Phosphoethanolamine Group Research Articles

Identification of a phosphoethanolamine transferase for lipid A modification in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a foodborne pathogen and naturally resistant to polymyxins, the last line of therapy for infections caused by multidrug-resistant bacteria. Polymyxins resistance is typically facilitated by the modification of phosphoethanolamine group on the lipid A of lipopolysaccharide in Gram-negative bacteria. In this study, we found that VP_RS21300 encodes a phosphoethanolamine transferase in V. parahaemolyticus ATCC33846. Deletion of VP_RS21300 in ATCC33846 resulted in the defection of phosphoethanolamine addition to lipid A. Furthermore, VP_RS21300 could recover the modification of phosphoethanolamine to lipid A in E. coli MG1655ΔeptA. In addition, when mouse RAW264.7 cells were infected by ATCC33846 and ATCC33846ΔRS2130, and the survival rate of ATCC33846ΔRS21300 was much higher than that of ATCC33846. The results suggest that VP_RS21300 encodes a phosphoethanolamine transferase for lipid A in V. parahaemolyticus and plays an important role on polymyxins resistance and pathogenicity.

Dissecting the Molecular Mechanism of Colistin Resistance in mcr-1 Bacteria.

Colistin or polymyxin B is the last resort antibiotic to treat infections of multidrug-resistant Gram-negative bacteria by disrupting their outer membranes. The recent emergence of Gram-negative bacteria that demonstrate colistin resistance, particularly plasmid-mediated mobile colistin resistance (mcr), poses a big challenge to the treatment of multidrug resistance infections. Using molecular dynamics simulations, we explore the mechanism of colistin resistance in a model lipid A bilayer mimicking the Gram-negative mcr-1 bacterial outer membrane. The simulation results reveal that the outer membrane of normal Gram-negative bacteria is stabilized by salt bridges between positively charged divalent ions and negatively charged phosphate groups of the membranes. In the presence of positively charged polymyxin B, these salt bridges are disrupted, and calcium is released into the aqueous phase, resulting in membrane disruption. In contrast, the lipid A in the outer membrane of mcr-1 bacteria has a novel modification, this being a covalently attached phosphoethanolamine group. This group enables the formation of a large number of hydrogen bonds between the amine and phosphate groups, resulting in an electrostatic net on the membrane. This extensive noncovalent electrostatic cross-linking between the lipid molecules collectively enhances the membrane stability and results in resistance to the action of cationic peptides such as polymyxin B. The simulation results shed new atomistic insights for understanding the mechanistic basis of colistin resistance and provide clues for the design of new membrane disruptors and permeabilizers to treat mcr-1 infections.

Oral administration of a synthetic vinyl-ether plasmalogen normalizes open field activity in a mouse model of rhizomelic chondrodysplasia punctata.

ABSTRACTRhizomelic chondrodysplasia punctata (RCDP) is a rare genetic disorder caused by mutations in peroxisomal genes essential for plasmalogen biosynthesis. Plasmalogens are a class of membrane glycerophospholipids containing a vinyl-ether-linked fatty alcohol at the sn-1 position that affect functions including vesicular transport, membrane protein function and free radical scavenging. A logical rationale for the treatment of RCDP is therefore the therapeutic augmentation of plasmalogens. The objective of this work was to provide a preliminary characterization of a novel vinyl-ether synthetic plasmalogen, PPI-1040, in support of its potential utility as an oral therapeutic option for RCDP. First, wild-type mice were treated with 13C6-labeled PPI-1040, which showed that the sn-1 vinyl-ether and the sn-3 phosphoethanolamine groups remained intact during digestion and absorption. Next, a 4-week treatment of adult plasmalogen-deficient Pex7hypo/null mice with PPI-1040 showed normalization of plasmalogen levels in plasma, and variable increases in plasmalogen levels in erythrocytes and peripheral tissues (liver, small intestine, skeletal muscle and heart). Augmentation was not observed in brain, lung and kidney. Functionally, PPI-1040 treatment normalized the hyperactive behavior observed in the Pex7hypo/null mice as determined by open field test, with a significant inverse correlation between activity and plasma plasmalogen levels. Parallel treatment with an equal amount of ether plasmalogen precursor, PPI-1011, did not effectively augment plasmalogen levels or reduce hyperactivity. Our findings show, for the first time, that a synthetic vinyl-ether plasmalogen is orally bioavailable and can improve plasmalogen levels in an RCDP mouse model. Further exploration of its clinical utility is warranted.This article has an associated First Person interview with the joint first authors of the paper.

