Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides

Shafer, Douglas E; Toll, Barbara; Schuman, Richard F.; Nelson, Brett L.; Mond, James J.; Lees, Andrew

doi:10.1016/s0264-410x(99)00370-9

Cited by 72 publications

(59 citation statements)

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“…5b). Confirmation of the phosphorylation of O-3 of the ␤-GlcNAc was obtained from the 13 C NMR spectrum of the B. pumilus Sh18 CWP, which displayed the C-2, C-3, and C-4 signals of the ␤-GlcNAc residue as doublets containing 31 P couplings (Table 4). 31 P NMR spectroscopy (1D spectrum at the left side of Fig.…”

Section: Resultsmentioning

confidence: 83%

“…An intense doublet was also observed for C-1,3 of 1,3-poly (glycerol phosphate) [1,3-poly(Gro-P)] and a weaker triplet for C-2, reflecting the presence of 31 P spin coupling to all of the Gro 13 C nuclei. Partial glycosylation of C-2 of 1,3-poly(Gro-P) by ␣-GalNAc and ␣-GlcNAc was confirmed by the observation of 2D 1 H/ 13 C HMBC connectivities for ␣-GalNAc H-1 and Gro-P C-2 and of ␣-GlcNAc H-1 and Gro-P C-2, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…1D 1 H NMR subspectra of individual residues were generated by extraction of F 2 slices from the 2D TOCSY spectra. For further confirmation of assignments, some 2D TOCSY experiments were conducted with either selective, digital, homonuclear 1 H decoupling or continuous, WALTZ-16 31 P decoupling. 2D heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) 1 H/ 13 C NMR spectra were recorded as 2,048-by 512-point or 800-point data sets, zero-filled to 2,048 by 2,048 points, using the GS, sensitivity-enhanced, phase-sensitive echoanti-echo mode for HSQC and a GS, low-pass-filtered, long-range, nondecoupled pulse sequence for HMBC, with an evolution delay of 83 ms, i.e., optimized for 2,3 Step I: bovine serum albumin (BSA; Sigma) was derivatized with adipic acid dihydrazide (ADH) as described previously (29).…”

mentioning

confidence: 99%

“…Step II: the Sh18 CWP was reacted with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (31) to form a cyanate ester derivative (CWP™O™C §N); CWP (10 mg) was dissolved in water at 5 mg/ml, the pH was adjusted to 9.0, and 10 mg of CDAP in 0.1 ml of acetonitrile was added. After 30 s of mixing, 0.1 ml of 0.2 M triethylamine was added, followed by 10 mg of rEPA-AH in 0.6 ml of PBS over the next 2.5 min.…”

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confidence: 99%

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Chemical Structure, Conjugation, and Cross-Reactivity ofBacillus pumilusSh18 Cell Wall Polysaccharide

2004

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Bacillus pumilus strain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-␣-galactopyranose (13%) and 2-acetamido-2-deoxy-␣-glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of 33-O-(2-acetamido-2-deoxy-␤-glucopyranosyl)-134-ribitol-1-OPO 3 3. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide ( Cell wall polysaccharides (CWP) of gram-positive bacteria, also referred to as teichoic acids, are structurally diverse linear polymers of polyols or carbohydrates linked through phosphodiester bonds, often substituted with different glycosyl or amino acid residues and terminally linked to the muramic acid of peptidoglycan (5). The functions and structures of different types of teichoic acids have been reviewed (23). These compounds comprise 20 to 50% of the weight of the cell wall and are major surface antigens in noncapsulated gram-positive bacteria. In capsulated bacteria, the capsule is the surface antigen covering other structures such as CWP in gram-positive bacteria and lipopolysaccharides in gram-negative bacteria. Antibodies to the capsular polysaccharides (CP) of pathogenic bacteria are protective, and the CP are used as vaccines, either alone or as protein conjugates (1).Bacillus pumilus strain Sh18, a nonpathogenic, enteric grampositive bacterium, produces a CWP, hitherto of unknown structure, reported to cross-react with the H. influenzae type b (Hib) CP. This cross-reactivity has been attributed to poly (ribitol phosphate), a known component of teichoic acids of bacilli. Ribitol phosphate, but not ribitol alone, inhibited precipitation of anti-Hib serum with B. pumilus Sh18 CWP (3, 9). No cross-reactivity, however, was observed with H. influenzae type a (Hia) CP, also containing ribitol phosphate in its subunit (Fig. 1). The induction of antibodies against Hia would be of interest, since this type has been reported in several countries to cause up to 10% of systemic infections due to H. influenzae (26,35,36). Here, we describe the purification of B. pumilus Sh18 CWP, the determination of its chemical structure, and the preparation and characterization of CWP-protein conjugates. MATERIALS AND METHODSBacteria and cultivation. B. pumilus Sh18 and Sh17, Hib strains Eagan and Rab, Hia strain Harding, Staphylococcus aureus type 5 strain Lowenstein, and Sta...

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Chemical Structure, Conjugation, and Cross-Reactivity ofBacillus pumilusSh18 Cell Wall Polysaccharide

2004

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“…A solution containing 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) (Sigma, USA) [8,9] and hydroxyl group activator (10 mg/0.1 mL acetonitrile) was added to an aqueous solution of YCP (30 mg/2 mL) while stirring. After 1 min, pH was raised to 9.0 by adding 0.25 mol/L NaOH.…”

Section: Preparation Of Fluorescence-labeled Ycpmentioning

confidence: 99%

Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108

et al. 2009

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Aim: YCP, a novel (1,4)-α-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation. Methods: Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production. Results: YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time-and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation. Conclusion: We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.Keywords: YCP; Phoma herbarum; macrophages; nitric oxide; receptor; p38 kinase Acta Pharmacologica Sinica (2009Sinica ( ) 30: 1008Sinica ( -1014 doi: 10.1038/aps.2009 Original Article Introduction YCP is a polysaccharide with a mean molecular weight of 2.4×10 3 kDa purified from the mycelium of Phoma herbarum YS4108, a marine filamentous fungus that inhabits the sediment at a depth of 50 m in the Yellow Sea near Sheyang Port, Yancheng, China. (YCP is the acronym of Yancheng and polysaccharide.) In an earlier study, YCP was found to be able to increase phagocytic activity of mice in vitro and in vivo, indicating that it may activate macrophages [1] . YCP could significantly increase the production of NO and interleukin-1 (IL-1) by macrophages. It also significantly increased phagocytic activity of macrophages [2] . However, the YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation are poorly understood.β-glucans are potent stimulators of the innate immune system in invertebrates, while in mammals they are potent activators of the complement system. Soluble and particulate β-glucans interact with cognate receptors on...

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Antibacterial Carbohydrate Vaccines

Compostella,

Morelli,

Lay

2023

Carbohydrate‐Based Therapeutics

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Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides

Cited by 72 publications

References 16 publications

Chemical Structure, Conjugation, and Cross-Reactivity ofBacillus pumilusSh18 Cell Wall Polysaccharide

Chemical Structure, Conjugation, and Cross-Reactivity ofBacillus pumilusSh18 Cell Wall Polysaccharide

Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108

Antibacterial Carbohydrate Vaccines

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