Structure of mycoside F, a family of trehalose-containing glycolipids of Mycobacterium fortuitum

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, gas chromatography-mass spectrometry, as well as chemical degradations were used to elucidate the structure of the major glycolipids of Mycobacterium fortuitum. Three main glycoconjugates were detected and their structures established as 2,3-diacyl, 2,3,4- and 2,3,6-triacyl trehalose. The characteristic infrared spectrum which led to their original designation as mycoside F, a family of glycolipids limited in distribution to M. fortuitum, was due to the nature of the fatty acyl substiuents identified primarily as 2-methyl-octadecen-2-oyl. The antigenic glycolipids typified the biovar. fortuitum, thus allowing its easy recognition from the C-mycoside glycopeptidolipid-containing biovar. peregrinum.     

Characterization of the specific antigenicity of Mycobacterium fortuitum.

Mycobacterium fortuitum, biovar, fortuitum, the cause of serious skin and soft-tissue infections, can be differentiated from M. fortuitum, biovar. peregrinum, and other rapidly growing opportunistic mycobacteria by the presence of a unique antigenic glycolipid. The glycolipid is among the simplest of the acyl-trehalose-containing lipooligosaccharide class. The application of 1H and 13C NMR, methylation analysis, FAB/MS, and other procedures demonstrated the structure, beta-D-Glcp-(1----6)-2-O-acyl-alpha-D-Glcp-(1 in equilibrium with 1)-3,4,6-tri-O-acyl-alpha-D-Glcp. Thus, practically all environmental mycobacteria, many of them opportunistic pathogens, can be differentiated serologically and chemically on the basis of unique sugar arrangements within a few classes of glycolipids. The simplicity of the structure in M. fortuitum fortuitum combined with the distinct roughness of the parent strain raises the intriguing possibility that it is a spontaneous rough variant of the other mycobacteria with more elaborate glycolipids.     

Antigenic glycolipids of Mycobacterium fortuitum based on trehalose acylated with 2-methyloctadec-2-enoic acid

Three characteristic glycolipids were identified in extracts of 10 representative strains of Mycobacterium fortuitum. The two most polar major lipids were shown to be antigens with strong reactions against homologous antiserum and sera raised against Mycobacterium tuberculosis H37Rv. Both of these lipids were shown to be acyl trehaloses, containing substantial amounts of 2-methyloctadec-2-enoic acid in addition to straight-chain and monounsaturated acids. These lipids correspond to 'mycoside F', lipids previously identified by infra-red spectroscopy in extracts of Myco. fortuitum.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

New-found phenolic glycolipids in Mycobacterium bovis BCG. Presence of a diglycosylated glycolipid

A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.     

Mycolipenates and mycolipanolates of trehalose from mycobacterium tuberculosis

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25- and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.     

Novel bacterial polar lipids containing ether-linked alkyl chains,the structures and biological properties of the four major glycolipids from Propionibacterium propionicum PCM 2431 (ATCC 14157T)

Propionibacterium propionicum belongs to the "acnes group" of propionibacteria, which is currently considered as clinically important because of its growing potential in infections, in particular with those connected with immune system dysfunctions. Propionibacteria are thought to be actinomycete-like microorganisms and may still cause diagnostic difficulties. The chloroform-methanol extracts of the cell mass of P. propionicum (type strain) gave in TLC analysis the characteristic glycolipid profile containing four major glycolipids, labeled G(1) through G(4). These polar lipids were found to be useful chemotaxonomic markers to differentiate P. propionicum from other cutaneous propionibacteria, in particular from strains of the acnes group. Glycolipids G(1)-G(4) were isolated and purified using gel-permeation chromatography, TLC, and high performance liquid chromatography, and their structures were elucidated by compositional and methylation analyses, specific chemical degradations, MALDI-TOF mass spectrometry, and (1)H NMR and (13)C NMR spectroscopy, including HMBC, TOCSY, HMQC, and NOESY experiments. Glycolipids G(2) and G(3) possess as backbone alpha-d-Glcp-(1 --> 3)-alpha-d-Glcp-(1 --> 1)-Gro (Gro, glycerol), in which position O-2 of the glycerol residue is acylated by a fatty acid (mainly C(15):0) while O-3 is substituted by an alkyl ether chain. In glycolipid G(3), an additional fatty acyl chain was linked to O-6 of the terminal glucose residue. Glycolipid G(4) was structurally related to G(2) but devoid of one glucose residue. Glycolipid G(1) was isolated in small amounts, and its structure was therefore deduced from MALDI-TOF-MS experiments alone, which revealed that it possessed the structure of G(2) but was lacking one fatty acid residue. In studies on the biological properties of P. propionicum glycolipids, the anti-P. propionicum rabbit antisera reacted in dot enzyme-immunoblotting test with G(2) and G(3). Glycolipid G(3) was able to induce the delayed type of hypersensitivity. The results indicated that these novel ether linkage-containing polar glycolipids are immunogenic and possibly active in hypersensitivity, and thus, in pathogenesis.     