Genetic and Functional Characterization of an MCR-3-Like Enzyme-Producing Escherichia coli Isolate Recovered from Swine in Brazil.

A collection of 126 pigs was screened for carriage of colistin-resistant Enterobacteriaceae in a farm in Minas Gerais, Brazil. Out of this collection, eight colistin-resistant Escherichia coli isolates were recovered, including one from Minas Gerais State producing a new MCR-3 variant (MCR-3.12). Analysis of the lipopolysaccharide revealed that MCR-3.12 had a function similar to that of MCR-1 and MCR-2 as a result of the addition of a phosphoethanolamine group to the lipid A moiety. Genetic analysis showed that the mcr-3.12 gene was carried by an IncA/C2 plasmid and was embedded in an original genetic environment. This study reports the occurrence of the MCR-3-like determinant in South America and is the first to demonstrate the functionality of this group of enzymes as a phosphoethanolamine transferase.

Bacteria make modified cellulose

Cellulose is an abundant biopolymer made of strands of β-1,4-linked glucose units. It provides mechanical strength to plants and biofilms made by bacteria. A team led by Lynette Cegelski of Stanford University and Regine Hengge of Humboldt University of Berlin now reports that the cellulose made by bacteria is a chemically modified version (Science 2018, DOI: 10.1126/science.aao4096). Using solid-state nuclear magnetic resonance spectroscopy to analyze the structure of the cellulosic material, the researchers determined that it contains a phosphoethanolamine group (shown in blue) attached to the C6 position of a glucose ring in the repeating unit. The modification had not been observed before because standard HCl hydrolysis degrades the modification, leaving only glucose, glucose-6-phosphate, and ethanolamine. The researchers also identified the genes behind the modification. They propose that a protein known as BcsG acts as an enzyme that installs phosphoethanolamine and that two other proteins in the sa...

Structure of an endotoxin modifying enzyme and virulence factor in Neisseria

Multiple drug resistance (MDR) in Gram-negative bacteria represents one of the most intractable problems facing modern medicine. Not only is antibiotic resistance incrementally increasing during clinical treatment of infections, but also the evolution and acquisition of new mechanisms of antibiotic resistance lead to the sudden loss of the capacity to treat infections. The most recent superbug, MDR-Neisseria gonorrhoeae, causes the untreatable sexually transmitted infection gonorrhoeae. Chronic gonococcal infections have a high morbidity rate and, due to the explosion in cases worldwide, the community burden is enormous. N. gonorrhoeae colonizes the mucosal surfaces of the human body and has a number of virulence mechanisms that prevent clearance by the human immune system. The most important of these mechanisms is decoration of the lipooligosaccharide lipid A headgroups with phosphoethanolamine (PEA) by the enzyme, lipid A PEA transferase (LptA). Inactivation of the LptA results in the complete loss of PEA groups from lipid A, loss of bacterial colonisation of epithelial cells (Takahashi et al., 2008), increased sensitivity to cationic antimicrobial peptides (Tzeng et al., 2005) and reduced resistance to human complement mediated killing (Lewis et al., 2013). LptA knockouts of N. gonorrhoeae also result in the complete loss of virulence in models of human and mouse infections. Based on these findings we have undertaken a structure-guided approach to develop inhibitors of LptA that will assist in controlling infection and transmission by this important human pathogen. LptA is a membrane protein that interacts with two different lipid substrates. We have determined the crystal structure of the enzyme to 2.75Å resolution. The structure provides insights into the mechanism of substrate binding and catalysis and suggests that significant conformational changes occur through its catalytic cycle.

