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Photogenerated Glycan Arrays Identify Immunogenic Sugar Moieties of Bacillus Anthracis Exosporium

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Photogenerated Glycan Arrays Identify Immunogenic Sugar Moieties of Bacillus Anthracis Exosporium 180 DOI 10.1002/pmic.200600478 Proteomics 2007, 7, 180–184 RAPID COMMUNICATION Photogenerated glycan arrays identify immunogenic sugar moieties of Bacillus anthracis exosporium Denong Wang1, 2, 3, Gregory T. Carroll4, Nicholas J. Turro4, Jeffrey T. Koberstein5, Pavol Kovácˇ 6, Rina Saksena6, Roberto Adamo6, Leonore A. Herzenberg2, Leonard A. Herzenberg2 and Lawrence Steinman3 1 Carbohydrate Microarray Laboratory, Stanford University School of Medicine, Stanford, CA, USA 2 Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA 3 Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA 4 Department of Chemistry, Columbia University, New York, NY, USA 5 Department of Chemical Engineering, Columbia University, New York, NY, USA 6 NIDDK, LMC, National Institutes of Health, Bethesda, MD, USA Using photogenerated glycan arrays, we characterized a large panel of synthetic carbohydrates Received: July 4, 2006 for their antigenic reactivities with pathogen-specific antibodies. We discovered that rabbit IgG Revised: September 2, 2006 antibodies elicited by Bacillus anthracis spores specifically recognize a tetrasaccharide chain that Accepted: October 17, 2006 decorates the outermost surfaces of the B. anthracis exosporium. Since this sugar moiety is highly specific for the spores of B. anthracis, it appears to be a key biomarker for detection of B. anthracis spores and development of novel vaccines that target anthrax spores. Keywords: Antibody / Bacteria / Biomarker / Carbohydrates / Sugar chips Anthrax is a fatal infectious disease caused by the Gram- the outermost surfaces of B. anthracis spores is of utmost positive, rod-shaped bacterium B. anthracis. Anthrax infec- importance. tion is initiated by the entry of spores into the mammalian Substantial effort has been made to study the structure host via intradermal inoculation, ingestion, or inhalation [1]. and antigenic elements of the outer layers of B. anthracis The most lethal form of human anthrax is the pulmonary spores. The mature B. anthracis spore contains a central infection caused by inhaled spores. In view of the risk of B. genome-containing core compartment and three adjacent anthracis spores as a biological weapon of mass destruction protective layers: the cortex, coat, and exosporium [7–9]. The (WMD) [2], it is necessary to achieve the capacity for the rapid exosporium is at the outermost surface and is fully exposed and specific detection of B. anthracis spores in various con- to the external environment. Morphologically, the exo- ditions [3, 4]. It is also important to develop new vaccines to sporium has two distinct structures, a paracrystalline basal block the anthrax infection at its initial stage before spore layer and an external hair-like nap [8]. Isolated B. anthracis germination takes place [5, 6]. In this context, identification exosporia contain as many as 20 protein components [9, 10]. of highly specific immunogenic targets that are displayed on The most prominent element is the BclA (for Bacillus collagen-like protein of anthracis) glycoprotein [11, 12]. The BclA glycoprotein displays a unique rhamnose-- containing tetrasaccharide. This carbohydrate moiety is cap- Correspondence: Dr. Denong Wang, Department of Genetics, ped at its upstream end with a previously unknown sugar Stanford University School of Medicine, 279 Campus Drive residue termed anthrose [2-O-methyl-4-(3-hydroxy-3-methyl- West, Beckman Center, B011 Stanford, California 94305, USA b D E-mail: [email protected] butamido)-4,6-dideoxy- - -glucose] [12]. The complete struc- Fax: 11-650-725-8564 ture of this tetrasaccharide has been determined to be 2-O- methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-b-D- Abbreviation: BclA, Bacillus collagen-like protein of anthracis glucopyranosyl-(1?3)-a-L-rhamnopyranosyl-(1?3)-a-L- © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2007, 7, 180–184 Protein Arrays 181 rhamnopyranosyl-(1?2)L-rhamnopyranose [12]. Impor- tantly, this sugar moiety is absent on the surfaces of B. anthracis vegetative cells or the spores of other bacillus strains, including B. cereus and B. thuringiensis, the two strains phylogenetically most close to B. anthracis [12]. Surface-exposed carbohydrate moieties that are char- acteristic of a given microbe may serve as key biomarkers for pathogen identification, diagnosis, and vaccine develop- ment. Therefore, the anthrose-containing tetrasaccharide of the BclA glycoprotein is an attractive target for immuno- logical investigations. BclA has been confirmed to be immu- nodominant [13]. However, whether its carbohydrate moi- eties contribute to