Supplementary Figure 1 from Enhanced Antitumor Effects by Chemical Modified IGb3 Analogues

Supplementary Figure 2 from Enhanced Antitumor Effects by Chemical Modified IGb3 Analogues

Unified wearable configurations have led to many natural advances, including write-ahead logging and wide-area networks. In this work, we disconfirm the analysis of Boolean logic. We disprove that while RAID and I/O automata can collaborate to answer this issue, the seminal Bayesian algorithm for the understanding of RPCs is optimal.

Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.

The glycosphingolipid α-GalCer has been found to influence mammalian immune system significantly through the natural killer T cells. Unfortunately, the pre-clinical and clinical studies revealed several critical disadvantages that prevented the therapeutic application of α-GalCer in treating cancer and other diseases. Recently, the detailed illustration of the CD1d/α-GalCer/NKT TCR complex crystal structural, together with other latest structural and biological understanding on glycolipid ligands and NKT cells, provided a new platform for developing novel glycolipid ligands with optimized therapeutic effects. Here, we designed a series of novel aromatic group substituted α-GalCer analogues. The biological activity of these analogues was characterized and the results showed the unique substitution group manipulated the immune responses of NKT cells. Computer modeling and simulation study indicated the analogues had unique binding mode when forming CD1d/glycolipid/NKT TCR complex, comparing to original α-GalCer.

The targeting of autologous vaccines toward antigen presenting cells (APCs) via the in vivo complexation between anti α-Gal (anti-Gal) antibodies and α-Gal antigens presents a promising cancer immunotherapy with enhanced immunogenicity. This strategy takes advantage of the ubiquitous anti-Gal antibody in human serum. In contrast to the α-Gal epitope, the recent identification of high titers of anti-l-rhamnose (anti-Rha) antibodies in humans reveals a new approach toward immunotherapy employing l-rhamnose (Rha) monosaccharides. In order to evaluate this simple antigen in preclinical applications, we have synthesized Rha-conjugated immunogens and successfully induced high titers of anti-Rha antibodies in wildtype mice. Moreover, our studies demonstrate for the first time that wildtype mice could replace α1,3galactosyltransferase knockout (α1,3GT KO) mice in such antigen/antibody-mediated vaccine design when developing cancer immunotherapies.

Abstract Certain glycolipid antigens for natural killer T (NKT) cells can direct the overall cytokine balance of the immune response. However, the molecular mechanism of Th1- or Th2-biased cytokine secretion by NKT cells is still unknown. Previously, we synthesized isoglobotrihexosylceramide (iGb3) analogues by introducing a hydroxyl group at C4 on the ceramide portion of iGb3 to produce 4-HO-iGb3 or to further deoxylation on the terminal galactose to produce 4‴-dh-iGb3. Both modified iGb3, especially 4‴-dh-iGb3, stimulated more IFN-γ production by hepatic NKT cells, and thus elicited preferential Th1 responses. Here, we found that 4‴-dh-iGb3–loaded bone marrow–derived dendritic cells (DC) could significantly inhibit growth of subcutaneous melanoma and suppress lung metastasis in C57BL/6 mice compared with unmodified iGb3-loaded DCs. In investigating the mechanisms of this improved activity, we found that 4‴-dh-iGb3 stimulation increased STAT1 signaling by NKT cells, whereas the phosphorylation of Th2 type cytokine–associated transcription factor STAT6 signaling was not affected. Analysis of the structures of iGb3 and 4‴-dh-iGb3 revealed that 4‴-dh-iGb3 provides greater stability and affinity between glycolipid and CD1d or NKT TCR complex than iGb3. Thus, 4‴-dh-iGb3 can improve the antitumor effects of a DC-based vaccine possibly by stabilizing the CD1d/glycolipid/TCR complex and stimulating IFN-γ signaling of NKT cells. Furthermore, chemical modification of iGb3 can elicit Th1-biased responses by NKT cells, and 4‴-dh-iGb3 combined with a DC vaccine may serve as a potent new NKT-based therapy against tumors and infectious diseases. Mol Cancer Ther; 10(8); 1375–84. ©2011 AACR.

