Linoleate-rich acylglucosylceramides of pig epidermis: structure determination by proton magnetic resonance

The structure of the linoleate-rich acylglycosylceramides isolated from pig epidermis has been reinvestigated. Gas-liquid chromatographic analysis of the alditol acetates produced from the sugar component indicated that 90% of the hexose is glucose while the remaining 10% is galactose. The predominance of the beta-D-glucosyl group was confirmed by 360 MHz proton magnetic resonance spectroscopy. The magnetic resonance method was also used to prove that the ester-linked linoleic acid is actually attached to the omega-hydroxyl group of the long chain hydroxyacid, not to the sugar as had been reported previously. A key spectral feature supporting this new structural assignment was a triplet at 3.82 ppm, which indicates methylene protons between another methylene and an ester linkage. After saponification, this signal moved to 3.33 ppm, a chemical shift expected for a methylene bearing a free hydroxyl group. Furthermore, all of the sugar ring protons could be accounted for both before and after acetylation. No evidence was found to suggest that an ester is attached to the sugar ring in the native material. It is concluded that the principal porcine epidermal acylglycosylceramide is 1-beta-D-glucosyl-N-(omega-O-linoleoyl)-triacontanoylsphingosine.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction

For the first time, the complete structure of the lipid A from the lipopolysaccharide of an Agrobacterium species is here reported. In particular, the structure of the lipid A from A. tumefaciens strain C58, a soil pathogen bacterium strictly related to Rhizobiaceae, was determined. The structural study, carried out by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy, revealed that lipid A fraction consisted of a mixture of species all sharing the bis-phosphorylated glucosamine disaccharide backbone that could be designated in two main structural motifs, according to the acylation pattern. The main species was a penta-acylated lipid A bearing two unsubstituted 14:0 (3-OH) fatty acids in ester linkage and two 16:0 (3-OH) in amide linkage; the one on GlcN II was O-acylated by a long chain fatty acid, 28:0 (27-OH). This in turn was esterified by a 3-hydroxy-butyroyl residue at its hydroxy group. The second species, in lesser amounts, was identified as a tetra-acylated lipid A and lacked the 14:0 (3-OH) residue on GlcN I. Other species deriving from these two lacked a phosphate group or 3-hydroxy-butyroyl residue or otherwise carried a 26:0 (25-OH) as long chain fatty acid. The lipid A structure of phytopathogen A. tumefaciens strain C58 presents deep structural analogies with lipid A of symbiotic Rhizobium, and the hypothesis is advanced that it can be a strategy of the bacterium to escape or attenuate the plant response.     

Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species.

Two strains of Bacteroides asaccharolyticus and two strains of Bacteroides fragilis were analyzed for total fatty acid, total lipid fatty acid, and total bound fatty acid profiles. Extracted lipids and defatted cell residues were subjected to sequential alkaline and acid methanolyses to distinguish ester- and amide-linked fatty acids in each fraction. In the lipid fractions, all the ester-linked fatty acids were nonhydroxylated, whereas all of the amide-linked fatty acids were hydroxylated. In the nonextractable fractions, both hydroxy and nonhydroxy fatty acids were found in both ester and amide linkage, although hydroxy acids predominated. The fatty acid profiles of the bound fractions differed widely from those of the lipid fractions. Bound fatty acid represented approximately 10% of the total cellular fatty acids.     

Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species

An analysis of the phospholipid ester-linked and the lipopolysaccharide (LPS) fatty acids and hydroxy fatty acids of six lactate-utilizing Desulfovibrio-type sulfate-reducing bacteria (SRB) has been performed using capillary gas-liquid chromatography-mass spectrometry (GLC-MS). The concentrations of normal fatty acids were essentially similar, with the possible exception of a high content of normal fatty acids in the LPS of Desulfovibrio gigas. Determination of monounsaturated acid double bond configuration was performed by GLC-MS analysis of the derivatized fatty acids. A total of nine branched chain and eight straight chain monounsaturated fatty acids was detected in the Desulfovibrio species analyzed. The major component detected in five Desulfovibrio was the 17-carbon iso-branched monoenoic acid which showed cis unsaturation [i17:1(n-7)c] seven carbons from the terminal methyl group of the fatty acid chain. D. gigas, in contrast, contained almost no unsaturated fatty acids and was greatly enriched in iso-branched 15:0. Major differences between strains were found in the phospholipid and LPS hydroxy fatty acids. These components, in addition to the i17:1(n-7)c and other characteristic branched chain unsaturated acids, can possibly be utilized as signatures of the lactate-utilizing SRB.     