the immunogenecity of this exosporium glycoprotein remains unknown [14]. This may be due to the lack of a method for the detection of antibodies specific for Figure 1. Photogenerated glycan arrays for rapid identification of the carbohydrate moieties of the glycoprotein. It has, there- pathogen-specific immunogenic sugar moieties. Saccharide fore, been difficult to decipher the anticarbohydrate specifi- preparations were dissolved in saline (0.9% NaCl) at a given cities and antiprotein specificities, even if the corresponding concentration and spotted using a high-precision robot (PIXSYS antibodies were present in the sera as a result of a polyclonal 5500C, Cartesian Technologies Irvine, CA) onto the phthalimide- antibody response to a natural infection or a vaccination amine monolayers (PAM)-coated slides. The printed PAM slides were subjected to UV irradiation (300 nm) for 1 h to activate the using immunogenic glycoconjugates. photocoupling of carbohydrates to the surface. Pathogen- We present here a high-throughput strategy to facilitate specific antisera were then applied on the glycan arrays to iden- identification and immunological characterization of patho- tify potential immunogenic sugar moieties of given pathogens. gen-specific carbohydrate moieties, including those of the B. anthracis exosporium. This strategy employs highly sensi- tive oligosaccharide microarrays to probe specific antibodies At the onset of this work, we assumed that if B. anthracis that were elicited by infections or immunizations. Our ratio- spores express potent immunogenic carbohydrate moieties, nale was that if a pathogen expressed immunogenic carbo- immunization with the spores would elicit antibodies spe- hydrate structures, then immunizing animals using this cific for these sugar structures. Such antibody reactivities microbe or its antigen preparations would have the possibil- would then be detected by glycan arrays that display the cor- ity to elicit antibodies specific for these structures. Char- responding sugar structures. Therefore, we incubated the acterizing these antibody responses using a broad-range gly- anthrax saccharide glycan arrays with rabbit anti-B. anthracis can array that displays the corresponding saccharides may spore antibodies and examined the potential presence of thus rapidly identify the immunogenic sugar moieties of the anticarbohydrate specificities. corresponding pathogens. A schematic overview of this bio- Figure 2 shows a glycan array image that was stained with marker identification strategy is shown in Fig. 1. a preparation of pooled rabbit polyclonal antianthrax spore Photogeneration of epitope-specific glycan arrays is a key IgG antibodies. Images (a-f) display a portion of the stained component of this strategy. We utilized a photoactive surface glycan arrays in the absence or presence of saccharide inhib- for covalent immobilization and micropatterning of carbo- itors: (a) no saccharide inhibitor; (b) anthrose; (c) D-glucose; hydrates onto glass substrates [15]. This method employs a (d) a-anthrose disaccharide; (e) a-anthrose tetrasaccharide; (f) glass slide coated with a self-assembled mixed monolayer b-anthrose tetrasaccharide. As highlighted with colored boxes that presents photoactive phthalimide chromophores at the in the image, a number of immobilized carbohydrate probes air–monolayer interface [15]. Upon exposure to UV radiation detected significant amounts of IgG antibodies. These (300 nm), the phthalimide end-groups graft the spotted car- include an anthrose-containing trisaccharide (White, array bohydrates by hydrogen abstraction followed by radical location B-1), and both the b-tetrasaccharide (Brown, array recombination. The efficacy of carbohydrate immobilization location C-1) and a-tetrasaccharide (Yellow, array location B-4) is independent of the molecular weights of spotted carbo- in the form of their methyl 6-hydroxyhexanoyl glycosides. hydrates [15]. Thus, a unique technical advantage of this The glycan array also shows other anticarbohydrate antibody method is the ability to produce epitope-specific glycan activities, such as antipolysaccharide antibodies for iso- arrays using unmodified mono- and oligo-saccharides. We lichenin [a(1,3)glucan] (positive spots in the upper-left cor- applied, therefore, this technology to display a large panel of ner) and Streptococcus pneumoniae type 23 (Pn 23) poly- saccharide structures, including synthetic fragments and saccharide (positive spots in the bottom-right corner). These derivatives of the anthrose-containing tetrasaccharide side reagents were spotted on the chips as positive controls for the chain of the B. anthracis exosporium [12, 16–18] and a num- assay system since we detected a significant amount of IgG ber of control carbohydrate antigens (Supplementary Table antibodies for these antigens in
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