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Journal of Lipid Research, Vol. 50, 424-438, March 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction^*

Constance M. John^*, Mingfeng Liu^* and Gary A. Jarvis^1,*,

^* Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, 94121
Department of Laboratory Medicine, University of California, San Francisco, CA 94143

^* This work was supported by Public Health Service Grant AI063927 from the National Institute of Allergy and Infectious Diseases and by the Research Service of the US Department of Veterans Affairs. The authors would like to acknowledge the UCSF Mass Spectrometry Core Facility that is supported by an NCI Cancer Center Support Grant (P30 CA82103) and the Sandler Family Foundation, and the UCSF Mass Spectrometry Facility (A.L. Burlingame, Director), which is supported by the Biomedical Research Technology Program of the National Center for Research Resources, NIH, NCRR P41RR001614. This is paper number 103 from the Center for Immunochemistry.

Published, JLR Papers in Press, October 2, 2008.

² Figures 2B, 3B, and 4B revised in press due to recent data (26).

¹ To whom correspondence should be addressed. e-mail: Gary.Jarvis{at}ucsf.edu

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.

Supplementary key words lipooligosaccharide • endotoxin • Neisseria gonorrhoeae • Neisseria meningitidis • MALDI • phosphorylation • acylation • structural • mass spectrometry

Abbreviations: Ac, acetyl; Gal, galactose; Glc, glucose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; Gly, glycine; Hep, L-glycero-D-manno-heptose; Hex, hexose; HexNAc, N-acetylhexosamine; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; LA, lipid A; LOS, lipooligosaccharide; LPS, lipopolysaccharide; OS, oligosaccharide; P, phosphate; PEA, phosphoethanolamine; THAP, 2,4,6-trihydroxyacetophonone; TLR4, toll-like receptor 4; TOF, time-of-flight; TREM, triggering receptor expressed on myeloid cells

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