Glycolipids of peripheral nerve: isolation and characterization of glycolipids from rabbit sciatic nerve

Besides cerebreside and sulfatide four other glycolipids were isolated from rabbit sciatic nerve and analyzed by chemical and chromatographic methods. Three of the glycolipids were shown to be fatty acid esters of cerebroside; the fourth was characterized as diacyl glycerol galactoside and its alkyl ether analog. In the ester linkage mainly unsubstituted acids with chain length C(16) to C(18) were present. Both hydroxy and unsubstituted acids were found in amide linkage. They varied in chain length from C(16) to C(24) and were typical of cerebrosides. The long-chain base fraction contained sphingosine and dihydrosphingosine as the main components.     

Nature, type of linkage, and absolute configuration of (hydroxy) fatty acids in lipopolysaccharides from Xanthomonas sinensis and related strains.

The fatty acids present in lipopolysaccharides from Xanthomonas sinensis were identified as decanoic, 9-methyl-decanoic, 2-hydroxy-9-methyl-decanoic, 2-hydroxy-9-methyl-decanoic, D-3-hydroxy-decanoic, D-3-hydroxy-9-methyl-decanoic, D-3-hydroxy-dodecanoic, and D-3-hydroxy-11-methyl-dodecanoic acid. These fatty acids occur in the lipid A component where they are bound through ester and amide linkages to glucosamine residues. All types of fatty acids are ester bound; however, part of D-3-hydroxy-dodecanoic and D-3-hydroxy-11-methyl-dodecanoic acid is also involved in amide linkage. The hydroxyl groups of ester-linked 3-hydroxy fatty acids are not substituted. Similar fatty acid patterns were obtained from lipopolysaccharides of nine other Xanthomonas species.     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.     

THE CHARACTERIZATION OF SPHINGOLIPIDS OF OLIGODENDROGLIA FROM CALF BRAIN

Abstract— Purified oligodendroglia isolated from bovine brain white matter were found to contain, in addition to galactosylceramide, sulfatide and sphingomyelin, significant quantities of glucosylcerai-mide, dihexosylceramide and esterified galactosylceramide. These sphingolipids were isolated and quan-titated and their fatty acid and long chain base patterns compared with those from sphingolipids isolated from bovine myelin, white matter and gray matter.
The minor glycosphingolipids, glucosylceramide, dihexosylceramide and esterified galactosylceramide, constituted a higher percentage of glial lipids than of myelin lipids. Glucosylceramide accounted for 12% of the total glial monohexosylceramide fraction and 0.8% of total lipids; dihexosylceramide was 0.9% of total glial lipids. Both of these lipids had small quantities of α-hydroxy fatty acids. The unsubstituted fatty acids of glucosylceramide were mostly short chain (16 and 18 carbons) and were different from those of the dihexosylceramides which were a mixture of short and long chain. The hydroxy acids of each of these lipids were, however, similar and resembled those of galactosylceramide.
The fatty acid patterns of galactosylceramide, sulfatide and sphingomyelin from glial cells resembled those of the corresponding lipids from myelin and white matter. The amide-linked acids of esterified galactosylceramide contained both unsubstituted and α-hydroxy chains. Their patterns were not identical to those of galactosylceramide, but were similar in all brain fractions.
With the exception of sphingomyelin and dihexosylceramide, which contained small amounts of C₂₀-sphingosine, all sphingolipids analyzed contained mostly sphingosine and dihydrosphingosine.
We conclude that the distribution of sphingolipids in the oligodendroglia is characteristic, but the lipophilic residues of these lipids are not cell-specific.     

Gangliosides of the starfish Aphelasterias japonica, evidence for a new linkage between two N-glycolylneuraminic acid residues through the hydroxy group of the glycolic acid residue

Mono- and disialogangliosides containing glucose, galactose and sialic acids were isolated from the total lipid extract of hepatopancreas of the starfish Aphelasterias japonica. Their structures were elucidated by total and partial acid hydrolysis, trideuteriomethylation analysis, neuraminidase treatment, chromium trioxide oxidation, methanolysis and periodate oxidation. The monosialoganglioside was identified as 8-O-methyl-N-glycolylneuraminosyl-alpha-(2-3)-galactosyl-beta-(1- 4)-glucosyl-beta-(1-1)-ceramide. The disialoganglioside has the additional N-glycolylneuraminic acid or its 8-O-methyl derivative residue at the subterminal position to which the terminal sialic acid residue is linked through the hydroxy group of the glycolic acid unit. The long-chain bases were found to be mixtures of phytosphingosines with both branched and linear chains, and the fatty acids were shown to be mixtures of normal and alpha-hydroxy fatty acids, the latter amounted to about 90% of the fatty-acid mixtures. The composition of the lipid moieties of the gangliosides was determined by GLC and GLC-MS.     

