Abstract
Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.
Highlights
Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM)
In this study we undertook the characterization of native LOS by MALDI mass spectrometry using a thin-layer method for deposition of the matrix, which is composed of a mixture of 2,4,6-trihydroxyacetophonone (THAP) with nitrocellulose to determine whether this methodology would provide a more thorough analysis of LOS structural variation than previous approaches [16]. We report that this method enables rapid profiling of the structural heterogeneity in native LOS and that the heterogeneity profile correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells
We demonstrate that MALDI mass spectrometry of native LOS provides a more thorough analysis of lipid A (LA) structural variability than previous approaches
Summary
Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2ulosonic acid (Kdo) and the lipid A (LA) Analyses of these data and comparison with spectra of the corresponding Odeacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. These differences in clotting activity were shown to be associated with the lipoidal portion of the LOS rather than the oligosaccharide (OS) terminus, as the purified lipid showed a similar potency to the parent LOS
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