Dissemination and serotype modification potential of pSFxv_2, an O-antigen PEtN modification plasmid in Shigella flexneri

The O-antigens of all Shigella flexneri serotypes, except serotype 6, share a linear tetrasaccharide repeat composed of one N-acetylglucosamine and three l-rhamnose residues, and differences between the serotypes are due to modification of various monosaccharide residues with glucosyl and/or O-acetyl and/or phosphoethanolamine (PEtN) groups. Plasmid-borne opt (formerly lpt-O) gene encoding a PEtN transferase which modifies the O-antigens of S. flexneri serotype X, 4a and Y strains and converts the hosts into MASF IV-1 (E1037) positive "variant" (v) Xv, 4av and Yv serotypes, respectively. In this study, we showed that the opt-carrying plasmid pSFxv_2 can transform strains of all S. flexneri serotypes (1-6) to confer them with the MASF IV-1 epitope recognized by monoclonal antibody MASF IV-1 and typing antiserum IV. The transformants possessed modified O-antigens with a PEtN group(s) at position 3 of one or two rhamnose residues. In some serotypes, the PEtN modification competed or/and interfered with glucosylation and O-acetylation at the same or its neighboring sugar residue. We also showed that the plasmid pSFxv_2 is mobilizable to other S. flexneri strains by conjugation. Although pSFxv_2-harboring S. flexneri strains found in clinical infections are restricted to serotypes Xv, 4av, Yv and, possibly, 6v, our results demonstrate a high potential of dissemination of this plasmid in S. flexneri and emergence of new S. flexneri serotypes.

A Novel Plasmid-Encoded Serotype Conversion Mechanism through Addition of Phosphoethanolamine to the O-Antigen of Shigella flexneri

Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O–antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.

Characterization of lipid A profiles from Shigella flexneri variant X lipopolysaccharide

In developing countries, Shigella flexneri (Sf) is the major causative agent of the endemic shigellosis (bacillary dysentery) responsible annually for one million fatalities mostly among infants. Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. Since lipid A is essential for the viability of the Gram-negative bacteria, it is subject to extensive chemical studies with new analytical techniques. Lipid A was released by mild acid hydrolysis from the lipopolysaccharide which was obtained via the phenol/water extraction, purified and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and matrix-assisted laser desorption/ionization laser-induced dissociation tandem mass spectrometry (MALDI-LID-MS/MS). A detailed structural study of the whole lipid A obtained from S. flexneri variant X was carried out for the first time. Thus, we have shown that lipid A is a heterogeneous mixture having different numbers of acylated and phosphoethanolamine groups attached to the diglucosamine backbone. Furthermore, we found in the phenol phase an unusual hepta-acylated lipid A species, although the abundance was very low. MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.

Oral bioavailability of the ether lipid plasmalogen precursor, PPI-1011, in the rabbit: a new therapeutic strategy for Alzheimer's disease

IntroductionDocosahexaenoic acid (DHA) and DHA-containing ethanolamine plasmalogens (PlsEtn) are decreased in the brain, liver and the circulation in Alzheimer's disease. Decreased supply of plasmalogen precursors to the brain by the liver, as a result of peroxisomal deficits is a process that probably starts early in the AD disease process. To overcome this metabolic compromise, we have designed an orally bioavailable DHA-containing ether lipid precursor of plasmalogens. PPI-1011 is an alkyl-diacyl plasmalogen precursor with palmitic acid at sn-1, DHA at sn-2 and lipoic acid at sn-3. This study outlines the oral pharmacokinetics of this precursor and its conversion to PlsEtn and phosphatidylethanolamines (PtdEtn).MethodsRabbits were dosed orally with PPI-1011 in hard gelatin capsules for time-course and dose response studies. Incorporation into PlsEtn and PtdEtn was monitored by LC-MS/MS. Metabolism of released lipoic acid was monitored by GC-MS. To monitor the metabolic fate of different components of PPI-1011, we labeled the sn-1 palmitic acid, sn-2 DHA and glycerol backbone with13C and monitored their metabolic fates by LC-MS/MS.ResultsPPI-1011 was not detected in plasma suggesting rapid release of sn-3 lipoic acid via gut lipases. This conclusion was supported by peak levels of lipoic acid metabolites in the plasma 3 hours after dosing. While PPI-1011 did not gain access to the plasma, it increased circulating levels of DHA-containing PlsEtn and PtdEtn. Labeling experiments demonstrated that the PtdEtn increases resulted from increased availability of DHA released via remodeling at sn-2 of phospholipids derived from PPI-1011. This release of DHA peaked at 6 hrs while increases in phospholipids peaked at 12 hr. Increases in circulating PlsEtn were more complex. Labeling experiments demonstrated that increases in the target PlsEtn, 16:0/22:6, consisted of 2 pools. In one pool, the intact precursor received a sn-3 phosphoethanolamine group and desaturation at sn-1 to generate the target plasmalogen. The second pool, like the PtdEtn, resulted from increased availability of DHA released during remodeling of sn-2. In the case of sn-1 18:0 and 18:1 plasmalogens with [13C3]DHA at sn-2, labeling was the result of increased availability of [13C3]DHA from lipid remodeling. Isotope and repeated dosing (2 weeks) experiments also demonstrated that plasmalogens and/or plasmalogen precursors derived from PPI-1011 are able to cross both the blood-retinal and blood-brain barriers.ConclusionsOur data demonstrate that PPI-1011, an ether lipid precursor of plasmalogens is orally bioavailable in the rabbit, augmenting the circulating levels of unesterified DHA and DHA-containing PlsEtn and PtdEtn. Other ethanolamine plasmalogens were generated from the precursor via lipid remodeling (de-acylation/re-acylation reactions at sn-2) and phosphatidylethanolamines were generated via de-alkylation/re-acylation reactions at sn-1. Repeated oral dosing for 2 weeks with PPI-1011 resulted in dose-dependent increases in circulating DHA and DHA-containing plasmalogens. These products and/or precursors were also able to cross the blood-retinal and blood-brain barriers.

