68 results on '"Wolfert MA"'
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2. Program and abstracts for the 2011 Meeting of the Society for Glycobiology
- Author
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Hollingsworth, MT, Hart, GW, Paulson, JC, Stansell, E, Canis, K, Huang, IC, Panico, M, Morris, H, Haslam, S, Farzan, M, Dell, A, Desrosiers, R, von Itzstein, M, Matroscovich, M, Luther, KB, Hülsmeier, AJ, Schegg, B, Hennet, T, Nycholat, C, McBride, R, Ekiert, D, Xu, R, Peng, W, Razi, N, Gilbert, M, Wakarchuk, W, Wilson, IA, Gahlay, G, Geisler, C, Aumiller, JJ, Moremen, K, Steel, J, Labaer, J, Jarvis, DL, Drickamer, K, Taylor, M, Nizet, V, Rabinovich, G, Lewis, C, Cobb, B, Kawasaki, N, Rademacher, C, Chen, W, Vela, J, Maricic, I, Crocker, P, Kumar, V, Kronenberg, M, Paulson, J, Glenn, K, Mallinger, A, Wen, H, Srivastava, L, Tundup, S, Harn, D, Menon, AK, Yamaguchi, Y, Mkhikian, H, Grigorian, A, Li, C, Chen, HL, Newton, B, Zhou, RW, Beeton, C, Torossian, S, Tatarian, GG, Lee, SU, Lau, K, Walker, E, Siminovitch, KA, Chandy, KG, Yu, Z, Dennis, JW, Demetriou, M, Pandey, MS, Baggenstoss, BA, Washburn, JL, Weigel, PH, Chen, CI, Keusch, JJ, Klein, D, Hofsteenge, J, Gut, H, Szymanski, C, Feldman, M, Schaffer, C, Gao, Y, Strum, S, Liu, B, Schutzbach, JS, Druzhinina, TN, Utkina, NS, Torgov, VI, Szarek, WA, Wang, L, Brockhausen, I, Hitchen, P, Peyfoon, E, Meyer, B, Albers, SV, Chen, C, Newburg, DS, Jin, C, Dinglasan, RD, Beverley, SM, Guo, H, Novozhilova, N, Hickerson, S, Elnaiem, DE, Sacks, D, Turco, SJ, McKay, D, Castro, E, Takahashi, H, Straus, AH, Stalnaker, SH, Live, D, Boons, GJ, Wells, L, Stuart, R, Aoki, K, Boccuto, L, Zhang, Q, Wang, H, Bartel, F, Fan, X, Saul, R, Chaubey, A, Yang, X, Steet, R, Schwartz, C, Tiemeyer, M, Pierce, M, Kraushaar, DC, Condac, E, Nakato, H, Nishihara, S, Sasaki, N, Hirano, K, Nasirikenari, M, Collins, CC, Lau, JT, Devarapu, SK, Jeyaweerasinkam, S, Albiez, RS, Kiessling, L, Gu, J, Clark, GF, Gagneux, P, Ulm, C, Mahavadi, P, Müller, S, Rinné, S, Geyer, H, Gerardy-Schahn, R, Mühlenhoff, M, Günther, A, Geyer, R, Galuska, SP, Shibata, T, Sugihara, K, Nakayama, J, Fukuda, M, Fukuda, MN, Ishikawa, A, Terao, M, Kimura, A, Kato, A, Katayama, I, Taniguchi, N, Miyoshi, E, Aderem, A, Yoneyama, T, Angata, K, Bao, X, Chanda, S, Lowe, J, Sonon, R, Ishihara, M, Talabnin, K, Wang, Z, Black, I, Naran, R, Heiss, C, Azadi, P, Hurum, D, Rohrer, J, Balland, A, Valliere-Douglass, J, Kodama, P, Mujacic, M, Eakin, C, Brady, L, Wang, WC, Wallace, A, Treuheit, M, Reddy, P, Schuman, B, Fisher, S, Borisova, S, Coates, L, Langan, P, Evans, S, Yang, SJ, Zhang, H, Hizal, DB, Tian, Y, Sarkaria, V, Betenbaugh, M, Lütteke, T, Agravat, S, Cholleti, S, Morris, T, Saltz, J, Song, X, Cummings, R, Smith, D, Hofhine, T, Nishida, C, Mialy, R, Sophie, D, Sebastien, F, Patricia, C, Eric, S, Stephane, H, Mokros, D, Joosten, RP, Dominik, A, Vriend, G, Nguyen, LD, Martinez, J, Hinderlich, S, Reissig, HU, Reutter, W, Fan, H, Saenger, W, Moniot, S, Asada, H, Nakahara, T, Miura, Y, Stevenson, T, Yamazaki, T, De Castro, C, Burr, T, Lanzetta, R, Molinaro, A, Parrilli, M, Sule, S, Gerken, TA, Revpredo, L, Thome, J, Cardenas, G, Almeida, I, Leung, MY, Yan, S, Paschinger, K, Bleuler-Martinez, S, Jantsch, V, Wilson, I, Yoshimura, Y, Adlercreutz, D, Mannerstedt, K, Wakarchuk, WW, Dovichi, NJ, Hindsgaul, O, Palcic, MM, Chandrasekaran, A, Bharadwaj, R, Deng, K, Adams, P, Singh, A, Datta, A, Konasani, V, Imamura, A, Lowry, T, Scaman, C, Zhao, Y, Zhou, YD, Yang, K, Zhang, XL, Leymarie, N, Hartshorn, K, White, M, Cafarella, T, Seaton, B, Rynkiewicz, M, Zaia, J, Acosta-Blanco, I, Ortega-Francisco, S, Dionisio-Vicuña, M, Hernandez-Flores, M, Fuentes-Romero, L, Newburg, D, Soto-Ramirez, LE, Ruiz-Palacios, G, Viveros-Rogel, M, Tong, C, Li, W, Kong, L, Qu, M, Jin, Q, Lukyanov, P, Zhang, W, Chicalovets, I, Molchanova, V, Wu, AM, Liu, JH, Yang, WH, Nussbaum, C, Grewal, PK, Sperandio, M, Marth, JD, Yu, R, Usuki, S, Wu, HC, O'Brien, D, Piskarev, V, Ramadugu, SK, Kashyap, HK, Ghirlanda, G, Margulis, C, Brewer, C, Gomery, K, Müller-Loennies, S, Brooks, CL, Brade, L, Kosma, P, Di Padova, F, Brade, H, Evans, SV, Asakawa, K, Kawakami, K, Kushi, Y, Suzuki, Y, Nozaki, H, Itonori, S, Malik, S, Lebeer, S, Petrova, M, Balzarini, J, Vanderleyden, J, Naito-Matsui, Y, Takematsu, H, Murata, K, Kozutsumi, Y, Subedi, GP, Satoh, T, Hanashima, S, Ikeda, A, Nakada, H, Sato, R, Mizuno, M, Yuasa, N, Fujita-Yamaguchi, Y, Vlahakis, J, Nair, DG, Wang, Y, Allingham, J, Anastassiades, T, Strachan, H, Johnson, D, Orlando, R, Harenberg, J, Haji-Ghassemi, O, Mackenzie, R, Lacerda, T, Toledo, M, Straus, A, Takahashi, HK, Woodrum, B, Ruben, M, O'Keefe, B, Samli, KN, Yang, L, Woods, RJ, Jones, MB, Maxwell, J, Song, EH, Manganiello, M, Chow, YH, Convertine, AJ, Schnapp, LM, Stayton, PS, Ratner, DM, Yegorova, S, Rodriguez, MC, Minond, D, Jiménez-Barbero, J, Calle, L, Ardá, A, Gabius, HJ, André, S, Martinez-Mayorga, K, Yongye, AB, Cudic, M, Ali, MF, Chachadi, VB, Cheng, PW, Kiwamoto, T, Na, HJ, Brummet, M, Finn, MG, Hong, V, Polonskaya, Z, Bovin, NV, Hudson, S, Bochner, B, Gallogly, S, Krüger, A, Hanley, S, Gerlach, J, Hogan, M, Ward, C, Joshi, L, Griffin, M, Demarco, C, Deveny, R, Aggeler, R, Hart, C, Nyberg, T, Agnew, B, Akçay, G, Ramphal, J, Calabretta, P, Nguyen, AD, Kumar, K, Eggers, D, Terrill, R, d'Alarcao, M, Ito, Y, Vela, JL, Matsumura, F, Hoshino, H, Lee, H, Kobayashi, M, Borén, T, Jin, R, Seeberger, PH, Pitteloud, JP, Cudic, P, Von Muhlinen, N, Thurston, T, von Muhlinen, N, Wandel, M, Akutsu, M, Foeglein, AÁ, Komander, D, Randow, F, Maupin, K, Liden, D, Haab, B, Dam, TK, Brown, RK, Wiltzius, M, Jokinen, M, Andre, S, Kaltner, H, Bullen, J, Balsbaugh, J, Neumann, D, Hardie, G, Shabanowitz, J, Hunt, D, Hart, G, Mi, R, Ding, X, Van Die, I, Chapman, AB, Cummings, RD, Ju, T, Aryal, R, Ashley, J, Feng, X, Hanover, JA, Wang, P, Keembiyehetty, C, Ghosh, S, Bond, M, Krause, M, Love, D, Radhakrishnan, P, Grandgenet, PM, Mohr, AM, Bunt, SK, Yu, F, Hollingsworth, MA, Ethen, C, Machacek, M, Prather, B, Wu, Z, Kotu, V, Zhao, P, Zhang, D, van der Wel, H, Johnson, JM, West, CM, Abdulkhalek, S, Amith, SR, Jayanth, P, Guo, M, Szewczuk, M, Ohtsubo, K, Chen, M, Olefsky, J, Marth, J, Zapater, J, Foley, D, Colley, K, Kawashima, N, Fujitani, N, Tsuji, D, Itoh, K, Shinohara, Y, Nakayama, K, Zhang, L, Ten