Confirmation of minor components of less polar neutral and acidic glycolipids in monkey brain tissue

Crude glycolipids, prepared without alkali treatment in advance, were separated into neutral and acidic glycolipids by DEAE-Sephadex A-25 (acetate form) column chromatography. Each glycolipid was further fractionated by a Silica gel 60-column chromatography. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS) of the intact glycolipid fractions, the less polar neutral glycolipids were found to contain alkali-labile ester cerebrosides and Galb-1-Diradylglycerols, whereas the less polar acidic glycolipids were found to contain alkali-labile ester sulfatide, HSO(3)-3Gal-1-Diradylglycerols, and novel alkali-stable plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols as minor components of glycolipids in monkey brain tissue.In conclusion, minor components of less polar neutral and acidic glycolipids in monkey brain tissue were confirmed as ester cerebrosides, Galb-1-Diradylglycerols, ester sulfatides, HSO(3)-3Galb-1-Diradylglycerols, and novel plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols by DE MALDI-TOF MS.     

The glycolipids of the causative agent of tularemia]

Up to 10 glycolipids were detected in F. tularensis with the use of thin-layer chromatographic techniques. These glycolipids were slime antigens of F. tularensis membrane. Attenuated F. tularensis strains were found to have defects in their glycolipid composition: in the vaccine strain glycolipid 8 was replaced by more polar lipid 8-a; the avirulent strain had only two glycolipids, and one of them was not typical for virulent strains. Considering that glycolipids differed from entero-bacterial Vi-antigen in their physical-chemical and biological properties, the suggestion was made that the use of the symbol "Vi" to denote the surface substances of F. tularensis should be abolished.     

Direct detection of Mycobacterium tuberculosis lipid antigens by thin-layer chromatography

Abstract Free lipids were extracted from Mycobacterium tuberculosis H37Rv, and their antigenicity was assessed directly on thin-layer chromatograms (TLC) by an immunostaining technique. A family of glycolipids, composed of trehalose acylated with multimethyl branched long-chain fatty acids, was investigated. The most polar of these glycolipids was identified as a possible specific surface antigen. A pair of novel polar glycolipids also showed positive antigenic reactions.     

Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii

As part of a study to identify novel lipids with immune adjuvant activity, a structural comparison was made between the polar lipids from two halophiles, an archaeon Haloferax volcanii and a eubacterium Planococcus H8. H. volcanii polar lipid extracts consisted of 44% archaetidylglycerol methylphosphate, 35% archaetidylglycerol, 4.7% of archaeal cardiolipin, 2.5% archaetidic acid, and 14% sulfated glycolipids 1 and 2. Nuclear magnetic resonance (NMR) and Fast atom bombardment mass spectrometry (FAB MS) data determined the glycolipids to be 6-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol] and a novel glycocardiolipin 6'-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-[sn-2,3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol]. The polar lipids of Planococcus H8 consisted of 49% saturated phosphatidylglycerol and cardiolipin (9:1, w/w), and surprisingly 51% of the photosynthetic membrane lipid sulfoquinovosyldiacylglycerol (SQDG). This study documents archaeal cardiolipin and a novel glycocardiolipin in H. volcanii (lacking purple membrane), and is the first report of SQDG in a non-photosynthetic, halophilic bacterium.     

Biological activity of polar lipids from bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Structural characterization of free glycolipids which are potential precursors for glycophosphatidylinositol anchors in mouse thymoma cell lines.