Sortase A-Catalyzed Transpeptidation of Glycosylphosphatidylinositol Derivatives for Chemoenzymatic Synthesis of GPI-Anchored Proteins

Several peptides/small proteins and glycosylphosphatidylinositol (GPI) derivatives were synthesized and compared as substrates of sortase A (SrtA), a bacterial transpeptidase, for enzymatic coupling. It was observed that peptides containing the LPKTGGS and LPKTGGRS sequences as sorting signals at the peptide C-terminus were effectively coupled to GPI derivatives having one or two glycine residues attached to the phosphoethanolamine group at the nonreducing end. This reaction was employed to prepare several analogues of the human CD52 and CD24 antigens, which are naturally GPI-anchored glycopeptides/glycoproteins. It was further observed that the trisaccharide GPI analogues 5 and 6 were better SrtA substrates than monosaccharide GPI analogue 4, suggesting that steric hindrance of the GPI analogues does not affect their peptidation reaction mediated by SrtA. Therefore, this synthetic strategy may be useful for the preparation of more complex GPI-anchored peptides, glycopeptides, proteins, and glycoproteins.

Natural Phosphoryl and Acyl Variants of Lipid A from Neisseria meningitidis Strain 89I Differentially Induce Tumor Necrosis Factor-α in Human Monocytes

The native lipooligosaccharide (LOS) from Neisseria meningitidis strain 89I was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and the spectrum compared with that of the LOS after O-deacylation and hydrogen fluoride treatment. The data are consistent with the presence of natural variations in the LOS, which include a triphosphorylated lipid A (LA) with and without a phosphoethanolamine group, and both hexa- and pentaacylated LA molecules. Thin-layer chromatography was performed on 89I LA produced by hydrolysis of the LOS, and the purified LA molecules were analyzed by MALDI-TOF and tested for their relative ability to induce the secretion of tumor necrosis factor-alpha by human monocytic THP-1 cells and primary human monocytes. The potency of tumor necrosis factor-alpha induction varied by approximately 2-10-fold, depending on the state of acylation and phosphorylation. The results highlight the significance of phosphorylation along with acylation of the LA component of LOS in stimulation of inflammatory signaling, and suggest that natural strain variation in these moieties may be a feature of meningococcal bacteria, which is of critical importance to the progression of the infection.

Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.