A library of 11 UDP-N-acetylglucosamine analogs were rapidly screened for their activities as donors for the Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase (LgtA) by direct on-chip reaction and detection with SAMDI-TOF mass spectrometry. Six of the analogs were active in this assay and were analyzed by SAMDI to characterize the kinetics toward LgtA. The analysis revealed that substitutions on C-2, C-4, and C-6 affect the activity of the donors, with bulky groups at these positions decreasing affinity of the donors for the enzyme, and also revealed that activity is strongly affected by the stereochemistry at C-3, but not C-4, of the donor. The study is also significant because it demonstrates that SAMDI can be used to both profile glycosyltransferase activities and to provide a quantitative assessment of enzyme activity.

Certain glycolipid antigens for natural killer T (NKT) cells can direct the overall cytokine balance of the immune response. However, the molecular mechanism of Th1or Th2-biased cytokine secretion by NKT cells is still unknown. Previously, we synthesized isoglobotrihexosylceramide (iGb3) analogues by introducing a hydroxyl group at C4 on the ceramide portion of iGb3 to produce 4-HO-iGb3 or to further deoxylation on the terminal galactose to produce 40 0 0-dh-iGb3. Both modified iGb3, especially 40 0 0-dh-iGb3, stimulated more IFN-g production by hepatic NKT cells, and thus elicited preferential Th1 responses. Here, we found that 40 0 0-dh-iGb3–loaded bone marrow–derived dendritic cells (DC) could significantly inhibit growth of subcutaneous melanoma and suppress lung metastasis in C57BL/6 mice compared with unmodified iGb3-loaded DCs. In investigating the mechanisms of this improved activity, we found that 40 0 0-dh-iGb3 stimulation increased STAT1 signaling by NKT cells, whereas the phosphorylation of Th2 type cytokine–associated transcription factor STAT6 signaling was not affected. Analysis of the structures of iGb3 and 40 0 0-dh-iGb3 revealed that 40 0 0-dh-iGb3 provides greater stability and affinity between glycolipid and CD1d or NKT TCR complex than iGb3. Thus, 40 0 0-dh-iGb3 can improve the antitumor effects of a DCbased vaccine possibly by stabilizing the CD1d/glycolipid/TCR complex and stimulating IFN-g signaling of NKT cells. Furthermore, chemical modification of iGb3 can elicit Th1-biased responses by NKT cells, and 40 0 0-dh-iGb3 combined with a DC vaccine may serve as a potent new NKT-based therapy against tumors and infectious diseases. Mol Cancer Ther; 10(8); 1–10. 2011 AACR.

Polysaccharides constitute one of the major classes of bio-macromolecules in living organisms. In the past several decades, tremendous advances in glycobiology and cell biology revealed that polysaccharides play essential roles in a variety of important biological processes. Various natural polysaccharides and their analogues have been synthesized via in vitro enzymatic polymerization. This chapter describes the in vitro biosynthesis of O-polysaccharides from Escherichia coli, as well as in vivo production of lipopolysaccharide and its analogues via biopathway engineering.

AbstractAn efficient synthesis of galactosylceramide analogues of type (VIII) for invariant natural killer T (iNKT) cell stimulation is described.

2-ketoGlc, which is the C-2-carbon isostere of GlcNAc, is a novel GlcNAc analogue with a ketone group. The corresponding glycosyltransferase donor substrate. UDP-2-ketoGlc, is necessary for synthesizing 2-ketoGlc-containing molecules and is thus highly important for metabolic polysaccharide remodeling and engineering. We report here the first chemoenzymatic synthesis of UDP-2-ketoGlc using our two-enzyme (NahK and GlmU) system in vitro.

Glycolipids are potential antigens for iNKT cells recognition and demonstrate important roles in both innate and adaptive immunity. However, the difficulties in the preparation of pure configuration defined glycolipids limit the exploration of their different profiles in activating iNKT cells. We report here a concise and stereospecific preparation of novel galactosylceramide analogues by oxime ligation. This strategy would provide an efficient way to generate varied glycolipid analogues with either synthetic or natural carbohydrates for biological evaluations.

