Phosphoethanolamine Residues Research Articles

The importance of side branches of glycosylphosphatidylinositol anchors: a molecular dynamics perspective.

Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors. These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated. GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others. All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers. Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent. This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses. Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer. This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases.

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2.

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.

Immunochemical characterization of the O antigens of two Proteus strains, O8-related antigen of Proteus mirabilis 12 B-r and O2-related antigen of Proteus genomospecies 5/6 12 B-k, infecting a hospitalized patient in Poland.

A hospitalized 73-year-old woman was infected with a Proteus mirabilis strain, 12 B-r, isolated from the place of injection of a blood catheter. Another strain, 12 B-k, recognized as Proteus genomospecies 5 or 6, was isolated from the patient's faeces, which was an example of a nosocomial infection rather than an auto-infection. Serological investigation using ELISA and Western blotting showed that strain 12 B-k from faeces belonged to the Proteus O2 serogroup. Strain 12 B-r from the wound displayed cross-reactions with several Proteus O serogroups due to common epitopes on the core or O-specific parts of the lipopolysaccharide. Studies of the isolated 12 B-r O-specific polysaccharide by NMR spectroscopy revealed its close structural similarity to that of Proteus O8. The only difference in 12 B-r was the presence of an additional GlcNAc-linked phosphoethanolamine residue, which creates a putative epitope responsible for the cross-reactivity with Pt. mirabilis O16. The new O-antigen form could appear as a result of adaptation of the bacterium to a changing environment. On the basis of the data obtained, we suggest division of the O8 serogroup into two subgroups: O8a for strains of various Proteus species that have been previously classified into the O8 serogroup, and O8a,b for Pt. mirabilis 12 B-r, where 'a' is a common epitope and 'b' is a phosphoethanolamine-associated epitope. These findings further confirm serological and structural heterogeneity of O antigens of Proteus strains isolated lately from patients in Poland.

More Than Just Oligomannose: An N-glycomic Comparison of Penicillium Species

N-glycosylation is an essential set of post-translational modifications of proteins; in the case of filamentous fungi, N-glycans are present on a range of secreted and cell wall proteins. In this study, we have compared the glycans released by peptide/N-glycosidase F from proteolysed cell pellets of three Penicillium species (P. dierckxii, P. nordicum and P. verrucosum that all belong to the Eurotiomycetes). Although the major structures are all within the range Hex(5-11)HexNAc(2) as shown by mass spectrometry, variations in reversed-phase chromatograms and MS/MS fragmentation patterns are indicative of differences in the actual structure. Hydrofluoric acid and mannosidase treatments revealed that the oligomannosidic glycans were not only in part modified with phosphoethanolamine residues and outer chain och1-dependent mannosylation, but that bisecting galactofuranose was present in a species-dependent manner. These data are the first to specifically show the modification of N-glycans in fungi with zwitterionic moieties. Furthermore, our results indicate that mere mass spectrometric screening is insufficient to reveal the subtly complex nature of N-glycosylation even within a single fungal genus.

Structure and Immunogenicity of the Rough-Type Lipopolysaccharide from the Periodontal Pathogen Tannerella forsythia

Tannerella forsythia is a Gram-negative anaerobic organism that inhabits subgingival plaque biofilms and is covered with a so far unique surface layer composed of two glycoproteins. It belongs to the so-called "red complex" of bacteria comprising species that are associated with periodontal disease. While the surface layer glycoprotein glycan structure had been elucidated recently and found to be a virulence factor, no structural data on the lipopolysaccharide (LPS) of this organism were available. In this study, the T. forsythia LPS structure was partially elucidated by a combined mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) approach and initial experiments to characterize its immunostimulatory potential were performed. The T. forsythia LPS is a complex, rough-type LPS with a core region composed of one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue, three mannose residues, and two glucosamine residues. MS analyses of O-deacylated LPS proved that, in addition, one phosphoethanolamine residue and most likely one galactose-phosphate residue were present, however, their positions could not be identified. Stimulation of human macrophages with T. forsythia LPS resulted in the production of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in a dose-dependent manner. The response to T. forsythia LPS was observed only upon stimulation in the presence of fetal calf serum (FCS), whereas no cytokine production was observed in the absence of FCS. This finding suggests that the presence of certain additional cofactors is crucial for the immune response induced by T. forsythia LPS.

