Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Cyclic ␤-1,2-glucans (C␤G) are osmolyte homopolysaccharides with a cyclic ␤-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the C␤G synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked ␤-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a ␤-1,2-glucan phosphorylase present at the C␤G synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the ␤-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this ''lengthcontrolling'' phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.cyclic ␤-1,2-glucan ͉ phosphorylase ͉ size control O smoregulated periplasmic glucans are oligosaccharides present in the periplasm of certain Gram-negative bacteria. Common features of these oligosaccharides are the presence of glucose as the sole sugar constituent and the regulation of their synthesis by the osmolarity of the growth media. Osmoregulated periplasmic glucans may be cyclic, branched cyclic, or branched linear and, depending on the species, may be substituted with a variety of nonglycosidic residues (1, 2). Agrobacterium, Rhizobium, Sinorhizobium, and Brucella species synthesize osmoregulated periplasmic glucans of family II. Glucans of this family have 17-25 glucose residue cyclic ␤-1,2-backbones substituted with sn-1-phosphoglycerol, succinic acid, methylmalonic acid, or a combination of them (1-3).Cyclic ␤-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of cyclic ␤-1,2-glucans (C␤G), is present in a restricted number of symbiotic or pathogenic bacteria, most of them belonging to the ␣-proteobacteria group, in which C␤G are a symbiotic or virulence factor required for successful host interaction (4-9). Brucella abortus Cgs is a 320-kDa (2,867 amino acid residues) polytopic integral inner membrane protein with six transmembrane-spanning segments (TMSs) and with the N and C termini located on the cytoplasmic side of the membrane (10). Cgs, an enzyme using UDP-glucose as sugar donor and Mg 2ϩ as cofactor, functions as an inverting processive ␤-1,2-glucosyltransferase that catalyzes the three enzymatic activities (initiation, elongation, and cyclization) required for synthesis of C␤G. Synthesis is initiated...

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