In this report, we have shown that the LOS structure of NMA Z2491 has an outer core oligosaccharide that consists of α-lactoneotetraose attached to O-4 of Hep I, and an inner core region in which Hep II is substituted at O-6 with a PEA group and at O-2 with α-GlcNAc. In addition, the α-GlcNAc residue is substituted with an acetyl group at O-3. A portion, approximately 40%, of the LOS consists of structures that are glycinylated in the inner core region, presumably at O-7 of the Hep II residue. As expected for an isolate without an endogenous supply of NeuNAc, the LOS is not sialylated. The structure of this oligosaccharide is shown in , structure L9. For the purposes of the following discussion, the structures of the oligosaccharides from L3, L4, and L7 immunotypes are also shown in .
A comparison of the structures of the oligosaccharides from immunotypes L3, L7, L4, and L9.
Jennings et al. 18
, reported that the LOSs from L3, L7, and L9 all had the same oligosaccharide structure as obtained after release from the lipid A by mild acid hydrolysis and after treatment with aqueous HF and O-deacetylation. Our results for the L9 LOS from NMA Z2491 also show the same oligosaccharide structure, after HF treatment, as reported by Jennings et al. 18
, with the exception that we show that the terminally linked α-GlcNAc residue is acetylated at O-3. This exception was likely due to the fact that Jennings et al. O-deacetylation the OS in addition to HF treatment, which removed any O
-acetyl groups. Therefore, it was possible that the L3, L7, and L9 LOSs could vary, not only in the location of PEA groups, but also in the presence or absence of the O
-acetyl group. Subsequent to the findings of Jennings et al. 18
, Kogan et al 19
reported that the L3 LOS was distinguished from L7 by the presence of a terminally linked NeuNAc residue. Similarly, comparison of the L4 structure with our results for the NMA Z2491 L9 structure reveals that the major distinction between these two structures is the presence or absence, respectively, of terminal NeuNAc on the lactoneotetraose portion of the LOS. In comparison to the L7 LOS structure, the L9 NMA Z2491 LOS structure differs in that it contains an acetyl group at the O-3 of the terminally linked α-GlcNAc residue, and it has a PEA substituent at O-6 rather than at O-3 of the Hep II residue.
The genetic basis for the structural differences between the L3, L4, L7, and L9 LOSs is due to the presence or absence or phase off or on status of lgtG, lpt3, lpt6
, and lot
, as well as the availability of CMP-NeuNAc for the addition of NeuNAc by the product of lst
. For LOSs that contain a complete α-lactoneotetraose, the expression of lgtG
prevents the addition of a PEA to O-3 of Hep II 14
. When both lgtG
are inactivated, the LOS contains the O-3 PEA substituent 12
. In the case of the L3 and L7 immunotypes, lgtG
are absent or not expressed, and the resulting LOS contains PEA at O-3 of Hep II and lacks the acetyl group at O-3 of the terminally linked α-GlcNAc residue. Based on the genome sequence of NMA Z2491 20
is absent, lpt3
contains a deletion, and an active lot
is present. This gene profile is consistent with the structure we observe; namely, a LOS that contains a PEA at O-6 of Hep II and not at O-3, and has an acetyl group at O-3 of the α-GlcNAc residue. The difference between this L9 structure and that reported for L4 is that the latter LOS contains a terminally linked NeuNAc. The lack of this terminally linked NeuNAc is due to the inability of serogroup A strains, and other strains that have a non-NeuNAc-containing capsule, to synthesize CMP-NeuNAc 19, 21
The L9, L10, L11 and L12 immunotypes are expressed by serogroup A strains, which are unable to synthesize NeuNAc-containing capsules, while immunotypes L1 to L8 are expressed in serogroup B and C isolates that have varying levels of NeuNAc capsule expression. In previous surveys, the L3, L7, and L9 LOS all react similarly with a panel of immunotyping antibodies 3, 22
. From the known structures of L3, L7 and L9, we can deduce that the immunotyping antibodies may be directed towards the common lactoneotetraose α-chain. Significantly, both immunotyping panels did not contain antibodies that cross-reacted between L4 and L9 structures, even though these structures share the same inner core with O-6 PEA and an O
-acetyl group, and differ only in the lack of sialylation of the L9 lactoneotetraose. For this to occur, we would predict that L4 and L9 structures represent different conformations based upon the state of sialylation of the lactoneotetraose, and that this difference is driven by the position of the O-6 PEA group on Hep II. Future molecular modeling of these structures will assess this prediction.
In summary, the L9 LOS structure of NMA Z2491 has the same structure as that reported for the L4 immunotype with the exception that it lacks the terminal NeuNAc residue. The finding has significance for understanding the genetic and immunological basis of meningococcal LOS structure and for design of meningococcal vaccines that contain LOS.