Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone

Abstract

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Eschel-icliici coli and Proteus mirahilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-0-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN 11) of the backbone carries, in S. minnesotu and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible.It was found that: (a) in all three lipids A GlcN I1 is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. rninnesota and P. mirabilis.In lipids A which were obtained from the P. mirahi1i.s Re mutant grown at low temperature (12 C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetradecanoic acid at GlcN I1 is positionally replaced by d9-hexadecenoic acid (palmitoleic acid).It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN I1 are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.

Nature and location of amide‐bound (R)‐3‐acyloxyacyl groups in lipid A of lipopolysaccharides from various gram‐negative bacteria

“…A limited variability, however, is seen with regard to the nature of acyl groups substituting the hydroxyl group of 3-hydroxy fatty acids amide-bound to GlcN 11: In Salmonella species and E. coli it carries 12:0, but in P. mirabilis it is acylated by 14:O. In addition, in Salmonella (or Proteus) lipid A, obtained from bacteria grown at 1 2 T , this very hydroxyl group carries cis-16: 1 (9) instead of 12:0 (or 14:O) [8,26,271. The results of the present paper indicate that in C. violaceum lipid A this hydroxyl group may be substituted by a nonhydroxylated fatty acid (1 2: 0) or, preferentially, by its a-oxidation product (2-OH-12: 0).…”

“…The same solvents, chemicals and reference compounds were used as in these previous studies. For laser desorption mass spectrometry the LAMMA 500 instrument (Leybold Heraeus, Koln, FRG) was used [8]. The preparation of samples for laser desorption mass spectrometry and the conditions of analysis were as described [8].…”

“…From these lipopolysaccharides free lipid A was prepared by hydrolysis in 0.1 M acetic acid as described, electrodialysed, converted to the triethylammonium salt, lyophilised and stored over P z 0 5 in vacuo [19]. For analysis with the laser microprobe mass analyser, (partly) dephosphorylated free lipid A was prepared [8] by treatment of lipopolysaccharide (50 mg) with H2F2 (48%, 1 ml, 48 h, 4°C) and subsequent acid hydrolysis (0.1 M HCl, 60 min, 100 "C). …”

Comparison of lipids A of several Salmonella and Escherichia strains by 252Cf plasma desorption mass spectrometry

“…Since the lipid portion (lipid A) of endotoxic lipopolysaccharide (LPS) has been shown to be responsible for most of the biological activities of these macromolecules, it is the focal point of such studies. However, for both biological and structural studies, two complications arise: the lipid moiety is often heterogeneous (1,20), and its composition can vary with bacterial growth conditions (11,13,14,18,25,27,30,33).…”

“…Few of these variants have been tested biologically, but some, especially those touching the phosphate groups (24,28,29), may well have an effect on the biological activities of lipid A. Several reports have already testified to the influence of bacterial growth temperature and medium on the LPS composition of endotoxins (11,13,14,18,25,27,30,33), and the observed variation may be attributed to differences in culture conditions. Thus, the analytical results obtained with one preparation should not be assumed to apply to all preparations obtained from a given strain.…”