Hagen, K, Koren, S, Yehezkel, G, Cohen, L, Kliger, A, Khalaila, I, Finkelstein, E, Parker, R, Kohler, J, Sacoman, J, Badish, L, Hollingsworth, R, Tian, E, Hoffman, M, Hou, X, Tashima, Y, Stanley, P, Kizuka, Y, Kitazume, S, Yoshida, M, Kunze, A, Nasir, W, Bally, M, Hook, F, Larson, G, Mahan, A, Alter, G, Zeidan, Q, Copeland, R, Pokrovskaya, I, Willett, R, Smith, R, Morelle, W, Kudlyk, T, Lupashin, V, Vasudevan, D, Takeuchi, H, Majerus, E, Haltiwanger, RS, Boufala, S, Lee, YA, Min, D, Kim, SH, Shin, MH, Gesteira, T, Pol-Fachin, L, Coulson-Thomas, VJ, Verli, H, Nader, H, Liu, X, Yang, P, Thoden, J, Holden, H, Tytgat, H, Sánchez-Rodríguez, A, Schoofs, G, Verhoeven, T, De Keersmaecker, S, Marchal, K, Ventura, V, Sarah, N, Joann, P, Ding, Y, Jarrell, K, Cook, MC, Gibeault, S, Filippenko, V, Ye, Q, Wang, J, Kunkel, JP, Arteaga-Cabello, FJ, Arciniega-Fuentes, MT, McCoy, J, Ruiz-Palacios, GM, Francoleon, D, Loo, RO, Loo, J, Ytterberg, AJ, Kim, U, Gunsalus, R, Costello, C, Soares, R, Assis, R, Ibraim, I, Noronha, F, De Godoy, AP, Bale, MS, Xu, Y, Brown, K, Blader, I, West, C, Chen, S, Ye, X, Xue, C, Li, G, Yu, G, Yin, L, Chai, W, Gutierrez-Magdaleno, G, Tan, C, Wu, D, Li, Q, Hu, H, Ye, M, Liu, D, Mink, W, Kaese, P, Fujiwara, M, Uchimura, K, Sakai, Y, Nakada, T, Mabashi-Asazuma, H, Toth, AM, Scott, DW, Chacko, BK, Patel, RP, Batista, F, Mercer, N, Ramakrishnan, B, Pasek, M, Boeggeman, E, Verdi, L, Qasba, PK, Tran, D, Lim, JM, Liu, M, Mo, KF, Kirby, P, Yu, X, Lin, C, Costello, CE, Akama, TO, Nakamura, T, Huang, Y, Shi, X, Han, L, Yu, SH, Zhang, Z, Knappe, S, Till, S, Nadia, I, Catarello, J, Quinn, C, Julia, N, Ray, J, Tran, T, Scheiflinger, F, Szabo, C, Dockal, M, Niimi, S, Hosono, T, Michikawa, M, Kannagi, R, Takashima, S, Amano, J, Nakamura, N, Kaneda, E, Nakayama, Y, Kurosaka, A, Takada, W, Matsushita, T, Hinou, H, Nishimura, S, Igarashi, K, Abe, H, Mothere, M, Leonhard-Melief, C, Johnson, H, Nagy, T, Nairn, A, Rosa, MD, Porterfield, M, Kulik, M, Dalton, S, Pierce, JM, Hansen, SF, McAndrew, R, Degiovanni, A, McInerney, P, Pereira, JH, Hadi, M, Scheller, HV, Barb, A, Prestegard, J, Zhang, S, Jiang, J, Tharmalingam, T, Pluta, K, McGettigan, P, Gough, R, Struwe, W, Fitzpatrick, E, Gallagher, ME, Rudd, PM, Karlsson, NG, Carrington, SD, Katoh, T, Panin, V, Gelfenbeyn, K, Freire-de-Lima, L, Handa, K, Hakomori, SI, Bielik, AM, McLeod, E, Landry, D, Mendoza, V, Guthrie, EP, Mao, Y, Wang, X, Moremen, KW, Meng, L, Ramiah, AP, Gao, Z, Johnson, R, Xiang, Y, Rosa, MDEL, Wu, SC, Gilbert, HJ, Karaveg, K, Chen, L, Wang, BC, Mast, S, Sun, B, Fulton, S, Kimzey, M, Pourkaveh, S, Minalla, A, Haxo, T, Wegstein, J, Murray, AK, Nichols, RL, Giannini, S, Grozovsky, R, Begonja, AJ, Hoffmeister, KM, Suzuki-Anekoji, M, Suzuki, A, Yu, SY, Khoo, KH, van Alphen, L, Fodor, C, Wenzel, C, Ashmus, R, Miller, W, Stahl, M, Stintzi, A, Lowary, T, Wiederschain, G, Saba, J, Zumwalt, A, Meitei, NS, Apte, A, Viner, R, Gandy, M, Debowski, A, Stubbs, K, Witzenman, H, Pandey, D, Repnikova, E, Nakamura, M, Islam, R, Kc, N, Caster, C, Chaubard, JL, Krishnamurthy, C, Hsieh-Wilson, L, Pranskevich, J, Rangarajan, J, Guttman, A, Szabo, Z, Karger, B, Chapman, J, Chavaroche, A, Bionda, N, Fields, G, Jacob, F, Tse, BW, Guertler, R, Nixdorf, S, Hacker, NF, Heinzelmann-Schwarz, V, Yang, F, Kohler, JJ, Losfeld, ME, Ng, B, Freeze, HH, He, P, Wondimu, A, Liu, Y, Zhang, Y, Su, Y, Ladisch, S, Grewal, P, Mann, C, Ditto, D, Lardone, R, Le, D, Varki, N, Kulinich, A, Kostjuk, O, Maslak, G, Pismenetskaya, I, Shevtsova, A, Takeishi, S, Okudo, K, Moriwaki, K, Terao, N, Kamada, Y, Kuroda, S, Li, Y, Peiris, D, Markiv, A, Dwek, M, Adamczyk, B, Thanabalasingham, G, Huffman, J, Kattla, J, Novokmet, M, Rudan, I, Gloyn, A, Hayward, C, Reynolds, R, Hansen, T, Klimes, I, Njolstad, P, Wilson, J, Hastie, N, Campbell, H, McCarthy, M, Rudd, P, Owen, K, Lauc, G, Wright, A, Goletz, S, Stahn, R, Danielczyk, A, Baumeister, H, Hillemann, A, Löffler, A, Stöckl, L, Jahn, D, Bahrke, S, Flechner, A, Schlangstedt, M, Karsten, U, Goletz, C, Mikolajczyk, S, Ulsemer, P, Gao, N, Cline, A, Flanagan-Steet, H, Sadler, KC, Lehrman, MA, Coulson-Thomas, YM, Gesteira, TF, Mader, AM, Waisberg, J, Pinhal, MA, Friedl, A, Toma, L, Nader, HB, Mbua, EN, Johnson, S, Wolfert, M, Dimitrievska, S, Huizing, M, Niklason, L, Perdivara, I, Petrovich, R, Tokar, EJ, Waalkes, M, Fraser, P, Tomer, K, Chu, J, Rosa, S, Mir, A, Lehrman, M, Sadler, K, Lauer, M, Hascall, V, Calabro, A, Cheng, G, Swaidani, S, Abaddi, A, Aronica, M, Yuzwa, S, Shan, X, Macauley, M, Clark, T, Skorobogatko, Y, Vosseller, K, Vocadlo, D, Banerjee, A, Baksi, K, Banerjee, D, Melcher, R, Kraus, I, Moeller, D, Demmig, S, Rogoll, D, Kudlich, T, Scheppach, W, Scheurlen, M, Hasilik, A, Steirer, L, Lee, J, Moe, G, Troy, FA, Wang, F, Xia, B, Wang, B, Yi, S, Yu, H, Suzuki, M, Kobayashi, T, Sato, Y, Zhou, H, Briscoe, A, Lee, R, Wolfert, MA, Matsumoto, Y, Hamamura, K, Yoshida, T, Akita, K, Okajima, T, Furukawa, K, Urano, T, Ruhaak, LR, Miyamoto, S, and Lebrilla, CB more...
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Embryogenesis ,Cancer screening ,Cancer research ,medicine ,Cell migration ,Neural cell adhesion molecule ,Biology ,medicine.disease ,Biochemistry ,Metastasis - Abstract
Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression. more...
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- 2016
Catalog
3. Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4
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Akimoto Yoshihiro, Miura Yuri, Toda Tosifusa, Wolfert Margreet A, Wells Lance, Boons Geert-Jan, Hart Gerald W, Endo Tamao, and Kawakami Hayato
- Subjects
O-GlcNAc modification ,Hexosamine biosynthetic pathway ,Kidney ,Glomerulus ,Cytoskeleton ,α-actinin ,GK Rat ,Mass spectrometry ,Proximity Ligation Assay ,Medicine - Abstract
Abstract Purpose The objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes. Methods O-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA). Results O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of actin, α-actinin 4 and myosin were significantly increased in the glomerulus and the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of α-actinin 4 is disturbed and increased in the foot process of podocytes of glomerulus and in the microvilli of proximal tubules. Conclusion These results suggest that changes in the O-GlcNAcylation of cytoskeletal proteins are closely associated with the morphological changes in the podocyte foot processes in the glomerulus and in microvilli of proximal tubules in the diabetic kidney. This is the first report to show that α-actinin 4 is O-GlcNAcylated. α-Actinin 4 will be a good marker protein to examine the relation between O-GlcNAcylation and diabetic nephropathy. more...
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- 2011
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4. Structure-Based Design and Synthesis of Lipid A Derivatives to Modulate Cytokine Responses.