Investigating the candidacy of lipopolysaccharide-based glycoconjugates as vaccines to combat Mannheimia haemolytica

Inner core lipopolysaccharide (LPS) has been shown to be conserved in the majority of veterinary strains from the species Mannheimia haemolytica, Actinobacillus pleuropneumoniae and Pasteurella multocida and as such is being considered as a possible vaccine antigen. The proof-in-principle that a LPS-based antigen could be considered as a vaccine candidate has been demonstrated from studies with monoclonal antibodies raised to the inner core LPS of Mannheimia haemolytica, which were shown to be both bactericidal and protective in a mouse model of disease. In this study we confirm and extend the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against Mannheimia haemolytica wild-type strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes a conjugation strategy that uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule. To protect the amino functionality on the phosphoethanolamine (PEtn) residue of the inner core, we developed a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy with the thiol linker on the carboxyl residues of the carrier protein and the maleimide linker on the carbohydrate resulted in a high loading of carbohydrates per carrier protein. Immunisation derived antisera from rabbits recognised fully extended Mannheimia haemolytica LPS and whole cells from serotypes 1 and 2, despite a somewhat immunodominant response to the linkers also being observed. Moreover, bactericidal activity was demonstrated to a strain elaborating the immunising carbohydrate antigen and crucially to wild-type cells of serotypes 1 and 2, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by Mannheimia haemolytica.

Synthesis of phosphorylated 3,4-branched trisaccharides corresponding to LPS inner core structures of Neisseria meningitidis and Haemophilus influenzae

2-( N-Benzyloxycarbonyl)aminoethyl 7- O-acetyl-6- O-allyl-2- O-benzoyl-4- O-benzyl-3- O-chloroacetyl- l- glycero-α- d- manno-heptopyranosyl-(1→3)-[2,3,4,6-tetra- O-benzoyl-β- d-glucopyranosyl-(1→4)]-6,7-di- O-acetyl-2- O-benzyl- l- glycero-α- d- manno-heptopyranoside, a spacer-equipped protected derivative of the common 3,4-branched diheptoside trisaccharide structure of the lipopolysaccharide core of Neisseria meningitidis and Haemophilus influenzae has been synthesized. The protecting group pattern installed allows regioselective introduction of phosphoethanolamine residues in the 3- and 6-position of the second heptose unit in accordance with native structures. From this intermediate the 3-and 6-monophosphoethanolamine as well as the non-phosphorylated deprotected trisaccharides have been synthesized to be used in evaluation of antibody binding specificity and in investigation of the substrate specificity of glycosyl transferases involved in the biosynthesis of LPS core structures.

Phosphoethanolamine is located at the 6-position and not at the 7-position of the distal heptose residue in the lipopolysaccharide from Neisseria meningitidis

Previous studies on LPS from Neisseria meningitidis strains M992B, the immunotype L6 strain, NMB, the type strain, a candidate LPS vaccine strain 6275z, and an extensively used clinical strain M986 had suggested that the location of the phosphoethanolamine (PEtn) residue was the 7-position of the distal heptose residue (HepII) of the inner-core oligosaccharide (OS). In all cases, this was only established by chemical methods, methylation linkage analyses. In this study, we have used standard NMR techniques to unequivocally show that the PEtn residue is actually located at the 6-position and not at the 7-position of the HepII residue in all of these strains. The 6-PEtn transferase genes were sequenced and their translated amino acid sequences were shown to be greater than 96% identical to that of the Lpt6 transferase from the L4 immunotype strain, which has been shown to transfer PEtn to the 6-position of the distal heptose residue. We discuss the implications of these findings with respect to the immunotyping scheme for the meningococci and in the context of LPS-based vaccine development.

The Fine Structure of Neisseria meningitidis Lipooligosaccharide from the M986 Strain and Three of Its Variants

Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose alpha-chain. The gamma-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl alpha-chain even though previous reports suggested that the presence of an extended alpha-chain hinders C3 PEA substitution of Hep II. Approximately 50% of gamma-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and gamma-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.

