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Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Palma AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W - Mol. Cell Proteomics (2015)

Bottom Line: The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans.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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.

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Validation of the glucome microarray with 12 glucan-recognizing proteins. (A) Murine monoclonal antibodies that recognize α- or β-glucans: MOPC-104E-IgM, 16.4.12.E-IgA, 3.4.1G6-IgG3, 2G8-IgG, 1H8-IgG and 1E12-IgM. (B) CBMs of microbial glycoside hydrolases of different CAZy families: TmCBM41, CtCBM11, TmCBM4–2, CmCBM32–2, BhCBM6 and CmCBM6–2. The microarrays included 153 gluco-oligosaccharide-NGLs for which the glucose linkages are indicated in the colored panels; these were linear sequences with homo linkages: 1,2-, 1,3-, 1,4-, or 1,6-linked, with α or β configurations in the size range mostly of DP-2 to DP-13, some up to DP-16; linear with hetero linkages: α1,4;1,6, ranging from DP-3 to DP-7 and β1,3;1,4 ranging from DP-3 to DP-16; branched with β1,3/β1,6 linkages, DP-2 to DP-13; and synthetic branched oligosaccharides with β1,3-linked linear DP-8 or DP-9 backbones, each with one or two β1,6-linked mono-glucosyl branches and a DP-4 backbone with an internal α1,3 linkage and two β1,6-linked glucose branches. Two xylo- and three manno-oligosaccharide NGLs were included as controls. Carbohydrate sequence information on these probes is in supplemental Table S7A. The binding signals are depicted as means of fluorescence intensities of duplicate spots at 5 fmol of oligosaccharide probe arrayed (with error bars) and are representative of at least two independent experiments; the numerical scores are given in supplemental Table S7B.
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Figure 5: Validation of the glucome microarray with 12 glucan-recognizing proteins. (A) Murine monoclonal antibodies that recognize α- or β-glucans: MOPC-104E-IgM, 16.4.12.E-IgA, 3.4.1G6-IgG3, 2G8-IgG, 1H8-IgG and 1E12-IgM. (B) CBMs of microbial glycoside hydrolases of different CAZy families: TmCBM41, CtCBM11, TmCBM4–2, CmCBM32–2, BhCBM6 and CmCBM6–2. The microarrays included 153 gluco-oligosaccharide-NGLs for which the glucose linkages are indicated in the colored panels; these were linear sequences with homo linkages: 1,2-, 1,3-, 1,4-, or 1,6-linked, with α or β configurations in the size range mostly of DP-2 to DP-13, some up to DP-16; linear with hetero linkages: α1,4;1,6, ranging from DP-3 to DP-7 and β1,3;1,4 ranging from DP-3 to DP-16; branched with β1,3/β1,6 linkages, DP-2 to DP-13; and synthetic branched oligosaccharides with β1,3-linked linear DP-8 or DP-9 backbones, each with one or two β1,6-linked mono-glucosyl branches and a DP-4 backbone with an internal α1,3 linkage and two β1,6-linked glucose branches. Two xylo- and three manno-oligosaccharide NGLs were included as controls. Carbohydrate sequence information on these probes is in supplemental Table S7A. The binding signals are depicted as means of fluorescence intensities of duplicate spots at 5 fmol of oligosaccharide probe arrayed (with error bars) and are representative of at least two independent experiments; the numerical scores are given in supplemental Table S7B.

Mentions: Hydrolysis of grifolan and lentinan was carried out with 0.02 m TFA at 100 °C for 12 h. After cooling to room temperature, the hydrolysate mixture was centrifuged. The supernatant was coevaporated with MeOH and fractionated by a short (1.6 × 36 cm) column of Bio-Gel P6. The high (>DP-20) and low (< DP-20) oligomer fractions, as determined by MALDI-MS, were kept separately. The precipitate and high oligomer fractions were combined and the TFA treatment procedure was repeated three times to increase the yield of low oligomers. The low oligomer fractions were combined and subjected to a final gel filtration to obtain, from grifolan, fractions 2 to 16 with Glc3 to Glc16 as major components (Grifo-3 to Grifo-16) and from lentinan fractions 2 to 13, with Glc2 to Glc13 as major components (Lenti-2 to Lenti-13). Microarray binding data with the α1,4-glucose specific TmCBM41 (Fig. 5B and supplemental Table S7B) indicated the presence of a minor α1,4-linked glucose contaminant in the lentinan fractions, particularly those containing oligomers with >DP-8 as major components. This was corroborated by MS/MS (footnote in supplemental Table S7A) and NMR (not shown).


Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Palma AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W - Mol. Cell Proteomics (2015)

Validation of the glucome microarray with 12 glucan-recognizing proteins. (A) Murine monoclonal antibodies that recognize α- or β-glucans: MOPC-104E-IgM, 16.4.12.E-IgA, 3.4.1G6-IgG3, 2G8-IgG, 1H8-IgG and 1E12-IgM. (B) CBMs of microbial glycoside hydrolases of different CAZy families: TmCBM41, CtCBM11, TmCBM4–2, CmCBM32–2, BhCBM6 and CmCBM6–2. The microarrays included 153 gluco-oligosaccharide-NGLs for which the glucose linkages are indicated in the colored panels; these were linear sequences with homo linkages: 1,2-, 1,3-, 1,4-, or 1,6-linked, with α or β configurations in the size range mostly of DP-2 to DP-13, some up to DP-16; linear with hetero linkages: α1,4;1,6, ranging from DP-3 to DP-7 and β1,3;1,4 ranging from DP-3 to DP-16; branched with β1,3/β1,6 linkages, DP-2 to DP-13; and synthetic branched oligosaccharides with β1,3-linked linear DP-8 or DP-9 backbones, each with one or two β1,6-linked mono-glucosyl branches and a DP-4 backbone with an internal α1,3 linkage and two β1,6-linked glucose branches. Two xylo- and three manno-oligosaccharide NGLs were included as controls. Carbohydrate sequence information on these probes is in supplemental Table S7A. The binding signals are depicted as means of fluorescence intensities of duplicate spots at 5 fmol of oligosaccharide probe arrayed (with error bars) and are representative of at least two independent experiments; the numerical scores are given in supplemental Table S7B.
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Related In: Results  -  Collection

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Figure 5: Validation of the glucome microarray with 12 glucan-recognizing proteins. (A) Murine monoclonal antibodies that recognize α- or β-glucans: MOPC-104E-IgM, 16.4.12.E-IgA, 3.4.1G6-IgG3, 2G8-IgG, 1H8-IgG and 1E12-IgM. (B) CBMs of microbial glycoside hydrolases of different CAZy families: TmCBM41, CtCBM11, TmCBM4–2, CmCBM32–2, BhCBM6 and CmCBM6–2. The microarrays included 153 gluco-oligosaccharide-NGLs for which the glucose linkages are indicated in the colored panels; these were linear sequences with homo linkages: 1,2-, 1,3-, 1,4-, or 1,6-linked, with α or β configurations in the size range mostly of DP-2 to DP-13, some up to DP-16; linear with hetero linkages: α1,4;1,6, ranging from DP-3 to DP-7 and β1,3;1,4 ranging from DP-3 to DP-16; branched with β1,3/β1,6 linkages, DP-2 to DP-13; and synthetic branched oligosaccharides with β1,3-linked linear DP-8 or DP-9 backbones, each with one or two β1,6-linked mono-glucosyl branches and a DP-4 backbone with an internal α1,3 linkage and two β1,6-linked glucose branches. Two xylo- and three manno-oligosaccharide NGLs were included as controls. Carbohydrate sequence information on these probes is in supplemental Table S7A. The binding signals are depicted as means of fluorescence intensities of duplicate spots at 5 fmol of oligosaccharide probe arrayed (with error bars) and are representative of at least two independent experiments; the numerical scores are given in supplemental Table S7B.
Mentions: Hydrolysis of grifolan and lentinan was carried out with 0.02 m TFA at 100 °C for 12 h. After cooling to room temperature, the hydrolysate mixture was centrifuged. The supernatant was coevaporated with MeOH and fractionated by a short (1.6 × 36 cm) column of Bio-Gel P6. The high (>DP-20) and low (< DP-20) oligomer fractions, as determined by MALDI-MS, were kept separately. The precipitate and high oligomer fractions were combined and the TFA treatment procedure was repeated three times to increase the yield of low oligomers. The low oligomer fractions were combined and subjected to a final gel filtration to obtain, from grifolan, fractions 2 to 16 with Glc3 to Glc16 as major components (Grifo-3 to Grifo-16) and from lentinan fractions 2 to 13, with Glc2 to Glc13 as major components (Lenti-2 to Lenti-13). Microarray binding data with the α1,4-glucose specific TmCBM41 (Fig. 5B and supplemental Table S7B) indicated the presence of a minor α1,4-linked glucose contaminant in the lentinan fractions, particularly those containing oligomers with >DP-8 as major components. This was corroborated by MS/MS (footnote in supplemental Table S7A) and NMR (not shown).

Bottom Line: The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans.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.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.

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Related in: MedlinePlus