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Verpalen ECJM, Brouwer AJ, Wolfert MA, and Boons GJ
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- Toll-Like Receptor 4 agonists, Toll-Like Receptor 4 metabolism, Toll-Like Receptor 4 chemistry, Humans, Lymphocyte Antigen 96 metabolism, Lymphocyte Antigen 96 chemistry, Drug Design, Structure-Activity Relationship, Signal Transduction drug effects, Lipid A chemistry, Lipid A pharmacology, Lipid A analogs & derivatives, Lipid A chemical synthesis, Cytokines metabolism, Lipopolysaccharides pharmacology
- Abstract
Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.) more...
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- 2024
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5. Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewis x Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.
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Li T, Spruit CM, Wei N, Liu L, Wolfert MA, de Vries RP, and Boons GJ
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- Humans, Oligosaccharides chemistry, Oligosaccharides chemical synthesis, Oligosaccharides metabolism, Receptors, Virus metabolism, Receptors, Virus chemistry, Epitopes chemistry, Epitopes metabolism, Animals, Polysaccharides chemistry, Polysaccharides metabolism, Influenza A virus metabolism, Sialyl Lewis X Antigen metabolism, Sialyl Lewis X Antigen chemistry, E-Selectin metabolism, E-Selectin chemistry
- Abstract
Sialyl-Lewis
x (SLex ) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex -Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2 -containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex -Lex . On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex ., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.) more...- Published
- 2024
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6. Antibacterial and anti-inflammatory properties of host defense peptides against Staphylococcus aureus .
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Cecotto L, van Kessel K, Wolfert MA, Vogely C, van der Wal B, Weinans H, van Strijp J, and Amin Yavari S
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Cationic host defense peptides (HDPs) are a promising alternative to antibiotics in the fight against Staphylococcus aureus infections. In this study, we investigated the antibacterial and immunomodulatory properties of three HDPs namely IDR-1018, CATH-2, and LL-37. Although all three HDPs significantly inhibited LPS-induced activation of human macrophages, only CATH-2 prevented S. aureus growth. When applied to different infection models focused on intracellularly surviving bacteria, only IDR-1018 showed a consistent reduction in macrophage bacterial uptake. However, this observation did not correlate with an increase in killing the efficiency of intracellular S. aureus . Here, we conclude that despite the promising antibacterial and anti-inflammatory properties of the selected HDPs, macrophages' intrinsic antibacterial functions were not improved. Future studies should either focus on combining different HDPs or using them synergistically with other antibacterial agents to improve immune cells' efficacy against S. aureus pathogenesis., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).) more...
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- 2022
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7. Heparan Sulfate Proteoglycans as Attachment Factor for SARS-CoV-2.
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Liu L, Chopra P, Li X, Bouwman KM, Tompkins SM, Wolfert MA, de Vries RP, and Boons GJ
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Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments, using an extensive heparan sulfate (HS) oligosaccharide library, showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. A hexasaccharide composed of IdoA2S-GlcNS6S repeating units was identified as the minimal binding epitope. Surface plasmon resonance showed the SARS-CoV-2 spike protein binds with a much higher affinity to heparin ( K
D = 55 nM) compared to the RBD ( KD = 1 μM) alone. It was also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. However, exogenous administered heparin or a highly sulfated HS oligosaccharide inhibited RBD binding to cells. Furthermore, an enzymatic removal of HS proteoglycan from physiological relevant tissue resulted in a loss of RBD binding. The data support a model in which HS functions as the point of initial attachment allowing the virus to travel through the glycocalyx by low-affinity high-avidity interactions to reach the cell membrane, where it can engage with ACE2 for cell entry. Microarray binding experiments showed that ACE2 and HS can simultaneously engage with the RBD, and it is likely no dissociation between HS and RBD is required for binding to ACE2. The results highlight the potential of using HS oligosaccharides as a starting material for therapeutic agent development., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.) more...- Published
- 2021
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8. The 3- O -sulfation of heparan sulfate modulates protein binding and lyase degradation.
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Chopra P, Joshi A, Wu J, Lu W, Yadavalli T, Wolfert MA, Shukla D, Zaia J, and Boons GJ
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- Acetylglucosamine chemistry, Acetylglucosamine metabolism, Antithrombin III chemistry, Antithrombin III genetics, Antithrombin III metabolism, Binding Sites, Binding, Competitive, Carbohydrate Sequence, Cell Line, Cornea cytology, Cornea metabolism, Epithelial Cells pathology, Epithelial Cells virology, Factor Xa chemistry, Factor Xa genetics, Factor Xa metabolism, Factor Xa Inhibitors chemistry, Factor Xa Inhibitors metabolism, Gene Expression, Glucuronic Acid chemistry, Glucuronic Acid metabolism, Heparin Lyase chemistry, Heparin Lyase genetics, Heparitin Sulfate chemistry, Herpesvirus 1, Human growth & development, Host-Pathogen Interactions genetics, Humans, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Microarray Analysis, Protein Binding, Proteolysis, Small Molecule Libraries, Substrate Specificity, Sulfates chemistry, Sulfotransferases chemistry, Sulfotransferases genetics, Viral Envelope Proteins chemistry, Viral Envelope Proteins genetics, Viral Envelope Proteins metabolism, Epithelial Cells metabolism, Heparin Lyase metabolism, Heparitin Sulfate metabolism, Herpesvirus 1, Human metabolism, Sulfates metabolism, Sulfotransferases metabolism
- Abstract
Humans express seven heparan sulfate (HS) 3- O -sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3- O -sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3- O -sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS., Competing Interests: The authors declare no competing interest. more...
- Published
- 2021
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9. Mono- and Di-Fucosylated Glycans of the Parasitic Worm S. mansoni are Recognized Differently by the Innate Immune Receptor DC-SIGN.
- Author
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Srivastava AD, Unione L, Wolfert MA, Valverde P, Ardá A, Jiménez-Barbero J, and Boons GJ
- Subjects
- Animals, Glycosylation, Humans, Immunity, Innate, Cell Adhesion Molecules immunology, Lectins, C-Type immunology, Polysaccharides immunology, Receptors, Cell Surface immunology, Schistosoma mansoni immunology
- Abstract
The parasitic worm, Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F and LDN-DF, respectively), which are epitopes expressed on glycolipids and glycoproteins of S. mansoni. It is based on the use of monosaccharide building blocks having carefully selected amino-protecting groups, facilitating high yielding and stereoselective glycosylations. The molecular interaction between the synthetic glycans and DC-SIGN was studied by NMR and molecular modeling, which demonstrated that the α1,3-fucoside of LDN-F can coordinate with the Ca
2+ -ion of the canonical binding site of DC-SIGN allowing for additional interactions with the underlying LDN backbone. The 1,2-fucoside of LDN-DF can be complexed in a similar manner, however, in this binding mode GlcNAc and GalNAc of the LDN backbone are placed away from the protein surface resulting in a substantially lower binding affinity. Glycan microarray binding studies showed that the avidity and selectivity of binding is greatly enhanced when the glycans are presented multivalently, and in this format Lex and LDN-F gave strong responsiveness, whereas no binding was detected for LDN-DF. The data indicates that S. mansoni has developed a strategy to avoid detection by DC-SIGN in a stage-dependent manner by the addition of a fucoside to a number of its ligands., (© 2020 Wiley-VCH GmbH.) more...- Published
- 2020
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10. Chemoenzymatic Synthesis of Campylobacter jejuni Lipo-oligosaccharide Core Domains to Examine Guillain-Barré Syndrome Serum Antibody Specificities.
- Author
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Li T, Wolfert MA, Wei N, Huizinga R, Jacobs BC, and Boons GJ
- Subjects
- Antibody Specificity, Biosensing Techniques, Cross Reactions, Gangliosides chemistry, Humans, Lectins chemistry, Protein Array Analysis, Serum chemistry, Small Molecule Libraries analysis, Antibodies, Bacterial blood, Biomimetic Materials chemistry, Campylobacter jejuni enzymology, Guillain-Barre Syndrome diagnosis, Lipopolysaccharides chemistry, Oligosaccharides chemistry
- Abstract
Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research. more...
- Published
- 2020
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11. Mutation of the second sialic acid-binding site of influenza A virus neuraminidase drives compensatory mutations in hemagglutinin.
- Author
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Du W, Wolfert MA, Peeters B, van Kuppeveld FJM, Boons GJ, de Vries E, and de Haan CAM
- Subjects
- Amino Acid Substitution, Animals, Binding Sites, Dogs, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Influenza A virus isolation & purification, Madin Darby Canine Kidney Cells, Neuraminidase chemistry, Neuraminidase metabolism, Orthomyxoviridae Infections genetics, Orthomyxoviridae Infections pathology, Protein Binding, Hemagglutinin Glycoproteins, Influenza Virus genetics, Influenza A virus genetics, Mutation, Neuraminidase genetics, Orthomyxoviridae Infections virology, Sialic Acids metabolism, Virus Replication
- Abstract
Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential., Competing Interests: The authors have declared that no competing interests exist. more...
- Published
- 2020
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12. Chemoenzymatic synthesis of the oligosaccharide moiety of the tumor-associated antigen disialosyl globopentaosylceramide.
- Author
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't Hart IME, Li T, Wolfert MA, Wang S, Moremen KW, and Boons GJ
- Subjects
- Antigens, Neoplasm, Carbohydrate Conformation, Enzyme Inhibitors chemistry, Globosides chemical synthesis, Globosides chemistry, HEK293 Cells, Humans, Microarray Analysis, Oligosaccharides chemistry, Sialyltransferases metabolism, beta-Galactoside alpha-2,3-Sialyltransferase, Carcinoma, Renal Cell chemistry, Enzyme Inhibitors pharmacology, Globosides pharmacology, Kidney Neoplasms chemistry, Oligosaccharides pharmacology, Sialyltransferases antagonists & inhibitors
- Abstract
Disialosyl globopentaosylceramide (DSGb5) is often expressed by renal cell carcinomas. To investigate properties of DSGb5, we have prepared its oligosaccharide moiety by chemically synthesizing Gb5 which was enzymatically sialylated using the mammalian sialyltransferases ST3Gal1 and ST6GalNAc5. Glycan microarray binding studies indicate that Siglec-7 does not recognize DSGb5, and preferentially binds Neu5Acα(2,8)Neu5Ac containing glycans. more...
