Extracellular structure of polysialic acid explored by on cell solution NMR

Abstract

The capsular polysaccharide of the pathogens Neisseria meningitidis serogroup B and of Escherichia coli K1, alpha(2 --> 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures in vivo. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on E. coli bacteria. Using 13C,15N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO, in vivo. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The 13C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for in vivo kinetic studies.

Fig. 1.

Chemical structure of PSA (poly-α(2 → 8)Neu5Ac). The numbers identify carbon positions, and n can reach 200. Blue and red are used to differentiate the two possible forms the linkage can take. At neutral pH, most α(2 → 8) linkages are like the blue one, resulting in a highly flexible linear molecule. At low pH, the number of lactones increases, resulting in a rigidified structure.

Fig. 2.

¹H⁻¹³C correlation spectra of PSA under several conditions. (A and B) Numbers identify carbons. m, monosaccharide; p, polysaccharide; eq, equatorial; ax, axial. (A) ¹H-¹³C HSQC spectrum of EV239^+neuS cells grown in LB media containing ¹³C,¹⁵N-Neu5Ac. To improve resolution a spectral width of 40 ppm, with carrier frequency at 68 ppm, was used in the ¹³C dimension, therefore the peaks between 1.50 and 3.10 ppm in the ¹H dimension are aliased in the ¹³C dimension. These peaks have ¹³C shifts that are 40 ppm lower than they appear. (B) The overlayed ¹H-¹³C HSQC spectra of ¹³C,¹⁵N-Neu5Ac (red) and ¹³C,¹⁵N-PSA at neutral pH (black) clearly show the difference between the monomer (in the β-configuration) and polymer (α-configuration). ∗, identifies the Tris buffer signal. (C) ¹H-¹³C HSQC spectrum of free ¹³C,¹⁵N-PSA at pH 4.0 (green), at which lactone formation is promoted, overlayed with spectrum (A). The number of green contours was purposely limited to better demonstrate the absence of lactones on cells.

Fig. 3.

Triple-resonance experiments correlating ¹H-¹⁵N-¹³C on EV239^+neuS cells grown with ¹³C,¹⁵N-Neu5Ac. The spectra show two sets of signals, which correspond to PSA (p) and Neu5Ac (m). (A) 2D-HNCO spectrum showing two signals correlating the amide ¹H with the ¹³CO chemical shifts on the N-acetyl group through the ¹⁵N atom. (B) 2D-HNCA spectrum correlating the amide ¹H chemical shift with ¹³CH₃ in the N-acetyl group and ¹³C5 through the ¹⁵N atom.

Fig. 4.

Effects of exo-neuraminidase addition on NMR spectra, monitored through the peak height of the ¹³CH-3 signals. (A) Region corresponding to ¹³CH-3 signals from ¹H-¹³C HSQC spectra of EV239^+neuS cells with ¹³C,¹⁵N-PSA before addition of the enzyme and at two representative times (indicated in the figure) after addition. The signals from PSA (labeled p) decrease in intensity as the monomer (m) increases. (B) Plot of the signal intensities from m3_eq (■) and p3_eq (●) as function of time after enzyme addition. Data fitting to single exponential functions (solid line) yielded identical rate constants (0.27 ± 0.01 h⁻¹) with opposite sign.