Biosynthesis of glycophosphatidylinositol-anchored membrane glycoproteins proceeds through the attachment of a preformed glycolipid onto a C-terminal amino acid rapidly after translation. Here we describe the structural analysis of two very polar glycolipids which can be observed after metabolic labeling of lymphoma cell lines S1A and EL-4 with either tritiated myo-inositol, mannose, or ethanolamine. These lipids are not made by mutant cells deficient in the biosynthesis of glycophosphatidylinositol anchors. The lipids were isolated, and their carbohydrate moiety was characterized using hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments, and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography. Results are compatible with the structure (X-->)Man alpha 1,2 Man alpha 1,6(Y-->)Man alpha-GlcN-acylinositol, X and Y being hydrofluoric acid-sensitive substituents (most likely phosphoethanolamine). The anchor oligosaccharide of the glycophosphatidylinositol protein anchors of S1A cells was isolated, similarly characterized, and found to contain the identical carbohydrate structure. Pulse-chase experiments indicate that the very polar glycolipids have half-lives which are much longer than the one of phosphatidylinositol. The results suggest that these very polar glycolipids represent supernumerary precursor glycolipids which did not get transferred onto proteins or represent processed forms of such precursors.     

Archaea contain a novel diether phosphoglycolipid with a polar head group identical to the conserved core of eucaryal glycosyl phosphatidylinositol.

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-GlcN. The polar head group was identified by means of chemical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucosaminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be 1-D-myo-inositol 1-phosphate by measuring optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. 1H NMR of the intact lipid showed that GlcN was linked to C-6 position of myo-inositol as an alpha-anomer. It is, finally, concluded that the complete structure of this lipid is 2,3-di-O-phytanyl-sn-glycero-1-phospho- 1'[6'-O-(2"-amino-2"-deoxy-alpha-D-glucopyranosyl)]-1'-D-myo-inositol. This lipid has a hybrid nature of an archaeal feature in alkyl glycerol diether core portion and an eucaryal feature in the polar head group identical to the conserved core structure (GlcNp(alpha 1-6)-myo-inositol 1-phosphate) of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eucaryal cells.     

Biological Activity of Polar Lipids from Bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Clostridium difficile contains plasmalogen species of phospholipids and glycolipids

Analysis of the polar lipids of many pathogenic and non-pathogenic clostridia has revealed the presence of plasmalogens, alk-1′-enyl ether-containing phospholipids and glycolipids. An exception to this finding so far has been Clostridium difficile, an important human pathogen which is the cause of antibiotic-associated diarrhea and other more serious complications. We have examined the polar lipids of three strains of C. difficile by thin-layer chromatography and have found acid-labile polar lipids indicative of the presence of plasmalogens. The lipids from one of these strains were subjected to further analysis by liquid chromatography coupled to electrospray ionization-mass spectrometry (LC/ESI-MS), which revealed the presence of phosphatidylglycerol, cardiolipin, monohexosyldiradylglycerol, dihexosyldiradylglycerol, and two unusual glycolipids identified as an aminohexosyl-hexosyldiradylglycerol, and a trihexosyldiradylglycerol. High resolution tandem mass spectrometry determined that monohexosyldiradylglycerol, cardiolipin and phosphatidylglycerol contained significant amounts of plasmalogens. C. difficile thus joins the growing list of clostridia that have plasmalogens. Since plasmalogens in clostridia are formed by an anaerobic pathway distinct from those in animal cells, their formation represents a potential novel target for antibiotic action.     

Polar glycosphingolipids in annelida. A novel series of glycosphingolipids containing choline phosphate from the earthworm, Pheretima hilgendorf.

A novel series of glycosphingolipids containing choline phosphate has been demonstrated in whole tissues of the earthworm, Pheretima hilgendorfi. The thin layer chromatographic pattern of the total polar glycolipids revealed the presence of more than three components with positive reactions toward orcinol-sulfuric acid (sugar), molybdate (phosphate), and Dragendorff's (choline) spray reagents. Two of these polar glycolipids (PGL1 and PGL2) were purified by the use of successive column chromatography on QAE-Sephadex A-25 and silicic acid (Iatrobeads) and detected during elution by the presence of galactose-bound choline phosphate. The structural elucidation of the oligosaccharide moieties was performed by compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and methylation analysis. Thus, the structures of PGL1 and PGL2 were deduced to be as follows: cholinephosphoryl-->6Gal beta 1-1Cer and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-1Cer. Although the oligosaccharide structures of both PGL1 and PGL2 have previously been found in other organisms, the presence of a choline phosphate group as an oligosaccharide substituent is the first finding in nature. The main molecular species of the ceramide moieties were composed of beheninyl- and lignocerinyloctadecasphingenines and their nonadecasphingenine homologues.     