- Published
- 2019
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13. Protecting-Group-Controlled Enzymatic Glycosylation of Oligo-N-Acetyllactosamine Derivatives.
- Author
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Gagarinov IA, Li T, Wei N, Sastre Toraño J, de Vries RP, Wolfert MA, and Boons GJ
- Abstract
We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri-N-acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH
2 ) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono- and bis-fucosylated oligosaccharides. Conversion of the GlcNH2 or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan-binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...- Published
- 2019
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14. N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses.
- Author
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Broszeit F, Tzarum N, Zhu X, Nemanichvili N, Eggink D, Leenders T, Li Z, Liu L, Wolfert MA, Papanikolaou A, Martínez-Romero C, Gagarinov IA, Yu W, García-Sastre A, Wennekes T, Okamatsu M, Verheije MH, Wilson IA, Boons GJ, and de Vries RP more...
- Subjects
- Animals, Chickens, Dogs, Erythrocytes metabolism, Erythrocytes virology, Hemagglutinins chemistry, Hemagglutinins metabolism, Horses, Influenza A virus pathogenicity, Neuraminic Acids chemistry, Orthomyxoviridae Infections veterinary, Protein Binding, Host Specificity, Influenza A virus metabolism, Neuraminic Acids metabolism, Orthomyxoviridae Infections metabolism
- Abstract
A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.) more...
- Published
- 2019
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15. Synthesis and Immunological Evaluation of a Multicomponent Cancer Vaccine Candidate Containing a Long MUC1 Glycopeptide.
- Author
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Supekar NT, Lakshminarayanan V, Capicciotti CJ, Sirohiwal A, Madsen CS, Wolfert MA, Cohen PA, Gendler SJ, and Boons GJ
- Subjects
- Animals, Cancer Vaccines chemistry, Glycopeptides chemistry, Humans, Mice, Mice, Inbred C57BL, Mice, Transgenic, Molecular Conformation, Mucin-1 chemistry, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Helper-Inducer immunology, Vaccines, Synthetic chemistry, Cancer Vaccines chemical synthesis, Cancer Vaccines immunology, Glycopeptides immunology, Mucin-1 immunology, Vaccines, Synthetic immunology
- Abstract
A fully synthetic MUC1-based cancer vaccine was designed and chemically synthesized containing an endogenous helper T-epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody-dependent cell-mediated cytotoxicity (ADCC). It also activated cytotoxic T-lymphocytes. Collectively, the immunological data demonstrate engagement of helper T-cells in immune activation. A synthetic methodology was developed for a penta-glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1-based cancer vaccines that elicited potent immune responses employed exogenous helper T-epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T-epitope will be more attractive, because it will activate cognate CD4
+ T-cells that will provide critical tumor-specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...- Published
- 2018
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16. Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements.
- Author
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Zong C, Venot A, Li X, Lu W, Xiao W, Wilkes JL, Salanga CL, Handel TM, Wang L, Wolfert MA, and Boons GJ
- Subjects
- Animals, Binding Sites, Carbohydrate Conformation, Cell Line, Cell Proliferation, Ligands, Mice, Surface Plasmon Resonance, Fibroblast Growth Factors chemistry, Heparitin Sulfate chemistry, Microarray Analysis
- Abstract
Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function. more...
- Published
- 2017
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17. Synthesis of asymmetrical multiantennary human milk oligosaccharides.
- Author
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Prudden AR, Liu L, Capicciotti CJ, Wolfert MA, Wang S, Gao Z, Meng L, Moremen KW, and Boons GJ
- Subjects
- Female, Humans, Microarray Analysis, Oligosaccharides chemistry, Glycosyltransferases chemistry, Milk, Human chemistry, Oligosaccharides chemical synthesis
- Abstract
Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability., Competing Interests: The authors declare no conflict of interest. more...
- Published
- 2017
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18. Mucin architecture behind the immune response: design, evaluation and conformational analysis of an antitumor vaccine derived from an unnatural MUC1 fragment.
- Author
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Martínez-Sáez N, Supekar NT, Wolfert MA, Bermejo IA, Hurtado-Guerrero R, Asensio JL, Jiménez-Barbero J, Busto JH, Avenoza A, Boons GJ, Peregrina JM, and Corzana F
- Abstract
A tripartite cancer vaccine candidate, containing a quaternary amino acid (α-methylserine) in the most immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to enzymatic degradation than the natural counter part. In spite of these features, the immune response elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural threonine. These observations were rationalized by conformational studies, indicating that the presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc and the threonine residues. The results described here will be helpful to the rational design of efficacious cancer vaccines. more...
- Published
- 2016
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19. Controlled Multi-functionalization Facilitates Targeted Delivery of Nanoparticles to Cancer Cells.
- Author
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Hudlikar MS, Li X, Gagarinov IA, Kolishetti N, Wolfert MA, and Boons GJ
- Subjects
- Antineoplastic Agents metabolism, Cell Line, Tumor, Cyclooctanes chemistry, Cyclooctanes metabolism, Doxorubicin pharmacology, Drug Carriers, Endocytosis, Humans, Hydrogen-Ion Concentration, Lymphoma, B-Cell metabolism, Antineoplastic Agents chemistry, Doxorubicin chemistry, Drug Delivery Systems methods, Gold chemistry, Lymphoma, B-Cell chemistry, Nanoparticles chemistry, Polyethylene Glycols chemistry, Polymers chemistry
- Abstract
A major objective of nanomedicine is to combine in a controlled manner multiple functional entities into a single nanoscale device to target particles with great spatial precision, thereby increasing the selectivity and potency of therapeutic drugs. A multifunctional nanoparticle is described for controlled conjugation of a cytotoxic drug, a cancer cell targeting ligand, and an imaging moiety. The approach is based on the chemical synthesis of polyethylene glycol that at one end is modified by a thioctic acid for controlled attachment to a gold core. The other end of the PEG polymers is modified by a hydrazine, amine, or dibenzocyclooctynol moiety for conjugation with functional entities having a ketone, activated ester, or azide moiety, respectively. The conjugation approach allowed the controlled attachment of doxorubicin through an acid-labile hydrazone linkage, an Alexa Fluor dye through an amide bond, and a glycan-based ligand for the cell surface receptor CD22 of B-cells using strain promoted azide-alkyne cycloaddition. The incorporation of the ligand for CD22 led to rapid entry of the nanoparticle by receptor-mediated endocytosis. Covalent attachment of doxorubicin via hydrazone linkage caused pH-responsive intracellular release of doxorubicin and significantly enhanced the cytotoxicity of nanoparticles. A remarkable 60-fold enhancement in cytotoxicity of CD22 (+) lymphoma cells was observed compared to non- targeted nanoparticles., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2016
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20. MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice.
- Author
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Lakshminarayanan V, Supekar NT, Wei J, McCurry DB, Dueck AC, Kosiorek HE, Trivedi PP, Bradley JM, Madsen CS, Pathangey LB, Hoelzinger DB, Wolfert MA, Boons GJ, Cohen PA, and Gendler SJ
- Subjects
- Animals, Antigens chemistry, Antigens immunology, Antigens therapeutic use, Cancer Vaccines chemistry, Cancer Vaccines immunology, Cell Line, Tumor, Glycosylation, Humans, Mice, Mice, Transgenic, Mucin-1 metabolism, Neoplasms, Experimental immunology, Peptides chemistry, Peptides immunology, T-Lymphocytes immunology, T-Lymphocytes metabolism, Tumor Escape, Cancer Vaccines therapeutic use, Mucin-1 genetics, Mucin-1 immunology, Neoplasms, Experimental prevention & control, Peptides therapeutic use
- Abstract
It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape. more...
- Published
- 2016
- Full Text
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21. Linear synthesis and immunological properties of a fully synthetic vaccine candidate containing a sialylated MUC1 glycopeptide.
- Author
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Thompson P, Lakshminarayanan V, Supekar NT, Bradley JM, Cohen PA, Wolfert MA, Gendler SJ, and Boons GJ
- Subjects
- Amino Acid Sequence, Animals, Chemistry Techniques, Synthetic, Humans, Molecular Sequence Data, Glycopeptides chemistry, Mucin-1 chemistry, N-Acetylneuraminic Acid chemistry, Vaccines, Synthetic chemistry, Vaccines, Synthetic immunology
- Abstract
A strategy for the linear synthesis of a sialylated glycolipopeptide cancer vaccine candidate has been developed using a strategically designed sialyl-Tn building block and microwave-assisted solid-phase peptide synthesis. The glycolipopeptide elicited potent humoral and cellular immune responses. T-cells primed by such a vaccine candidate could be restimulated by tumor-associated MUC1. more...
- Published
- 2015
- Full Text
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22. A multifunctional anomeric linker for the chemoenzymatic synthesis of complex oligosaccharides.
- Author
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Prudden AR, Chinoy ZS, Wolfert MA, and Boons GJ
- Subjects
- Chromatography, High Pressure Liquid, Fluorenes chemistry, Galectin 3 chemistry, Polysaccharides chemistry
- Abstract
A new anomeric linker has been developed that facilitates the purification of glycans prepared by chemoenzymatic approaches and can readily give compounds that are appropriately modified for microarray development or glycan derivatives with a free reducing end that are needed as standards for the development of analytical protocols. more...
