NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus

Abstract

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.

Keywords: Capnocytophaga canimorsus; DHPC Micelles; Glycolipid Structure; Gram-negative Bacteria; High-performance Liquid Chromatography (HPLC); Lipopolysaccharide (LPS); Nuclear Magnetic Resonance (NMR).

FIGURE 1.

LPS and part structures of C. canimorsus. Shown are structures of the core hexasaccharide (1) obtained after acid hydrolysis (AcOH), isolated core backbone octasaccharide (2) obtained after strong hydrazinolysis (N₂H₄), and the complete rough-type LPS (3).

FIGURE 2.

Reversed phase HPLC profile obtained from the C. canimorsus Y1C12 mutant LPS after mild acetate buffer hydrolysis. Note that only ∼10% of the free lipid A (pool 3) was obtained under the conditions used, whereas the intact rough-type LPS (3, pool 2) predominated (>60%) and could be separated to homogeneity. ELS Detector, evaporative light-scattering detector.

FIGURE 3.

Charge-deconvoluted mass spectrum of 3 obtained by ESI Fourier transform ion cyclotron resonance MS in the negative ion mode. The monoisotopic mass found (2976.680 Da) is in excellent agreement with structure 3 (Fig. 1). The inlet shows an enlargement of the isotopic peaks obtained from the most abundant peak. The calculated ion intensity profile (red) is nearly identical to the measured one (blue), indicating the heterogeneity in the hybrid lipid A backbone (GlcN3N-GlcN) versus E. coli-type backbone (GlcN-GlcN) to be minor (∼1–2%). Mass values in the inset represent the calculated masses for 3.

FIGURE 4.

HSQC-contour plot of the complete spectrum (¹H 6.0–0.5 ppm, ¹³C 120–5 ppm) in which all three distinct regions of 3 can be clearly distinguished. Ring protons/carbon signals and those of fatty acids are enlarged in Fig. 5 and 6, to demonstrate good signal resolution. Signals from the DHPC-d₄₀ (98% D) detergent are shown in orange. The ¹H NMR spectrum is shown along the F2 axis at the top.

FIGURE 5.

Section of the ¹H,¹³C-HSQC NMR (700 MHz) spectrum showing ring proton/carbon signals of 3. The spectrum was recorded at 325 K in D₂O containing 1.5% DHPC-d₄₀. Selected ¹H,¹³C ring signals and their assignments are indicated. ¹H,¹³C signals from the DHPC-d₄₀ detergent (98% D) are shown in orange. The one-dimensional ¹H spectrum is shown as a projection of the F2 axis.

FIGURE 6.

HSQC contour plot showing the region with fatty acid signals H-2/C-2 to H-ω/C-ω as well as H-3_ax,eq/C-3 Kdo and H-6/C-6 of l-Rha sugars. Signals assigned to one acyloxyacyl residue (R2″-R2′), (3R)-3-(15-methyl-hexadecanoyloxy)-13-methyl-tetradecanoic acid, attached to position 2′ of GlcpN3N (II) as well as those originating from the (R)-3-hydroxy-hexadecanoic acid (R3′) are indicated. Signals from the DHPC-d₄₀ detergent are shown in orange.

FIGURE 7.

NOE connectivities detected in 3 by which the linkages of sugars and P-Etn residues were assigned. Some of the anomeric protons (e.g. H-1 of l-Rha and Man^II) show NOE cross-peaks (Table 2) not only to the substituting but also to the neighboring sugar protons to which they are bound, indicating a rather twisted conformation of the branched oligosaccharide. The fatty acid residues attached to the lipid A backbone (R3′, R2″-R2′, R3, and R2) are abbreviated for clarity.

FIGURE 8.

Special conformation adopted by the intact core when bound to the lipid A. Shown is part of the ROESY spectrum of 3 with NOE signals showing interresidual connectivities between H-3_ax of Kdo and H-3/H-5 of Man^I. This indicates a free rotation in the core oligosaccharide of about 180° (gray arrow) around the Man^I-C1-O-C5-Kdo bond axis as shown in the bottom part together with the membrane matrix of the DHPC micelles. Indicated are the two disaccharides attached to Man^I (i.e. α-l-Rhap-(1→3)-β-d-Galp-(1→ (OR¹) and α-d-GalNp-(1→6)-α-d-Manp^II-(1→4) (OR²)).