The structure of the L9 immunotype lipooligosaccharide from Neisseria meningitidis NMA Z2491

Abstract

The lipooligosaccharide (LOS) from the Neisseria meningitidis prototype serogroup A strain NMA Z2491, an L9 immunotype LOS, was isolated and structurally characterized using glycosyl composition and linkage determination, mass spectrometry, and both 1- and 2-D nuclear resonance spectroscopy. The results show that the L9 LOS has an identical structure to that of an L4 LOS structure with the exception that it does not contain a sialic acid residue linked to position 3 of the lactoneotetraose terminal galactosyl residue. Further, two oligosaccharides are present in the Z2491 LOS preparation, OS1 and OS2. They differ from one another only in that OS2 contains an added glycine moiety, presumably at O-7 on the inner core Hep II residue. The structures of these oligosaccharides are as follows: where R=H or Gly.

Figure 1

The inner core structure of N. meningitidis LOS is shown with the various substituents that can be attached, their location and the genes responsible for these substituents. The gene responsible for the addition of glycine has not yet been identified or reported.

Figure 2

A comparison of the GC–MS profile of the partially methylated aldtitol acetates obtained from the LOS of (A.) N. meningitidis NMA Z2491, and (B.) N. meningitidis NMB. t-Glc = terminally linked glucose; t-Gal = terminally linked galactose, t-GlcNAc = terminally linked N-acetylglucosamine; →4)Glc = 4-linked glucose; →3)Gal = 3-linked galactose; →3,4)Hep = 3,4-linked heptose; and →4)GlcNAc = 4-linked N-acetylglucosamine.

Figure 3

MALDI-TOF/TOF mass spectra of the oligosaccharides isolated from NMA-LOS and purified by Bio-Gel P4 column. (a.) The mass spectrum of the OS1 fraction. (b.) The mass spectrum of the OS2 fraction. The spectra were acquired in positive mode and the proposed molecular compositions are shown in Table 1.

Figure 4

¹H NMR spectrum of OS1 isolated from (A.) NMA Z2491 LOS and (B.) NMB LOS. The data were acquired at 27 °C on Varian 600-MHz instrument. The anomeric region and structural reporter signals are as labeled.

Figure 5

¹H-¹³C HSQC NMR spectrum of OS1 isolated from (A.) NMA Z2491 LOS and (B.) NMB LOS. The data were acquired at 27 °C on a Varian 600-MHz instrument. The anomeric and some of the non-anomeric protons are labeled, the carbon chemical shifts of major signals are marked.

Figure 6

¹H-³¹P HMQC NMR spectrum of OS1 isolated from NMA-LOS and purified by a Bio-Gel P4 column. The data were acquired at 27 °C at pH 6.8 on a Varian 500-MHz instrument. The projection on top represents 1D proton spectrum and the projection on left hand side the 1D phosphorus indicating the connectivity between the phosphate and sugar ring.

Figure 7

A comparison of the structures of the oligosaccharides from immunotypes L3, L7, L4, and L9.