- Published
- 2014
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23. Immune and anticancer responses elicited by fully synthetic aberrantly glycosylated MUC1 tripartite vaccines modified by a TLR2 or TLR9 agonist.
- Author
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Abdel-Aal AB, Lakshminarayanan V, Thompson P, Supekar N, Bradley JM, Wolfert MA, Cohen PA, Gendler SJ, and Boons GJ
- Subjects
- Adjuvants, Immunologic chemistry, Amino Acid Sequence, Animals, Breast immunology, Breast Neoplasms immunology, Cancer Vaccines chemistry, Cancer Vaccines immunology, Cell Line, Tumor, Female, Glycopeptides chemistry, Glycopeptides immunology, Glycosylation, Humans, Immunity, Cellular, Immunization, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Mucin-1 chemistry, Mucin-1 immunology, T-Lymphocytes, Cytotoxic immunology, Vaccines, Synthetic chemistry, Vaccines, Synthetic immunology, Vaccines, Synthetic therapeutic use, Adjuvants, Immunologic therapeutic use, Breast Neoplasms therapy, Cancer Vaccines therapeutic use, Glycopeptides therapeutic use, Mucin-1 therapeutic use, Toll-Like Receptor 2 agonists, Toll-Like Receptor 9 agonists
- Abstract
The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3 CysSK4 ) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3 CysSK4 -containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3 CysSK4 , which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2014
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24. Selective exo-enzymatic labeling of N-glycans on the surface of living cells by recombinant ST6Gal I.
- Author
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Mbua NE, Li X, Flanagan-Steet HR, Meng L, Aoki K, Moremen KW, Wolfert MA, Steet R, and Boons GJ
- Subjects
- Click Chemistry methods, Fibroblasts chemistry, Fibroblasts metabolism, Humans, N-Acetylneuraminic Acid, Polysaccharides metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sialic Acids chemical synthesis, Sialic Acids chemistry, Sialyltransferases genetics, Sialyltransferases metabolism, beta-D-Galactoside alpha 2-6-Sialyltransferase, Polysaccharides chemistry, Sialyltransferases chemistry
- Abstract
A game of tag: N-Glycans on the surface of living cells were selectively tagged by exogenously administering recombinant ST6Gal I sialyltransferase and azide-modified CMP-Neu5Ac. This modification was followed by a strain-promoted cycloaddition using a biotin-modified dibenzylcyclooctynol (red star=biotin). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2013
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25. Adaptive immune activation: glycosylation does matter.
- Author
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Wolfert MA and Boons GJ
- Subjects
- Glycoproteins, Glycosylation, Vaccines, Adaptive Immunity physiology, Antigens metabolism, Major Histocompatibility Complex physiology
- Abstract
Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered. more...
- Published
- 2013
- Full Text
- View/download PDF
26. Abnormal accumulation and recycling of glycoproteins visualized in Niemann-Pick type C cells using the chemical reporter strategy.
- Author
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Mbua NE, Flanagan-Steet H, Johnson S, Wolfert MA, Boons GJ, and Steet R
- Subjects
- Azides pharmacokinetics, Carrier Proteins genetics, Cell Line, Cholesterol metabolism, Dextrans pharmacokinetics, Endosomes metabolism, Fibroblasts drug effects, Fibroblasts metabolism, Fibroblasts pathology, Galactosamine pharmacokinetics, Glycoconjugates genetics, Glycoconjugates metabolism, Glycoproteins genetics, Humans, Intracellular Signaling Peptides and Proteins, Lysosomes metabolism, Membrane Glycoproteins genetics, Niemann-Pick C1 Protein, Niemann-Pick Disease, Type C genetics, Protein Transport physiology, Sialoglycoproteins metabolism, Vesicular Transport Proteins, beta-Cyclodextrins pharmacology, Carrier Proteins metabolism, Glycoproteins metabolism, Membrane Glycoproteins metabolism, Niemann-Pick Disease, Type C metabolism, Niemann-Pick Disease, Type C pathology
- Abstract
Niemann-Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins. more...
- Published
- 2013
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27. Chemical synthesis and immunological evaluation of the inner core oligosaccharide of Francisella tularensis.
- Author
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Boltje TJ, Zhong W, Park J, Wolfert MA, Chen W, and Boons GJ
- Subjects
- Animals, Antibody Formation, Bacterial Vaccines chemistry, Female, Francisella tularensis chemistry, Immunization, Immunoconjugates chemistry, Immunoconjugates immunology, Lipopolysaccharides chemistry, Mice, Mice, Inbred BALB C, Bacterial Vaccines chemical synthesis, Bacterial Vaccines immunology, Francisella tularensis immunology, Lipopolysaccharides chemical synthesis, Lipopolysaccharides immunology, Tularemia immunology, Tularemia prevention & control
- Abstract
Francisella tularensis, which is a Gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics, and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All), and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the (1)H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis . The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate. more...
- Published
- 2012
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28. Polar dibenzocyclooctynes for selective labeling of extracellular glycoconjugates of living cells.
- Author
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Friscourt F, Ledin PA, Mbua NE, Flanagan-Steet HR, Wolfert MA, Steet R, and Boons GJ
- Subjects
- Cycloparaffins chemical synthesis, Cycloparaffins metabolism, Fibroblasts cytology, Fibroblasts metabolism, Glycoconjugates chemical synthesis, Glycoconjugates metabolism, Humans, Jurkat Cells, Kinetics, Molecular Structure, Cycloparaffins chemistry, Fibroblasts chemistry, Glycoconjugates chemistry, Staining and Labeling
- Abstract
Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represent a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location, and in particular it has been shown that DIBO derivative 4 can enter cells, thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking. more...
- Published
- 2012
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29. Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine.
- Author
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Lakshminarayanan V, Thompson P, Wolfert MA, Buskas T, Bradley JM, Pathangey LB, Madsen CS, Cohen PA, Gendler SJ, and Boons GJ
- Subjects
- Amino Acid Sequence, Animals, Antibody-Dependent Cell Cytotoxicity immunology, Antigens, Neoplasm chemistry, Cancer Vaccines chemistry, Cytokines biosynthesis, Enzyme-Linked Immunosorbent Assay, Glycosylation, Humans, Immunity, Cellular immunology, Immunity, Humoral immunology, Mammary Tumor Virus, Mouse immunology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Molecular Sequence Data, Neoplasms pathology, T-Lymphocytes, Cytotoxic immunology, Tumor Burden immunology, Vaccines, Synthetic chemistry, Antigens, Neoplasm immunology, Cancer Vaccines immunology, Mucin-1 immunology, Neoplasms immunology, Vaccines, Synthetic immunology
- Abstract
The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens. more...
- Published
- 2012
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30. Strain-promoted alkyne-azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.
- Author
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Mbua NE, Guo J, Wolfert MA, Steet R, and Boons GJ
- Subjects
- Alkynes metabolism, Animals, Azides metabolism, CHO Cells, Cricetinae, Cricetulus, Glycoproteins chemistry, Glycoproteins metabolism, Glycosylation, Humans, Jurkat Cells, Lectins metabolism, N-Acetylneuraminic Acid metabolism, Polysaccharides metabolism, Protein Transport physiology, Spectrometry, Fluorescence, Triazoles chemistry, Alkynes chemistry, Azides chemistry, Click Chemistry methods, Glycoconjugates biosynthesis, Golgi Apparatus metabolism, Molecular Probe Techniques, Staining and Labeling methods
- Abstract
We have shown that 4-dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthesis approach, reacts exceptionally fast in the absence of a Cu(I) catalyst with azido-containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide-alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts; this provides the first example of a metal-free alkyne-azide fluoro-switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, and thereby facilitate biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido-containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the conserved oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O-glycan sialylation than galactosylation, whereas sialylation and galactosylation of N-glycans was similarly impacted. These results highlight the fact that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells might have a means to selectively alter specific glycan structures of glycoproteins., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2011
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31. Multifunctional surface modification of gold-stabilized nanoparticles by bioorthogonal reactions.
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Li X, Guo J, Asong J, Wolfert MA, and Boons GJ
- Subjects
- Cell Survival drug effects, Drug Carriers adverse effects, Drug Carriers pharmacokinetics, HeLa Cells, Humans, Lactones chemistry, Nanoparticles adverse effects, Nanoparticles ultrastructure, Polyethylene Glycols chemistry, Surface Properties, Thioctic Acid chemistry, Drug Carriers chemistry, Gold chemistry, Nanoparticles chemistry
- Abstract
Nanocarriers that combine multiple properties in an all-in-one system hold great promise for drug delivery. The absence of technology to assemble highly functionalized devices has, however, hindered progress in nanomedicine. To address this deficiency, we have chemically synthesized poly(ethylene oxide)-β-poly(ε-caprolactone) (PEO-b-PCL) block polymers modified at the apolar PCL terminus with thioctic acid and at the polar PEO terminus with an acylhydrazide, amine, or azide moiety. The resulting block polymers were employed to prepare nanoparticles that have a gold core, an apolar polyester layer for drug loading, a polar PEO corona to provide biocompatibility, and three different types of surface reactive groups for surface functionalization. The acylhydrazide, amine, or azide moieties of the resulting nanoparticles could be reacted with high efficiencies with modules having a ketone, isocyanate, or active ester and alkyne function, respectively. To demonstrate proof of principle of the potential of multisurface functionalization, we prepared nanoparticles that have various combinations of an oligo-arginine peptide to facilitate cellular uptake, a histidine-rich peptide to escape from lysosomes, and an Alexa Fluor 488 tag for imaging purposes. It has been shown that uptake and subcellular localization of the nanoparticles can be controlled by multisurface modification. It is to be expected that the modular synthetic methodology provides unique opportunities to establish optimal configurations of nanocarriers for disease-specific drug delivery. more...