Phospholipid and lipopolysaccharide normal and hydroxy fatty acids as potential signatures for methane-oxidizing bacteria

Abstract The extractable ester-linked and the lipopolysaccharide (LPS) normal and hydroxy fatty acids of the methylotrophic bacteria Methylosinus trichosporium 0B3B, Methylobacterium organophilum XX, grown on methane and methanol, Mb. organophilum RG and Methylomonas sp. were analysed by capillary gas chromotography-mass spectrometry (GC-MS). Precise monounsaturated double bond position and geometry was determined by GC-MS analysis of the derivatized fatty acids. The three species were readily distinguished based on the extractable fatty acid and LPS hydroxy acid profiles. Type I and Type II methylotrophs can be separated based on the presence of 16-carbon and 18-carbon monoenoic fatty acids in the two groups of organisms, respectively. Relatively novel components, 18: 1ω8c, 18: 1ω8t, 18: 1ω7t and 18: 1ω6c were present in Ms. trichosporium , and 16: 1ω8c, 16: 1ω8t, 16: 1ω7t, 16: 1ω5c and 16: 1ω5t were detected in Methylomonas sp. These specific lipids may be used, together with other components, as signatures for these methylotrophic bacteria in manipulated laboratory and environmental samples.     

Existence of carotenoids in Acholeplasma axanthum.

The neutral lipids of Acholeplasma axanthum contain carotenoid pigments, as evidenced by spectral characteristics, visual color, color reactions, and labeling with [2-14C-A1mevalonic acid. Approximately 80% of the label from [2-14C]mevalonic acid appeared in esterified fatty acids of the glycolipids and polar lipids. These carboxylic acids behaved as hydroxy acids of varying chain length.     

Study of amphibian lipids. I. Characterization of monoglycosylceramides from the skin of Rana nigromaculata (Japanese pond frog).

1. Monoglycosylceramide was isolated from the skin of Rana nigromaculata (Japanese pond frog), and further fractionated into three subgroups (Fraction I, Fraction II and Fraction III) by borate-impregnated Florisil column chromatography. 2. Fraction I and Fraction II contained mainly glucose as their hexose components, while Fraction III contained galactose. 3. Major long chain bases of Fraction I and Fraction III were D-erythro-1, 3-dihydroxy-2-amino-4-trans-octadecene (4-sphingenine) and D-erythro-1, 3-dihydroxy-2-aminooctadecane (sphinganine), whereas those of Fraction II were D-ribo-1, 3, 4-trihydroxy-2-aminooctadecane (4D-hydroxysphinganine) and 1, 3, 4-trihydroxy-2-aminoeicosane (C20 homologues of 4D-hydroxysphinganine). This is the first evidence of the presence of trihydroxy base-containing glycolipids in the skin of vertebrates. 4. All three subgroups of monoglycosylceramide contained both hydroxy and nonhydroxy fatty acids ranging from C14 and C26. Saturated fatty acids represented more than 90% of the total. Some differences of the fatty acid composition in the three subgroups were also observed.     

A method for fractionation of cerebrosides into classes with different fatty acid compositions

A method is described for the separation of beef brain cerebrosides into three fractions containing different classes of fatty acids: nonhydroxy (I), unsaturated nonhydroxy (II), and hydroxy fatty acid cerebrosides (III). The procedure consists of benzoylation of either crude or purified cerebrosides, followed by column chromatographic separation of benzoylated derivatives containing nonhydroxy acids from those containing hydroxy fatty acids. The benzoyl groups are removed by sodium methoxide-catalyzed transesterification; from the reaction mixtures, fractions I and III precipitate. The fraction II present in mother liquor of I was shown to contain mainly short-chain and unsaturated nonhydroxy fatty acid cerebrosides. The fatty acid composition of each fraction was obtained by gas-liquid chromatography.     

Head‐group acylation of monogalactosyldiacylglycerol is a common stress response,and the acyl‐galactose acyl composition varies with the plant species and applied stress

Formation of galactose‐acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap‐freezing. Here, lipidomic analysis using mass spectrometry showed that galactose‐acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl‐galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl‐galactose components in response to the same stress. Additionally, the composition of the acyl‐galactose component of Arabidopsis acMGDG (galactose‐acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub‐lethal freezing treatment, acMGDG contained mainly non‐oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid‐containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose‐acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.     