ChemMedChemVolume 4, Issue 11 p. 1810-1815 Communication The Roles of 3′ and 4′ Hydroxy Groups in α-Galactosylceramide Stimulation of Invariant Natural Killer T Cells Chengfeng Xia Prof. Dr., Chengfeng Xia Prof. Dr. [email protected] State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204 (China), Fax: (+86) 871-522-3354 Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106 These authors contributed equally to this work.Search for more papers by this authorWenpeng Zhang, Wenpeng Zhang Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106 These authors contributed equally to this work.Search for more papers by this authorYalong Zhang Dr., Yalong Zhang Dr. Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorWenlan Chen, Wenlan Chen Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorJanos Nadas, Janos Nadas Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorRyan Severin, Ryan Severin Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121 (USA)Search for more papers by this authorRobert Woodward, Robert Woodward Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorBin Wang Dr., Bin Wang Dr. College of Pharmacy and State Key Laboratory of Element-Organic, Chemistry, Nankai University, Tianjin 300071 (China)Search for more papers by this authorXin Wang, Xin Wang College of Pharmacy and State Key Laboratory of Element-Organic, Chemistry, Nankai University, Tianjin 300071 (China)Search for more papers by this authorMitchell Kronenberg Prof. Dr., Mitchell Kronenberg Prof. Dr. Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121 (USA)Search for more papers by this authorPeng G. Wang Prof. Dr., Peng G. Wang Prof. Dr. [email protected] Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this author Chengfeng Xia Prof. Dr., Chengfeng Xia Prof. Dr. [email protected] State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204 (China), Fax: (+86) 871-522-3354 Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106 These authors contributed equally to this work.Search for more papers by this authorWenpeng Zhang, Wenpeng Zhang Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106 These authors contributed equally to this work.Search for more papers by this authorYalong Zhang Dr., Yalong Zhang Dr. Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorWenlan Chen, Wenlan Chen Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorJanos Nadas, Janos Nadas Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorRyan Severin, Ryan Severin Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121 (USA)Search for more papers by this authorRobert Woodward, Robert Woodward Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this authorBin Wang Dr., Bin Wang Dr. College of Pharmacy and State Key Laboratory of Element-Organic, Chemistry, Nankai University, Tianjin 300071 (China)Search for more papers by this authorXin Wang, Xin Wang College of Pharmacy and State Key Laboratory of Element-Organic, Chemistry, Nankai University, Tianjin 300071 (China)Search for more papers by this authorMitchell Kronenberg Prof. Dr., Mitchell Kronenberg Prof. Dr. Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121 (USA)Search for more papers by this authorPeng G. Wang Prof. Dr., Peng G. Wang Prof. Dr. [email protected] Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12 th Avenue, Columbus, OH 43210 (USA), Fax: (+1) 614-688-3106Search for more papers by this author First published: 23 October 2009 https://doi.org/10.1002/cmdc.200900350Citations: 16 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract NKT cell stimulation: C3′- and C4′-modified α-galactosylceramide (α-GalCer) analogues show that the 3′-OH group is crucial for maintaining α-GalCer immunogenicity; any modifications at this position lead to loss of activity. However, the C4′ position is less sensitive and tolerates minor modifications. Moreover, C4′-substituted analogues can induce the release of biased Th2 cytokines. Supporting Information Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Filename Description cmdc_200900350_sm_miscellaneous_information.pdf197.3 KB miscellaneous_information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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The semi‐invariant natural killer (NK) T‐cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the α‐chain of the NKTcr is invariant, the β‐chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an α‐linked monosaccharide (α‐galactosylceramide and α‐galactosyldiacylglycerol) and the β‐linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their β‐chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including α‐galactosylceramide, the structurally similar α‐galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr β‐chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr β‐chain allows these cells to recognise unique aspects of structurally diverse CD1d‐restricted ligands.

Identification of uncommon targets for Inhibition, such as virulence factors, represents an emerging approach for combating the problem of antibiotic resistance among bacteria. Unfortunately, the lack of effective systems for the discovery and evaluation of inhibitors for such targets has considerably slowed progress. A recent article in ACS Chemical Biology, however, details the development of an in vivo based high-throughput screening strategy for Identification of small molecule inhibitors of wall techoic acid biosynthesis.