- Published
- 2011
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32. Surface modification of polymeric micelles by strain-promoted alkyne-azide cycloadditions.
- Author
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Guo J, Chen G, Ning X, Wolfert MA, Li X, Xu B, and Boons GJ
- Subjects
- Cyclization, Drug Screening Assays, Antitumor, Female, Humans, Microscopy, Atomic Force, Molecular Structure, Polymers chemical synthesis, Azides chemistry, Cyclooctanes chemistry, Cycloparaffins chemistry, Micelles, Polymers chemistry
- Abstract
Organomicelles modified by surface dibenzylcyclooctyne moieties can conveniently be functionalized by strain-promoted alkyne-azide cycloadditions. The ligation approach is highly efficient, does not require toxic reagents and is compatible with a wide variety of functional modules. Interactions of proteins with surface ligands of the micelles have been studied by AFM, which revealed that it leads to disassembly of the particles thereby providing a mechanism for triggered drug release. more...
- Published
- 2010
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33. Surface functionalization using catalyst-free azide-alkyne cycloaddition.
- Author
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Kuzmin A, Poloukhtine A, Wolfert MA, and Popik VV
- Subjects
- Click Chemistry, Cyclization, Molecular Structure, Surface Properties, Alkynes chemistry, Aza Compounds chemistry, Azides chemistry, Azocines chemistry
- Abstract
The utility of catalyst-free azide-alkyne [3 + 2] cycloaddition for the immobilization of a variety of molecules onto a solid surface and microbeads was demonstrated. In this process, the surfaces are derivatized with aza-dibenzocyclooctyne (ADIBO) for the immobilization of azide-tagged substrates via a copper-free click reaction. Alternatively, ADIBO-conjugated molecules are anchored to the azide-derivatized surface. Both immobilization techniques work well in aqueous solutions and show excellent kinetics under ambient conditions. We report an efficient synthesis of aza-dibenzocyclooctyne (ADIBO), thus far the most reactive cyclooctyne in cycloaddition to azides. We also describe convenient methods for the conjugation of ADIBO with a variety of molecules directly or via a PEG linker. more...
- Published
- 2010
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34. Differential induction of innate immune responses by synthetic lipid a derivatives.
- Author
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Gaekwad J, Zhang Y, Zhang W, Reeves J, Wolfert MA, and Boons GJ
- Subjects
- Animals, Cell Line, Cytokines genetics, Cytokines immunology, Enzyme-Linked Immunosorbent Assay, Immunity, Innate genetics, Lipid A analogs & derivatives, Lipid A chemical synthesis, Mice, Polymerase Chain Reaction, Immunity, Innate immunology, Lipid A immunology
- Abstract
Recent studies have indicated that lipopolysaccharides (LPS) isolated from particular bacterial strains can bias innate immune responses toward different signal transduction pathways thereby eliciting unique patterns of cytokines. Heterogeneity in the structure of lipid A (the active component of LPS) and possible contaminations with other inflammatory components have made it difficult to confirm these observations and dissect molecular motifs that may be responsible for modulatory properties. To address these issues, we have examined, for the first time, the ability of a range of well defined synthetic lipid As and isolated LPS and lipid A preparations to induce the production of a wide range of cytokines in three different mouse cell types. It was found that, for a given compound, the potencies of production of the various cytokines differed significantly. An additive model, in which a chemical change in the structure of a compound effects the potencies of all cytokines in the same manner, could describe the potencies of the cytokines for all compounds. Thus, no evidence was found that the structure of lipid A can modulate the pattern of cytokine production. In addition, the statistical analysis showed that the relative ordering of the potencies of the compounds was identical in the different cell types and that structural features such as the presence of a 3-deoxy-D-manno-octulosonic acid moiety, anomeric phosphate, lipid length, and acylation pattern were important for pro-inflammatory activity. Finally, it was found that transcriptional and post-transcription control mechanisms determine potencies and efficacies of cytokine production in cell-specific manners. more...
- Published
- 2010
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35. Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4.
- Author
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Berghaus LJ, Moore JN, Hurley DJ, Vandenplas ML, Fortes BP, Wolfert MA, and Boons GJ
- Subjects
- Animals, Bone Marrow Cells immunology, Cell Line, Cells, Cultured, Chemokine CCL5 biosynthesis, Dendritic Cells immunology, Immunophenotyping, In Vitro Techniques, Ligands, Lipopeptides immunology, Lipopolysaccharides immunology, Macrophages microbiology, Mice, Mice, Inbred C57BL, Poly I-C immunology, Spleen cytology, Spleen immunology, Tumor Necrosis Factor-alpha biosynthesis, Immunity, Innate, Macrophages immunology, Toll-Like Receptor 2 immunology, Toll-Like Receptor 3 immunology, Toll-Like Receptor 4 immunology
- Abstract
Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone marrow-derived macrophages, and bone marrow-derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture., (Copyright 2009 Elsevier Ltd. All rights reserved.) more...
- Published
- 2010
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36. Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.
- Author
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Teo CF, Ingale S, Wolfert MA, Elsayed GA, Nöt LG, Chatham JC, Wells L, and Boons GJ
- Subjects
- Acetylglucosamine immunology, Enzyme-Linked Immunosorbent Assay, Immunoprecipitation, Acetylglucosamine chemistry, Antibodies, Monoclonal immunology, Glycopeptides immunology
- Abstract
Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model. more...
- Published
- 2010
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37. Protein modification by strain-promoted alkyne-nitrone cycloaddition.
- Author
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Ning X, Temming RP, Dommerholt J, Guo J, Ania DB, Debets MF, Wolfert MA, Boons GJ, and van Delft FL
- Subjects
- Cyclization, Alkynes chemistry, Nitrogen Oxides chemistry, Proteins chemistry
- Published
- 2010
- Full Text
- View/download PDF
38. Chemical synthesis and proinflammatory responses of monophosphoryl lipid A adjuvant candidates.
- Author
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Maiti KK, Decastro M, El-Sayed AB, Foote MI, Wolfert MA, and Boons GJ
- Abstract
Lipopolysaccharides (LPS), which are structural components of the outer surface membrane of Gram-negative bacteria, trigger innate immune responses through activation of Toll-like receptor 4 (TLR4). Such responses may be exploited for the development of adjuvants and in particular monophosphoryl lipid A (MPLA) obtained by controlled hydrolysis of LPS of Salmonella minnesota, exhibits low toxicity yet possesses beneficial immuno-stimulatory properties. We have developed an efficient synthetic approach for the preparation of a major component of MPLA (1), which has as a key feature the use of allyloxycarbonates (Alloc) as permanent protecting groups for the C-3 and C-4 hydroxyls of the proximal glucosamine unit. The latter protecting groups greatly facilitated deprotection of the fully assembled compound. Furthermore, the amino functions were protected as N-2,2,2-trichloroethoxycarbamates (Troc), which performed efficient neighboring group participation to give selectively 1,2-trans-glycosides and could easily be removed under mild conditions without affecting the permanent Alloc carbonates and anomeric dimethylthexylsilyl (TDS) ether. The synthetic methodology was also employed for the preparation of a monophosphoryl lipid A (2) derivative that has the anomeric center of the proximal sugar modified as a methyl glycoside. Compound 1 was not able to induce cytokine production in mouse macrophages whereas methyl glycoside 2 displayed activity, however it has a lower potency and efficacy than lipid A obtained by controlled hydrolysis S. minnesota. This indicates compound 2 is an attractive candidate for adjuvant development and that 1 is not the active substance of MPLA obtained by controlled hydrolysis of LPS. more...
- Published
- 2010
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39. Selective labeling of living cells by a photo-triggered click reaction.
- Author
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Poloukhtine AA, Mbua NE, Wolfert MA, Boons GJ, and Popik VV
- Subjects
- Cyclopropanes pharmacology, Humans, Jurkat Cells, Magnetic Resonance Spectroscopy, Cyclopropanes chemistry, Photochemistry
- Abstract
Phototriggering of the metal-free azide to acetylene cycloaddition reaction was achieved by masking the triple bond of dibenzocyclooctynes as cyclopropenone. Such masked cyclooctynes do not react with azides in the dark. Irradiation of cyclopropenones results in the efficient (Phi(355) = 0.33) and clean regeneration of the corresponding dibenzocyclooctynes, which then undergo facile catalyst-free cycloadditions with azides to give corresponding triazoles under ambient conditions. In situ light activation of a cyclopropenone linked to biotin made it possible to label living cells expressing glycoproteins containing N-azidoacetyl-sialic acid. The cyclopropenone-based phototriggered click chemistry offers exciting opportunities to label living organisms in a temporally and spatially controlled manner and may facilitate the preparation of microarrays. more...
- Published
- 2009
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40. Binding and Cellular Activation Studies Reveal That Toll-like Receptor 2 Can Differentially Recognize Peptidoglycan from Gram-positive and Gram-negative Bacteria.