Characterization of a waaF mutant of Helicobacter pylori strain 26695 provides evidence that an extended lipopolysaccharide structure has a limited role in the invasion of gastric cancer cells

An LD-heptosyltransferase gene, HP1191 (waaF), involved in biosynthesis of the inner-core region of Helicobacter pylori strain 26695 lipopolysaccharide (LPS), has been cloned and its function established by complementation of Salmonella enterica serovar Typhimurium waaF mutant strain, strain 3789. Insertional inactivation of the HP1191 open reading frame in strain 26695 resulted in the formation of a deeply truncated LPS molecule, as observed using SDS-PAGE. Subsequent compositional and fatty acid analyses, followed by capillary electrophoresis - mass spectrometry and nuclear magnetic resonance studies established its structure as the following: PE-->7)-L-alpha-D-Hepp-(1-->5)-alpha-Kdop-(2-->6)-Lipid A, where PE represents a phosphoethanolamine group, LD-Hep represents L-glycero-D-manno-heptose, and Kdo represents 3-deoxy-D-manno-oct-2-ulosonic acid. This structural analysis identifies the activity of HP1191 as a heptosyltransferase and a waaF homolog. In vitro invasion assays using human cultured gastric adenocarcinoma cells as a host cell model confirmed that the level of invasion was unaffected for an H. pylori HP1191::Kan deep-rough mutant strain compared with the wild-type strain 26695 expressing the O-chain polysaccharide, providing evidence that LPS is not a critical factor for invasion.

O-Acetylation of the Terminal N-Acetylglucosamine of the Lipooligosaccharide Inner Core in Neisseria meningitidis: INFLUENCE ON INNER CORE STRUCTURE AND ASSEMBLY

O-Acetylation is a common decoration on endotoxins derived from many Gram-negative bacterial species, and it has been shown to be instrumental (e.g. in Salmonella typhimurium) in determining the final tertiary structure of the endotoxin and the immunogenicity of the molecule. Structural heterogeneity of endotoxins produced by mucosal pathogens such as Neisseria meningitidis is determined by decorations on the heptose inner core, including O-acetylation of the terminal N-acetylglucosamine (GlcNAc) attached to HepII. In this report, we show that O-acetylation of the meningococcal lipooligosaccharide (LOS) inner core has an important role in determining inner core assembly and immunotype expression. The gene encoding the LOS O-acetyltransferase, lot3, was identified by homology to NodX from Rhizobium leguminosarum. Inactivation of lot3 in strain NMB resulted in the loss of the O-acetyl group located at the C-3 position of the terminal GlcNAc of the LOS inner core. Inactivation of either lot3 or lgtG, which encodes the HepII glucosyltransferase, did not result in the appearance of the O-3-linked phosphoethanolamine (PEA) groups on the LOS inner core. Construction of a double mutant in which both lot3 and lgtG were inactivated resulted in the appearance of O-3-linked PEA groups on the LOS inner core. In conclusion, O-acetylation status of the terminal GlcNAc of the gamma-chain of the meningococcal LOS inner core is an important determinant for the appearance or exclusion of the O-3-linked PEA group on the LOS inner core and contributes to LOS structural diversity. O-Acetylation also likely influences resistance to complement-mediated lysis and may be important in LOS conjugate vaccine design.

Proton Transfer in Gramicidin Water Wires in Phospholipid Bilayers: Attenuation by Phosphoethanolamine

The transfer of protons in water wires was studied in native gramicidin A (gA), and in the SS- and RR- diastereoisomers of dioxolane-linked gA channels (SS and RR channels). These peptides were incorporated into membranes comprised of distinct combinations of phospholipid headgroups and acyl chains. Quantitative relationships between single channel conductances to H + (g H) and [H +] were determined in distinct phospholipid membranes, and are in remarkable contrast with results previously obtained in monoglyceride membranes. In particular: 1), g H-[H +] relationships for the various gA channels in distinct phospholipid membranes are well fitted by single adsorption isotherms. A simple kinetic model assuming mono-occupancy of channels by protons fits said relationships. This does not occur with monoglyceride membranes. 2), Under nonsaturating [H +], g H is ∼1 order of magnitude larger in phospholipid than in monoglyceride membranes. 3), Differences between rates of H + transfer in various gA channels are still present but considerably attenuated in phospholipid relative to monoglyceride membranes. 4), Charged phospholipid headgroups affect g H via changes in [H +] at the membrane/solution interfaces. 5), Phosphoethanolamine groups caused a marked attenuation of g H relative to membranes with other phospholipid headgroups. This attenuation is voltage-dependent and tends to saturate H + currents at voltages larger than 250 mV. This effect is likely to occur by limiting the access and exit of H + in and out of the channel due to relatively strong oriented H bonds between waters and phosphoethanolamine groups at channel interfaces. The differential effects of phospholipids on proton transfer could be reasoned by considering solvation effects of side chain residues of gramicidin channels by double acyl chains and by the presence of polar headgroups facilitating the entrance/exit of protons through the channel mouths.