Structural Requirements for Monoclonal Antibody 2-1-L8 Recognition of Neisserial Lipooligosaccharides

Monoclonal antibodies (MAbs) that bind neisserial lipooligosaccharides (LOS) have been widely used in structural studies of these glycolipids. MAb 2-1-L8 binds LOS with a lactosyl a chain (Gal beta1-4 Glc beta1-4 [Glc-NAc alpha1-2 Hep2 alpha1-3] Hep1 alpha1-KDO) and at least one phosphoethanolamine (PEA) substitution of Hep2, but the requirement for PEA substitution and/or the exact position of this substitution, cyclic or exocyclic, remains unclear. In order to clarify the exact specificity of this MAb, we engineered an isogenic family of lpt mutants that each make LOS with a lactosyl a chain, but that lacked cyclic (-3Hep2), exocyclic (-6Hep2), or any PEA residues. Mass spectrometry showed that mutants that lack either Lpt3 or Lpt6 make small amounts of LOS with two PEA substitutions. Thus, each enzyme is able to phosphoethanolaminylate the alternate site, albeit with low efficiency. LOS made by the mutant that lacked both Lpt3 and Lpt6 was devoid of PEA. LOS made by the deltalpt3 mutant did not bind MAb 2-1-L8 on Western blot analysis, whereas delta pt6 LOS did. Analysis of intact mutants by fluorescence-activated cell sorting confirmed that PEA substitution at 3Hep2, but not at 6Hep2, is needed for optimal binding of MAb 2-1-L8. These data confirm that the MAb 2-1-L8 epitope requires a -3Hep2 cyclic PEA substitution for optimal conformation and that this MAb specifies the PEA-3Hep2 lactosyl LOS structure.

The Lipid A 1-Phosphatase ofHelicobacter pyloriIs Required for Resistance to the Antimicrobial Peptide Polymyxin

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

Glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

GPI-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT), a penta-subunit membrane-bound enzyme. The enzyme recognizes a C-terminal signal sequence in the pro-protein and replaces it with a preformed GPI lipid. We developed detergent-solubilization conditions that permit detection of interactions between GPIT and its GPI and pro-protein substrates in pull-down assays. Using these procedures we showed that the polytopic Gaa1 subunit of GPIT is functionally important for recognition of GPI by the enzyme (JBC 279: 6540–6545 (2004)). We now extend these studies to establish the structural features of GPI that are required for GPI::GPIT interaction in the pull down assay. Our results indicate that GPI structures must contain a single phospho-ethanolamine (P-EtN) residue anywhere on the mannose core in order to be recognized by GPIT. Thus GPI anchor precursors such as H7 (EtN-P-6Manα1,2Manα1-6(EtN-P-2)Manα1,4GlcN-acyl PI) and even P-EtN-containing GPI molecules that cannot serve as protein anchors such as H6 (Manα1,2Manα1-6(EtN-P-2)Manα1,4GlcN-acyl PI) are pulled down by GPIT, whereas GPIs such as H4 (Manα1,2Manα1-6Manα1,4GlcN-acylPI) that lack P-EtN are not. The implications of these data will be discussed. Supported by NIH GM55427.

Structural analysis of the oligosaccharide of Histophilus somni ( Haemophilus somnus) strain 2336 and identification of several lipooligosaccharide biosynthesis gene homologues

The structure of the core oligosaccharide from a pneumonic Histophilus somni ( Haemophilus somnus) strain 2336 was elucidated. The lipooligosaccharide (LOS) was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core oligosaccharide was determined on the basis of the combined data from these experiments: ▪ The structural elucidation was intriguing as it suggested several differences in the LOS structures between strain 2336 and the related strain 738. Strain 738 originated following passaging of strain 2336 through a calf. The differences between the two structures are a different linkage between Gal II and GlcNAc (1→4 here; 1→3 in 738), the absence of phosphocholine (PCho) from 2336 and the presence of two phosphoethanolamine (PEtn) residues and Gal III (at the 2-position) of Hep II in 2336. Although pulse-field gel electrophoresis data following digest with only one restriction enzyme showed identical profiles suggesting that strains 738 and 2336 are the same strain, the structural data does suggest that, if strain 738 is indeed a phase variant of strain 2336, considerable variation occurred on calf passaging and could therefore be an intriguing example of how broadly this bacterium can adapt itself in the host.

