been identified as an endogenous ligand recognized by NKT cells; however, it is a weak agonist compared to the exogenous alpha-galactosylceramide. Modification of the structure of iGb3 might improve its stimulatory activity. In this study, we assessed the stimulating activity of chemically-modified iGb3 analogues on murine hepatic NKT cells. We analyzed the percentage of IFN-gamma- or IL-4-producing cells in hepatic iNKT cell population and found that two chemically-modified iGb3 analogues, especially 4'''-dh-iGb3, induced significantly greater intracellular IFN-gamma+ NKT cells in liver by flow cytometry. In vivo experiments also showed that 4-HO-iGb3 and 4'''-dh-iGb3 are selectively strong inducer for rapid serum IFN-gamma production compared with unmodified iGb3. Comparing the structure of iGb3 and its two iGb3 analogues, 4-HO-iGb3 has an extra hydroxy group on C4, suggesting that the additional hydroxy group of phytosphingosine might augment the stability of the CD1d/glycoceramide complex forming and thereby possibly promote IFN-gamma producing. By further modifying the polysaccharide of glycolipid as did in 4'''-dh-iGb3, we found that 4'''-dh-iGb3 elicited more Th1-biased responses than iGb3 and 4-HO-iGb3. This modification might more strongly strengthen the affinity of the TCR/glycoceramide complex and ultimately polarize iNKT cells to release more Th1 cytokines. Our data suggests that a combination modification on both polysaccharide and sphingosine chain of iGb3 elicits preferential Th1-biased responses.
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Affiliation(s)
- Pingping Shang
- Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, China
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18
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Li Y, Teneberg S, Thapa P, Bendelac A, Levery SB, Zhou D. Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry. Glycobiology 2007; 18:158-65. [PMID: 18056651 DOI: 10.1093/glycob/cwm129] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Glycosphingolipids serve as ligands for receptors involved in signal transduction and immune recognition, as exemplified by isoglobotrihexosylceramide, an antigenic ligand for T cell receptors. Mechanistic studies on the regulation of isoglobotrihexosylceramide require biochemical measurement of its lysosomal precursor, isoglobotetraglycosylceramide. It remains a challenge to distinguish between complex tetraglycosylceramide glycosphingolipid isomers with the same sugar components but diverse internal linkages. Here we established a simple and sensitive method to separate globo- and isoglobotetraglycosylceramide by MS5 ion trap mass spectrometry, and report the identification of isoglobotetraglycosylceramide in a CHO cell line transfected by iGb3 synthase, as well as in human thymus.
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Affiliation(s)
- Yunsen Li
- Department of Melanoma Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77054, USA
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El Haj M, Ya'acov AB, Lalazar G, Ilan Y. Potential role of NKT regulatory cell ligands for the treatment of immune mediated colitis. World J Gastroenterol 2007; 13:5799-804. [PMID: 17990345 PMCID: PMC4205426 DOI: 10.3748/wjg.v13.i44.5799] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Natural killer T lymphocytes (NKT) have been implicated in the regulation of autoimmune processes in both mice and humans. In response to stimuli, this subset of cells rapidly produces large amounts of cytokines thereby provoking immune responses, including protection against autoimmune diseases. NKT cells are present in all lymphoid compartments, but are most abundant in the liver and bone marrow. They are activated by interaction of their T-cell receptor with glycolipids presented by CD1d, a nonpolymorphic, major histocompatibility complex class I-like molecule expressed by antigen presenting cells. Several possible ligands for NKT cells have recently been suggested. β-glucosylceramide, a naturally occurring glycolipid, is a metabolic intermediate in the anabolic and catabolic pathways of complex glycosphingolipids. Like other β-glycolipids, β-glucosylceramide has an immunomodulatory effect in several immune mediated disorders, including immune mediated colitis. Due to the broad impact that NKT cells have on the immune system, there is intense interest in understanding how NKT cells are stimulated and the extent to which NKT cell responses can be controlled. These novel ligands are currently being evaluated in animal models of colitis. Here, we discuss strategies to alter NKT lymphocyte function in various settings and the potential clinical applications of natural glycolipids.
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Ya'acov AB, Lalazar G, Ilan Y. Sulfatides for the treatment of autoimmune disorders. Expert Opin Ther Pat 2007. [DOI: 10.1517/13543776.17.9.1191] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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Zajonc DM, Kronenberg M. CD1 mediated T cell recognition of glycolipids. Curr Opin Struct Biol 2007; 17:521-9. [PMID: 17951048 PMCID: PMC2121122 DOI: 10.1016/j.sbi.2007.09.010] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2007] [Revised: 07/24/2007] [Accepted: 09/18/2007] [Indexed: 10/22/2022]
Abstract
Specialized subsets of T lymphocytes can distinguish the carbohydrate portions of microbial and self-glycolipids when they are presented by proteins in the CD1 family of antigen presenting molecules. Recent immunochemical and structural analyses indicate that the chemical composition of the presented carbohydrate, together with its precise orientation above the CD1 binding groove, determines if a particular T cell is activated. More recently, however, it has been shown that the lipid backbone of the glycolipid, buried inside the CD1 protein, also can have an impact on T cell activation. While glycolipid recognition is a relatively new category of T cell specificity, the powerful combination of microbial antigen discovery and structural biochemistry has provided great insight into the mechanism of carbohydrate recognition.
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Affiliation(s)
- Dirk M. Zajonc
- Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA.
| | - Mitchell Kronenberg
- Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA.
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22
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Xia C, Zhou D, Liu C, Lou Y, Yao Q, Zhang W, Wang PG. Thio-isoglobotrihexosylceramide, an agonist for activating invariant natural killer T cells. Org Lett 2007; 8:5493-6. [PMID: 17107055 DOI: 10.1021/ol062199b] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Thio-isoglobotrihexosylceramide (S-iGb3) might be resistant to alpha-galactosidases in antigen-presenting cells and have a longer retaining time in the lysosome before being loaded to CD1d. The biological assay showed that S-iGb3 demonstrates a much higher increase as a stimulatory ligand toward invariant natural killer T (iNKT) cells as compared to iGb3. [structure: see text].
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Affiliation(s)
- Chengfeng Xia
- Departments of Biochemistry and Chemistry, The Ohio State University, Columbus, Ohio 43210, USA
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