A Role for Human SPα as a Pattern Recognition Receptor

A Role for Human SPα as a Pattern Recognition Receptor

Heteromeric Protein Complexes Mediate Zinc Transport into the Secretory Pathway of Eukaryotic Cells

The cation diffusion facilitator (CDF) family of metal ion transporters plays important roles in zinc transport at all phylogenetic levels. In this report, we describe a novel interaction between two members of the CDF family in Saccharomyces cerevisiae. One CDF member in yeast, Msc2p, was shown recently to be involved in zinc transport into the endoplasmic reticulum (ER) and required for ER function. We describe here a newly recognized CDF family member in yeast, Zrg17p. ZRG17 was previously identified as a zinc-regulated gene controlled by the zinc-responsive Zap1p transcription factor. A zrg17 mutant exhibits the same zinc-suppressible phenotypes as an msc2 mutant, including an induction of the unfolded protein response in low zinc. Moreover, a significant fraction of the total Zrg17p protein appears to localize to the ER. Their common phenotypes and localization suggested that these two proteins function together to mediate zinc transport into the ER. Consistent with this hypothesis, Msc2p and Zrg17p physically interact with each other, as determined by co-immunoprecipitation. Therefore, we propose that Msc2p and Zrg17p form a heteromeric zinc transport complex in the ER membrane. We also demonstrate that ZnT5 and ZnT6, mammalian homologues of Msc2p and Zrg17p, functionally interact as well. These results suggest that heteromeric complexes formed by different CDF members may be a common phenomenon for this ubiquitous family of metal ion transporters.

Inner core assembly and structure of the lipooligosaccharide of Neisseria meningitidis: capacity of strain NMB to express all known immunotype epitopes

Neisseria meningitidis expresses a heterogeneous population of lipooligosaccharide (LOS) inner cores variously substituted with alpha1-3-linked glucose and O-3, O-6, and O-7 linked phosphoethanolamine (PEA), as well as glycine, attached to HepII. Combinations of these attachments to the LOS inner core represent immunodominant epitopes that are being exploited as future vaccine candidates. Historically, each LOS immunotype was structurally assessed and prescribed a certain unique inner core epitope. We report that a single isolate, strain NMB, possesses the capacity to produce all of the known neisserial LOS inner core immunotype structures. Analysis of the inner cores from parental LOS revealed the presence or absence of alpha1,3-linked glucose, O-6 and/or O-7 linked PEA, in addition to glycine attached at the 7 position of the HepII inner core. Identification and inactivation of lpt-6 in strain NMB resulted in the loss of both O-6 and O-7 linked PEA groups from the LOS inner core, suggesting that Lpt-6 of strain NMB may have bifunctional transferase activities or that the O-6 linked PEA groups once attached to the inner core undergo nonenzymatic transfer to the O-7 position of HepII. Although O-3 linked PEA was not detected in parental LOS inner cores devoid of alpha1-3-linked glucose residues, LOS glycoforms bearing O-3 PEA groups accumulated in a truncated mutant, NMBlgtK (Hep2Kdo2-lipid A). Because these structures disappeared upon inactivation of the lpt-3 locus, strain NMB expresses a functional O-3 PEA transferase. The LOS glycoforms expressed by NMBlgtK were also devoid of glycine attachments, indicating that glycine was added to the inner core after the completion of the gamma-chain by LgtK. In conclusion, strain NMB has the capability to express all known inner core structures, but in in vitro culture L2 and L4 immunotype structures are predominantly expressed.

Convergent synthesis of a GPI containing an acylated inositol.

A GPI of sperm CD52 was synthesized by a highly convergent procedure, representing the first chemical synthesis of a complex GPI having an acylated inositol. The presence of a large acyl group resulted in unusual properties and reactions of the relevant intermediates, which gave rise to a number of problems. To overcome the problems and achieve the target molecule, a new synthetic strategy was developed. First, the pseudodisaccharide of 2-O-palmitoylinositol was phospholipidated, and then the trimannose segment and the phosphoethanolamine group were sequentially attached. Global deprotection eventually afforded the sperm CD52 GPI. The method may be useful for the synthesis of other GPIs having an acylated inositol.