Structures of polar lipids from the thermophilic, deep-sea archaeobacterium Methanococcus jannaschii

Cells of Methanococcus jannaschii, grown at 65 degrees C in a defined medium, contained 7% of lipid composed of 87% polar and 13% neutral components. Within the polar fraction 16 lipids were resolved by thin-layer chromatography, 4 of which were present in trace amounts. Staining reactions demonstrated that the more abundant lipids were glycolipids, aminophospholipids, and an aminophosphoglycolipid. Most of the polar fraction (82%) consisted of five diether lipids, which were purified and their structures were resolved largely through nuclear magnetic resonance, mass spectrometry, and optical rotation methods. Macrocyclic diethers had the head groups phosphoethanolamine-(1----6)-beta-D-glucopyranose, beta-D-glucopyranose, and beta-D-glucopyranosyl-(1----6)-beta-D-glucopyranose. Phosphoethanolamine was identified as a head group for both the noncyclized and macrocylic diether core lipids. The neutral lipids were mainly acyclic C30 isoprenoids, predominantly dihydro-, hexahydro, and octahydro-squalenes.     

Polar lipids in cosmetics: recent trends in extraction,separation, analysis and main applications

Demand for active, natural, safe and biomimetic (similar to human molecules) plantderived cosmetic ingredients is always greater because consumers are increasingly suspicious of the potential toxicity of current ingredients. In this context, interest has increased for polar lipids like ceramides, sphingolipids or glycolipids that share structural properties with the skin lipids. In the same manner, processes to obtain such lipids should be driven by the principles of green chemistry and sustainable development. The identified needs are biodegradability, biocompatibility, efficiency, quality and profitability. In this research for new and novel natural or ‘green’ compounds, the development of bioactive lipids thanks to ecofriendly processes has obviously intensified, especially for cosmetic and agro-food industries. This paper reviews extraction methods for polar lipids (glycolipids and phospholipids), especially ecoprocesses (supercritical fluid extraction, microwaves, sonication, enzyme extraction…), and promising chromatographic methods like countercurrent chromatography, supercritical fluid chromatography or high performance chromatography coupled to mass spectrometry Interests of polar lipids for cosmetic industries are exemplified to show their broad applications mostly relying on their amphiphilic properties allowing them to play functional roles (liposome or micelle structures for example) or physiological roles (skin barrier function or anti-ageing effect).     

Blood group A glycolipid antigen expression in kidney, ureter, kidney artery, and kidney vein from a blood group A1Le(a-b+) human individual. Evidence for a novel blood group A heptaglycosylceramide based on a type 3 carbohydrate chain

Kidney, ureter, kidney artery, and kidney vein tissue were obtained from a single human transplant specimen. The donors erythrocyte blood group phenotype was A1Le(a-b+). Total non-acid glycolipid fractions were isolated and individual glycolipid components were identified by immunostaining thin layer plates with a panel of monoclonal antibodies and by mass spectrometry of the permethylated and permethylated-reduced total glycolipid fractions. The dominating glycolipids in all tissues were mono- to tetraglycosylceramides. In the kidney, ureter, and artery tissue less than 1% of the glycolipids were of blood group type, having more than 4 sugar residues. In contrast, 14% of the vein glycolipids were of blood group type, and the dominating components were type 1 chain blood group H pentaglycosylceramides and A hexaglycosylceramides. Trace amounts of structurally different blood group A glycolipids (type 1 to 4 core saccharide chains) with up to 10 sugar residues were found in the kidney, ureter, and vein tissues, including evidence for a novel blood group A heptaglycosylceramide based on the type 3 chain in the vein. The only detected A glycolipid antigen in the artery tissue was the blood group A difucosyl type 1 chain heptaglycosylceramide (ALeb) structure. Blood group Lewis and related antigens (Lea, Leb, and ALeb) were expressed in the kidney, ureter, and artery, but were completely lacking in the vein, indicating that the Le gene-coded alpha 1-4-fucosyltransferase was not expressed in this tissue. The X and Y antigens (type 2 chain isomers of the Lea and Leb antigens) were detected only in the kidney tissue.