- Author
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Asong J, Wolfert MA, Maiti KK, Miller D, and Boons GJ
- Subjects
- Cell Line, Gram-Negative Bacteria immunology, Gram-Positive Bacteria immunology, Host-Pathogen Interactions physiology, Humans, Peptides immunology, Peptidoglycan immunology, Structure-Activity Relationship, Surface Plasmon Resonance, Toll-Like Receptor 2 immunology, Gram-Negative Bacteria chemistry, Gram-Positive Bacteria chemistry, Immunity, Innate physiology, Peptides chemistry, Peptidoglycan chemistry, Toll-Like Receptor 2 chemistry
- Abstract
Although much progress has been made toward the identification of innate immune receptors, far less is known about how these receptors recognize specific microbial products. Such studies have been hampered by the need to purify compounds from microbial sources and a reliance on biological assays rather than direct binding to monitor recognition. We have employed surface plasmon resonance (SPR) binding studies using a wide range of well defined synthetic muropeptides derived from Gram-positive (lysine-containing) and Gram-negative (diaminopimelic acid (DAP)-containing) bacteria to demonstrate that Toll-like receptor 2 can recognize peptidoglycan (PGN). In the case of lysine-containing muropeptides, a limited number of compounds, which were derived from PGN remodeled by bacterial autolysins, was recognized. However, a wider range of DAP-containing muropeptides was bound with high affinity, and these compounds were derived from nascent and remodeled PGN. The difference in recognition of the two classes of muropeptides is proposed to be a strategy by the host to respond appropriately to Gram-negative and -positive bacteria, which produce vastly different quantities of PGN. It was also found that certain modifications of the carboxylic acids of isoglutamine and DAP can dramatically reduce binding, and thus, bacterial strains may employ such modifications to evade innate immune detection. Cellular activation studies employing highly purified PGN from Bacillus licheniformis, Bacillus subtilis, Escherichia coli, Lactobacillus plantarum, Micrococcus luteus, and Staphylococcus aureus support the structure binding relationship. The data firmly establish Toll-like receptor 2 as an innate immune sensor for PGN and provides an understanding of host-pathogen interactions at the molecular level. more...
- Published
- 2009
- Full Text
- View/download PDF
41. Increasing the antigenicity of synthetic tumor-associated carbohydrate antigens by targeting Toll-like receptors.
- Author
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Ingale S, Wolfert MA, Buskas T, and Boons GJ
- Subjects
- Amino Acid Sequence, Animals, Antigens, Tumor-Associated, Carbohydrate chemistry, Antigens, Tumor-Associated, Carbohydrate genetics, Cell Line, Epitopes, Female, Humans, Macrophages cytology, Macrophages immunology, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Molecular Structure, Mucin-1 genetics, Mucin-1 immunology, Protein Engineering, Recombinant Proteins genetics, Toll-Like Receptors genetics, Antigens, Tumor-Associated, Carbohydrate immunology, Cancer Vaccines chemical synthesis, Cancer Vaccines immunology, Recombinant Proteins immunology, Toll-Like Receptors immunology
- Abstract
SYNTHETIC CANCER VACCINES: A number of fully synthetic vaccine candidates have been designed, chemically synthesized, and immunologically evaluated to establish a strategy to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides and to determine the importance of Toll-like receptor (TLR) engagement for antigenic responses against these compounds.Epithelial cancer cells often overexpress mucins that are aberrantly glycosylated. Although it has been realized that these compounds offer exciting opportunities for the development of immunotherapy for cancer, their use is hampered by the low antigenicity of classical immunogens composed of a glycopeptide derived from a mucin conjugated to a foreign carrier protein. We have designed, chemically synthesized, and immunologically evaluated a number of fully synthetic vaccine candidates to establish a strategy to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. The compounds were also designed to allow study of the importance of Toll-like receptor (TLR) engagement for these antigenic responses in detail. We have found that covalent attachment of a TLR2 agonist, a promiscuous peptide T-helper epitope, and a tumor-associated glycopeptide gives a compound (1) that elicits in mice exceptionally high titers of IgG antibodies that recognize MCF7 cancer cells expressing the tumor-associated carbohydrate. Immunizations with glycolipopeptide 2, which contains lipidated amino acids instead of a TLR2 ligand, gave significantly lower titers of IgG antibodies; this demonstrates that TLR engagement is critical for optimum antigenic responses. Although mixtures of compound 2 with Pam(3)CysSK(4) (3) or monophosphoryl lipid A (4) elicited titers of IgG antibodies similar to those seen with 1, the resulting antisera had impaired ability to recognize cancer cells. It was also found that covalent linkage of the helper T-epitope to the B-epitope is essential, probably because internalization of the helper T-epitope by B-cells requires assistance of the B-epitope. The results presented here show that synthetic vaccine development is amenable to structure-activity relationship studies for successful optimization of carbohydrate-based cancer vaccines. more...
- Published
- 2009
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42. Synthetic tetra-acylated derivatives of lipid A from Porphyromonas gingivalis are antagonists of human TLR4.
- Author
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Zhang Y, Gaekwad J, Wolfert MA, and Boons GJ
- Subjects
- Acylation, Animals, Cell Line, Escherichia coli chemistry, Humans, Mice, Molecular Structure, Monocytes drug effects, Toll-Like Receptor 4 metabolism, Lipid A chemistry, Lipid A pharmacology, Porphyromonas gingivalis chemistry, Toll-Like Receptor 4 antagonists & inhibitors
- Abstract
Tetra-acylated lipid As derived from Porphyromonas gingivalis LPS have been synthesized using a key disaccharide intermediate functionalized with levulinate (Lev), allyloxycarbonate (Alloc) and anomeric dimethylthexylsilyl (TDS) as orthogonal protecting groups and 9-fluorenylmethoxycarbamate (Fmoc) and azido as amino protecting groups. Furthermore, an efficient cross-metathesis has been employed for the preparation of the unusual branched R-(3)-hydroxy-13-methyltetradecanic acid and (R)-3-hexadecanoyloxy-15-methylhexadecanoic acid of P. gingivalis lipid A. Biological studies have shown that the synthetic lipid As cannot activate human and mouse TLR2 and TLR4 to produce cytokines. However, it has been found that the compounds are potent antagonist of cytokine secretion by human monocytic cells induced by enteric LPS. more...
- Published
- 2008
- Full Text
- View/download PDF
43. Chemical synthesis and immunological properties of oligosaccharides derived from the vegetative cell wall of Bacillus anthracis.
- Author
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Vasan M, Rauvolfova J, Wolfert MA, Leoff C, Kannenberg EL, Quinn CP, Carlson RW, and Boons GJ
- Subjects
- Animals, Anthrax Vaccines metabolism, Antibodies immunology, Bacillus anthracis metabolism, Cattle, Enzyme-Linked Immunosorbent Assay, Hemocyanins metabolism, Oligosaccharides metabolism, Rabbits, Spores, Bacterial immunology, Spores, Bacterial metabolism, Anthrax Vaccines chemical synthesis, Anthrax Vaccines immunology, Bacillus anthracis cytology, Bacillus anthracis immunology, Cell Wall chemistry, Cell Wall immunology, Oligosaccharides chemical synthesis, Oligosaccharides immunology
- Published
- 2008
- Full Text
- View/download PDF
44. Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions.
- Author
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Ning X, Guo J, Wolfert MA, and Boons GJ
- Subjects
- Animals, CHO Cells, Cell Survival, Copper chemistry, Cricetinae, Cricetulus, Glycoconjugates chemistry, Macrocyclic Compounds chemical synthesis, Macrocyclic Compounds chemistry, Molecular Structure, Surface Properties, Time Factors, Azides chemistry, Glycoconjugates analysis, Glycoconjugates metabolism
- Published
- 2008
- Full Text
- View/download PDF
45. Innate immune responses of synthetic lipid A derivatives of Neisseria meningitidis.
- Author
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Zhang Y, Gaekwad J, Wolfert MA, and Boons GJ
- Subjects
- Animals, Carbohydrate Conformation, Escherichia coli chemistry, Escherichia coli immunology, Immunologic Tests, Interferon-beta biosynthesis, Interferon-beta drug effects, Interferon-beta immunology, Lipid A analogs & derivatives, Lipopolysaccharides chemistry, Lipopolysaccharides pharmacology, Macrophages chemistry, Macrophages immunology, Mice, Molecular Structure, Neisseria meningitidis immunology, Stereoisomerism, Tumor Necrosis Factor-alpha biosynthesis, Tumor Necrosis Factor-alpha drug effects, Tumor Necrosis Factor-alpha immunology, Immunity, Innate drug effects, Lipid A chemical synthesis, Lipid A pharmacology, Macrophages drug effects, Neisseria meningitidis chemistry, Sugar Acids chemistry
- Abstract
Differences in the pattern and chemical nature of fatty acids of lipid A of Neisseria meningitides lipooligosaccharides (LOS) and Escherichia coli lipopolysaccharides (LPS) may account for differences in inflammatory properties. Furthermore, there are indications that dimeric 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) moieties of LOS and LPS enhance biological activities. Heterogeneity in the structure of lipid A and possible contaminations with other inflammatory components have made it difficult to confirm these observations. To address these problems, a highly convergent approach for the synthesis of a lipid A derivative containing KDO has been developed, which relies on the ability to selectively remove or unmask in a sequential manner an isopropylidene acetal, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc), azide, and thexyldimethylsilyl (TDS) ether. The strategy was employed for the synthesis of N. meningitidis lipid A containing KDO (3). Mouse macrophages were exposed to the synthetic compound and its parent LOS, E. coli lipid A (2), and a hybrid derivative (4) that has the asymmetrical acylation pattern of E. coli lipid A, but the shorter lipids of meningococcal lipid A. The resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha) and interferon beta (IFN-beta) production. The lipid A derivative containing KDO was much more active than lipid A alone and just slightly less active than its parent LOS, indicating that one KDO moiety is sufficient for full activity of TNF-alpha and IFN-beta induction. The lipid A of N. meningitidis was a significantly more potent inducer of TNF-alpha and IFN-beta than E. coli lipid A, which is due to a number of shorter fatty acids. The compounds did not demonstrate a bias towards a MyD88- or TRIF-dependent response. more...