Ornithine lipid of Mycobacterium tuberculosis: its distribution in some slow- and fast-growing mycobacteria

An ornithine-amide lipid is present in Mycobacterium tuberculosis. Its structure was established by a combination of chemical analysis and mass spectrometry. 3-Hydroxyoctadecanoic and 3-hydroxyeicosanoic acids (and homologues) were found to be linked through an amide bond to the alpha-amino group of L-ornithine, the hydroxyl group of the fatty acid being esterified mainly by tuberculostearic acid (10-methyloctadecanoic acid). This ornithine-amide lipid was detected in several other slow-growing pathogenic mycobacteria by thin layer chromatography, but not in an avirulent strain (H37 Ra) of M. tuberculosis. In each case mass spectrometry showed that all the structures were identical, thus revising an earlier reported structure for the lipid from M. bovis.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L2 and Chlamydophila psittaci 6BC.

The lipopolysaccharide (LPS) of Chlamydia trachomatis serotype E was isolated from tissue culture-grown elementary bodies and analyzed structurally by mass spectrometry and 1H, 13C and 31P nuclear magnetic resonance. The LPS is composed of the same pentasaccharide bisphosphate alphaKdo-(2-8)-alphaKdo-(2-4)-alphaKdo-(2-6)-betaGlcN-4P-(1-6)-alphaGlcN-1P (Kdo is 3-deoxy-alpha-d-manno-oct-2-ulosonic acid) as reported for C. trachomatis serotype L2[Rund, S., Lindner, B., Brade, H. and Holst, O. (1999) J. Biol. Chem. 274, 16819-16824]. The glucosamine disaccharide backbone is substituted with a complex mixture of fatty acids with ester or amide linkage whereby no ester-linked hydroxy fatty acids were found. The LPS was purified carefully (with contaminations by protein or nucleic acids below 0.3%) and tested for its ability to induce proinflammatory cytokines in several readout systems in comparison to LPS from C. trachomatis serotype L2 and Chlamydophila psittaci strain 6BC as well as enterobacterial smooth and rough LPS and synthetic hexaacyl lipid A. The chlamydial LPS were at least 10 times less active than typical endotoxins; specificity of the activities was confirmed by inhibition with the LPS antagonist, B1233, or with monoclonal antibodies against chlamydial LPS. Like other LPS, the chlamydial LPS used toll-like receptor TLR4 for signalling, but unlike other LPS activation was strictly CD14-dependent.     

Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals

The gram-negative metal-reducing microorganism, previously known as strain GS-15, was further characterized. This strict anaerobe oxidizes several short-chain fatty acids, alcohols, and monoaromatic compounds with Fe(III) as the sole electron acceptor. Furthermore, acetate is also oxidized with the reduction of Mn (IV), U (VI), and nitrate. In whole cell suspensions, the c-type cytochrome(s) of this organism was oxidized by physiological electron acceptors and also by gold, silver, mercury, and chromate. Menaquinone was recovered in concentrations comparable to those previously found in gram-negative sulfate reducers. Profiles of the phospholipid ester-linked fatty acids indicated that both the anaerobic desaturase and the branched pathways for fatty acid biosynthesis were operative. The organism contained three lipopolysaccharide hydroxy fatty acids which have not been previously reported in microorganisms, but have been observed in anaerobic freshwater sediments. The 16S rRNA sequence indicated that this organism belongs in the delta proteobacteria. Its closest known relative is Desulfuromonas acetoxidans. The name Geobacter metallireducens is proposed.     

Metabolism of hydroxy fatty acids in dogs with steatorrhea secondary to experimentally produced intestinal blind loops

Several aspects of the metabolism of hydroxy fatty acids were studied in dogs with steatorrhea resulting from an experimentally produced jejunal blind loop. In these animals hydroxy acids were present in the stool in amounts far above normal. These acids disappeared from the feces during tetracycline administration and after exclusion of the blind loop-both procedures that corrected the steatorrhea apparently by reducing bacterial overgrowth. Hydroxy acids persisted in higher than normal amounts, however, after administration of taurocholic acid, which also corrected the steatorrhea, but by a different mechanism. Both in normal dogs and in those with blind loops, hydroxy acid constituted a higher percentage of total fatty acids in the jejunum. A possible conclusion is that hydroxy fatty acids have an enterohepatic circulation via the portal system. When hydroxy acids were fed to normal dogs, steatorrhea was not produced and absorption in amounts similar to that of unsubstituted stearic acid was observed. Isotopic oleic and linoleic acids were converted to hydroxy acids both in vivo and during in vitro incubation with feces; stearic acid was not. These findings support the idea that hydroxy acids arise by the addition of water across double bonds, this addition being catalyzed by enzymes of intestinal bacteria.