Neisserial Lipooligosaccharide Is a Target for Complement Component C4b: INNER CORE PHOSPHOETHANOLAMINE RESIDUES DEFINE C4b LINKAGE SPECIFICITY

We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement.

Phosphorylation of the lipid A region of meningococcal lipopolysaccharide: identification of a family of transferases that add phosphoethanolamine to lipopolysaccharide.

A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A."

Immunohistochemical localization and differentiation of phosphocholine-containing antigens of the porcine, parasitic nematode, Ascaris suum.

The glycolipids of Ascaris suum represent either neutral, zwitterionic or acidic structures. The acidic fraction comprises a sulphatide and an unusual phosphoinositolglycosphingolipid (Lochnit et al. 1998b). The sulphatide was previously localized to the hypodermis, contractile zone of somatic muscle cells and the external musculature of the female uterus, whereas the presence of the phosphoinositolglycosphingolipid species was restricted to the intestine. The neutral and zwitterionic components belong to the arthro-carbohydrate series, which are substituted in their zwitterionic structures by phosphocholine (PC) and in one glycolipid by an additional phosphoethanolamine residue. In previous immunohistochemical localization studies, however, the chemical nature of the PC-substituted biomolecules has not been investigated in detail. Here, we report on the immunohistochemical localization and differentiation of phosphocholine-containing structures into lipid- and protein-bound species in adult A. suum. The patterns of immunostaining, obtained with a PC-specific monoclonal antibody and anti-zwitterionic glycolipid hyperimmune serum in the female worm, indicated a parallel organ distribution for glycolipid- and protein-bound PC-epitopes. Immunoreactivity was localized to specific tissues of the body wall, intestine and reproductive tract. This is the first report of surface-located PC-epitopes for ascarids. The patterns of immunolabelling obtained with antibodies directed against the unsubstituted arthro-carbohydrate series backbone suggested that the glycolipid-bound epitope was restricted to the hypodermis, whilst the protein-bound antigenic determinant resembled that for PC.

Glycosphingolipids in insects

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two-dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica-gel column chromatography and characterized by the presence of a N-acetyl-glucosamine-bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing-4-enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as: (PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1- 4)Glc beta Cer; Gal(beta 1-3)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1- 3)Man(beta 1-4)Glc beta Cer; GlcNAc(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn- 6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer.

Characterization of Two Glucuronic Acid-Containing Glycosphingolipids in Larvae of the Green-Bottle Fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.

Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds. MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties. The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking. We now report that phosphatidylethanolamine, exogenously added to intact cells of E. coli, provides a source of phosphoethanolamine residues that are transferred to MDO. The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.

Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium.

Temperature-sensitive mutants of Salmonella typhimurium that are defective in the biosynthesis of 3-deoxy-D-manno-octulosonate are known to accumulate disaccharide precursor(s) of lipid A at 42 degrees C (Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912). We have devised new methods for purifying this material by chromatography on DEAE-cellulose and silicic acid columns and have fractionated it into eight related anionic components that fall into four sets, as judged by their charge. Substances IA and IB have an apparent net charge of -1, IIA and IIB of -2, IIIA and IIIB of -3, and IVA and IVB of -4. Negative ion fast atom bombardment mass spectrometry reveals that the simplest component is IVA [( M - H]- at m/z 1404). Compound IVA is also the most abundant, representing 30-50% of the accumulated lipids after 3 h at 42 degrees C. Structural studies of IVA, including NMR spectroscopy described in the accompanying paper, reveal that it consists of O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-alpha - D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl moieties and bearing phosphate groups at positions 1 and 4'. Compound IIIA ([M - H]- at m/z 1527) contains an additional phosphoethanolamine residue, while IIA ([M - H]- m/z 1535) bears an aminodeoxypentose substituent, presumably 4-amino-4-deoxy-L-arabinose. Compound IA ([M - H]- at m/z 1658) bears both a phosphoethanolamine and an aminodeoxypentose. The compounds of the less abundant B series are further derivatized with an ester-linked palmitoyl moiety. Our results demonstrate that these precursors are far more heterogeneous than previously suspected.