- Published
- 2008
- Full Text
- View/download PDF
46. Robust immune responses elicited by a fully synthetic three-component vaccine.
- Author
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Ingale S, Wolfert MA, Gaekwad J, Buskas T, and Boons GJ
- Subjects
- Amino Acid Sequence, Animals, Antigens, Tumor-Associated, Carbohydrate chemistry, Cancer Vaccines chemical synthesis, Cancer Vaccines therapeutic use, Cytokines metabolism, Dendritic Cells immunology, Dendritic Cells metabolism, Dose-Response Relationship, Drug, Epitopes immunology, Epitopes metabolism, Glycopeptides chemical synthesis, Immunoglobulin G immunology, Macrophages immunology, Macrophages metabolism, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments immunology, T-Lymphocytes, Helper-Inducer immunology, T-Lymphocytes, Helper-Inducer metabolism, Toll-Like Receptor 2 agonists, Toll-Like Receptor 2 immunology, Toll-Like Receptor 2 metabolism, Up-Regulation, Antigens, Tumor-Associated, Carbohydrate immunology, Cancer Vaccines immunology, Glycopeptides immunology
- Abstract
The overexpression of saccharides such as Globo-H, Lewis(Y) and Tn antigen is a common feature of oncogenic transformed cells. Endeavors to exploit this aberrant glycosylation for cancer vaccine development have been complicated by difficulties in eliciting high titers of IgG antibodies against classical conjugates of tumor-associated carbohydrates to carrier proteins. We have designed, chemically synthesized and immunologically evaluated a number of fully synthetic vaccine candidates to establish strategies to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. We have found that a three-component vaccine composed of a TLR2 agonist, a promiscuous peptide T-helper epitope and a tumor-associated glycopeptide can elicit in mice exceptionally high titers of IgG antibodies that can recognize cancer cells expressing the tumor-associated carbohydrate. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells and avoidance of epitope suppression. more...
- Published
- 2007
- Full Text
- View/download PDF
47. The influence of the long chain fatty acid on the antagonistic activities of Rhizobium sin-1 lipid A.
- Author
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Zhang Y, Wolfert MA, and Boons GJ
- Subjects
- Cell Line, Fatty Acids chemical synthesis, Humans, Lipid A metabolism, Molecular Structure, Monocytes drug effects, Monocytes metabolism, Structure-Activity Relationship, Fatty Acids chemistry, Fatty Acids pharmacology, Lipid A antagonists & inhibitors, Rhizobium metabolism
- Abstract
The lipid A from nitrogen-fixing bacterial species Rhizobium sin-1 is structurally unusual due to lack of phosphates and the presence of a 2-aminogluconolactone and a very long chain fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0), moiety. This structurally unusual lipid A can antagonize TNF-alpha production by human monocytes induced by Escherichia coli LPS. To establish the relevance of the unusual long chain 27-hydroxyoctacosanoic acid for antagonistic properties, a highly convergent strategy for the synthesis of several derivatives of the lipid A of R. sin-1 has been developed. Compound 1 is a natural R. sin-1 lipid A having a 27-hydroxyoctacosanoic acid at C-2', compound 2 contains an octacosanoic acid moiety at this position, and compound 3 is modified by a short chain tetradecanoic acid. Cellular activation studies with a human monocytic cell line have shown that the octacosanoic acid is important for optimal antagonistic properties. The hydroxyl of the natural 27-hydroxyoctacosanoic moiety does, however, not account for inhibitory activity. The resulting structure-activity relationships are important for the design of compounds for the treatment of septic shock. more...
- Published
- 2007
- Full Text
- View/download PDF
48. Agonistic and antagonistic properties of a Rhizobium sin-1 lipid A modified by an ether-linked lipid.
- Author
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Vasan M, Wolfert MA, and Boons GJ
- Subjects
- Animals, Cell Line, Cross-Linking Reagents chemistry, Escherichia coli chemistry, Humans, Interferon-beta biosynthesis, Lipopolysaccharides pharmacology, Macrophages immunology, Macrophages metabolism, Mice, Molecular Structure, Species Specificity, Toll-Like Receptors metabolism, Tumor Necrosis Factor-alpha biosynthesis, Ether chemistry, Lipid A chemistry, Lipid A pharmacology, Lipids chemistry, Lipopolysaccharides agonists, Lipopolysaccharides antagonists & inhibitors, Macrophages drug effects, Rhizobium chemistry
- Abstract
LPS from Rhizobium sin-1 (R. sin-1) can antagonize the production of tumor necrosis factor alpha (TNF-alpha) by E. coli LPS in human monocytic cells. Therefore these compounds provide interesting leads for the development of therapeutics for the prevention or treatment of septic shock. Detailed structure activity relationship studies have, however, been hampered by the propensity of these compounds to undergo beta-elimination to give biological inactive enone derivatives. To address this problem, we have chemically synthesized in a convergent manner a R. sin-1 lipid A derivative in which the beta-hydroxy ester at C-3 of the proximal sugar unit has been replaced by an ether linked moiety. As expected, this derivative exhibited a much-improved chemical stability. Furthermore, its ability to antagonize TNF-alpha production induced by enteric LPS was only slightly smaller than that of the parent ester modified derivative demonstrating that the ether-linked lipids affect biological activities only marginally. Furthermore, it has been shown for the first time that R. sin-1 LPS and the ether modified lipid A are also able to antagonize the production of the cytokine interferon-inducible protein 10, which arises from the TRIF-dependent pathway. The latter pathway was somewhat more potently inhibited than the MyD88-dependent pathway. Furthermore, it was observed that the natural LPS possesses much greater activity than the synthetic and isolated lipid As, which indicates that di-KDO moiety is important for optimal biological activity. It has also been found that isolated R. sin-1 LPS and lipid A agonize a mouse macrophage cell line to induce the production of TNF-alpha and interferon beta in a Toll-like receptor 4-dependent manner demonstrating species specific properties. more...
- Published
- 2007
- Full Text
- View/download PDF
49. Modulation of innate immune responses with synthetic lipid A derivatives.
- Author
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Zhang Y, Gaekwad J, Wolfert MA, and Boons GJ
- Subjects
- Animals, Cytokines analysis, Cytokines metabolism, Escherichia coli immunology, Lipopolysaccharides chemical synthesis, Macrophages drug effects, Macrophages immunology, Mice, Salmonella typhimurium immunology, Immunity, Innate drug effects, Lipid A analogs & derivatives, Lipopolysaccharides chemistry, Lipopolysaccharides pharmacology
- Abstract
The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting with Toll-like receptor 4 (TLR4), which results in the production of a wide range of cytokines. Derivatives of lipid A show potential for use as immuno-modulators for the treatment of a wide range of diseases and as adjuvants for vaccinations. Development to these ends requires a detailed knowledge of patterns of cytokines induced by a wide range of derivatives. This information is difficult to obtain by using isolated compounds due to structural heterogeneity and possible contaminations with other inflammatory components. To address this problem, we have developed a synthetic approach that provides easy access to a wide range of lipid A's by employing a common disaccharide building block functionalized with a versatile set of protecting groups. The strategy was employed for the preparation of lipid A's derived from E. coli and S. typhimurium. Mouse macrophages were exposed to the synthetic compounds and E. coli 055:B5 LPS, and the resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha), interferon beta (IFN-beta), interleukin 6 (IL-6), interferon-inducible protein 10 (IP-10), RANTES, and IL-1beta. It was found that for each compound, the potencies (EC50 values) for the various cytokines differed by as much as 100-fold. These differences did not follow a bias toward a MyD88- or TRIF-dependent response. Instead, it was established that the observed differences in potencies of secreted TNF-alpha and IL-1beta were due to differences in the processing of respective pro-proteins. Examination of the efficacies (maximum responses) of the various cytokines showed that each synthetic compound and E. coli 055:B5 LPS induced similar efficacies for the production of IFN-beta and IP-10. However, lipid A's 1-4 gave lower efficacies for the production of RANTES and IL-6 as compared to LPS. Collectively, the presented results demonstrate that cytokine secretion induced by LPS and lipid A is complex, which can be exploited for the development of immuno-modulating therapies. more...
- Published
- 2007
- Full Text
- View/download PDF
50. Modification of the structure of peptidoglycan is a strategy to avoid detection by nucleotide-binding oligomerization domain protein 1.
- Author
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Wolfert MA, Roychowdhury A, and Boons GJ
- Subjects
- Cell Line, Diaminopimelic Acid analysis, Enzyme-Linked Immunosorbent Assay, Humans, Immunity, Innate, Lipopolysaccharides immunology, Lipopolysaccharides metabolism, Molecular Structure, NF-kappa B analysis, Nod2 Signaling Adaptor Protein metabolism, Peptidoglycan metabolism, Protein Binding, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Tumor Necrosis Factor-alpha genetics, Nod1 Signaling Adaptor Protein metabolism, Peptidoglycan chemistry, Peptidoglycan immunology, Tumor Necrosis Factor-alpha biosynthesis
- Abstract
Nucleotide-binding oligomerization domain (NOD) protein 1 (NOD1) and NOD2 are pathogen recognition receptors that sense breakdown products of peptidoglycan (PGN) (muropeptides). It is shown that a number of these muropeptides can induce tumor necrosis factor alpha (TNF-alpha) gene expression without significant TNF-alpha translation. This translation block is lifted when the muropeptides are coincubated with lipopolysaccharide (LPS), thereby accounting for an apparently synergistic effect of the muropeptides with LPS on TNF-alpha protein production. The compounds that induced synergistic effects were also able to activate NF-kappaB in a NOD1- or NOD2-dependent manner, implicating these proteins in synergistic TNF-alpha secretion. It was found that a diaminopimelic acid (DAP)-containing muramyl tetrapeptide could activate NF-kappaB in a NOD1-dependent manner, demonstrating that an exposed DAP is not essential for NOD1 sensing. The activity was lost when the alpha-carboxylic acid of iso-glutamic acid was modified as an amide. However, agonists of NOD2, such as muramyl dipeptide and lysine-containing muramyl tripeptides, were not affected by amidation of the alpha-carboxylic acid of iso-glutamic acid. Many pathogens modify the alpha-carboxylic acid of iso-glutamic acid of PGN, and thus it appears this is a strategy to avoid recognition by the host innate immune system. This type of immune evasion is in particular relevant for NOD1. more...
- Published
- 2007
- Full Text
- View/download PDF
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