Galactosamine Residues Research Articles

Eplontersen: First Approval.

Eplontersen (Wainua™) is a ligand-conjugated antisense oligonucleotide directed to TTR, which is being developed by Ionis Pharmaceuticals and AstraZeneca for the treatment of TTR-mediated amyloidosis (ATTR). Eplontersen, which is targeted to the liver by a ligand containing three N-acetyl galactosamine residues, binds to wild-type and variant TTR mRNA, thus reducing the levels of circulating TTR protein and amyloid deposition. Subcutaneous eplontersen reduced serum TTR levels, inhibited neuropathy progression and improved health-related quality of life in patients with polyneuropathy of hereditary ATTR (ATTRv-PN; v for variant) in a phase III trial. Based on these results, eplontersen was approved in the USA for the treatment of ATTRv-PN on 21 December 2023 and is currently undergoing regulatory review for a similar indication in the EU, the UK, Switzerland and Canada. Eplontersen is also undergoing phase III development for ATTR cardiomyopathy. This article summarizes the milestones in the development of eplontersen leading to this first approval for ATTRv-PN.

The absorption of bovine milk small extracellular vesicles largely depends on galectin 3 and galactose ligands in human intestinal cells and C57BL/6J mice.

Small extracellular vesicles in milk (sMEVs) have attracted attention in drug delivery and as bioactive food compounds. Previous studies implicate galactose residues on the sMEV surface in sMEV transport across intestinal and endothelial barriers in humans, but details of glycoprotein-dependent transport are unknown. We used a combination of cell biology and genetics protocols to identify glycoproteins on the sMEV surface that facilitate sMEV absorption. We identified 256 proteins on the bovine sMEVs surface by using LC-MS/MS, and bioinformatics analysis suggested that 42, 13, and 13 surface proteins were N-, O-, and 13 C-glycosylated, respectively. Lectin blots confirmed the presence of mannose, galactose, N-acetyl galactose, fucose, and neuraminate. When surface proteins were removed by various treatment with various proteases, sMEV uptake decreased by up to 58% and 67% in FHs-74 Int and Caco-2 cells, respectively, compared with controls (P < 0.05). When glycans were removed by treatment with various glycosidases, sMEV uptake decreased by up to 54% and 74% in FHs-74 Int and Caco-2 cells, respectively (P < 0.05). When galactose and N-acetyl galactosamine residues were blocked with agglutinins, sMEV uptake decreased by more than 50% in FHs-74 Int cells (P < 0.05). When bovine sMEVs were administered to Galectin-3 knockout mice by oral gavage, hepatic sMEV accumulation decreased by 56% compared with wild-type mice (P < 0.05), consistent with a role of β-galactoside glycan structures in the absorption of sMEVs. We conclude that sMEVs are decorated with glycoproteins, and Galectin-3 and its galactose ligands are particularly important for sMEV absorption.NEW & NOTEWORTHY This is the first paper to assess the role of unique glycans and their Galectin-3 receptor in the transport and distribution of small extracellular vesicles ("exosomes") from milk in mammals. The research assessed milk exosome transport and distribution by using multiple approaches and platforms including cell cultures, various exosome labels, knockout and mutant mice, enzymatic removal of surface proteins and glycans, and lectin blocking of glycans.

The Structure of Sulfated Polysaccharides from the Sea Cucumber &lt;i&gt;Holothuria&lt;/i&gt; (&lt;i&gt;Stauropora&lt;/i&gt;)&lt;i&gt; fuscocinerea&lt;/i&gt;

Fucosylated chondroitin sulfate FCS-Hf and preparations of fucan sulfates Hf-Fuc1 and Hf-Fuc2 were isolated from the Vietnamese sea cucumber Holothuria (Stauropora) fuscocinerea. Separation of the polysaccharides was carried out using anion-exchange chromatography on DEAE-Sephacel. The structure of polysaccharides was established by determinations of the content of monosaccharides and sulfate, as well as by NMR spectra. It was shown that FCS-Hf was built of the repeating trisaccharide fragments, with alternating 3‑linked N-acetyl-β-D-galactosamine and 4-linked β-D-glucuronic acid residues forming the main polymer chain, which carries α-L-fucose residues as side branches attached to O3 of glucuronic acid. The regular structure of polymer is masked by an uneven distribution of sulfate groups attached to fucose residues (2,4-disulfate, 3,4-disulfate and 4-monosulfate in a ratio of 2 : 2 : 1) and galactosamine residues (4,6-disulfate and 4-monosulfate in a ratio of 3 : 1). It was also shown that fucan sulfate Hf-Fuc1 contained predominantly linear molecules built of 4-linked α-L-fucose 3-sulfate residues, while Hf-Fuc2 appeared to be a mixture of several related linear and branched fucan sulfates containing 3-linked and 4-linked α-L-Fuc residues sulfated at different positions.

Crystal Structure of Methyl 3-O-α-d-Glucopyranosyl 2-Acetamido-2-Deoxy-α-d-Galactopyranoside Hydrate

Methyl 3-O-α-d-glucopyranosyl 2-acetamido-2-deoxy-α-d-galactopyranoside as a monohydrate, C15H27NO11·H2O, crystallizes in space group P212121, with four molecules in the unit cell. It constitutes the methyl glycoside of the carbohydrate part of the teichoic acid type polysaccharide from Micrococcus sp. A1, in which the disaccharides are joined through phosphodiester linkages. The conformation of the disaccharide is described by the glycosidic torsion angles ϕ = − 31° and ψ = + 1°, and the hydroxymethyl groups of the constituent monosaccharides are present in the gg and gt conformations for the sugar residues having the gluco- and galacto-configuration, respectively. For the N-acetyl group at C2 of the galactosamine residue the torsion angle τH = 147°, i.e., the amide proton has an antiperiplanar relationship to H2 of the sugar ring. The structure shows extensive hydrogen bonding along the a-direction, including the water molecule, and forms sheets with hydrophilic interactions within the sheets as a result of hydrogen bonding between disaccharides as well as hydrophobic interactions between the sheets, in particular, amongst methyl groups of the N-acetyl group of the α-d-GalpNAc residue in the disaccharides.Graphical The structure of the title disaccharide as a monohydrate, C15H27NO11·H2O, which shows extensive hydrogen bonding as well as hydrophobic interactions, was determined from minute crystals using synchrotron radiation and verified by solid state DFT calculations using plane waves.

Boron-doped carbon nanowalls for fast and direct detection of cytochrome C and ricin by matrix-free laser desorption/ionization mass spectrometry

Detecting proteins via surface assisted laser desorption/ionization mass spectrometry (SALDI-MS) method is still highly challenging, and only few examples of nanomaterials have been demonstrated to perform such detection so far. In this study, carbon nanowalls (CNWs), vertically aligned graphene sheet-based materials, presenting specific morphology, dimensions, and boron doping levels have shown improved performances for both qualitative and quantitative detection of Cytochrome C under optimized experimental conditions. Boron doped carbon nanowalls (B-CNWs) with a [B]/[C] ratio of 5000 ppm and growing time of 4 h have shown the best performance in terms of signal intensity and reliability. Then, the detection of ricin, a ribosomal-inhibiting protein (RIP) classified as category B bioterrorism agent by CDC (Centre of Disease and Control and Prevention), was performed. For the first time, direct SALDI-MS detection of ricin B chain was reported without tedious sample preparation steps or database interrogation, and results were obtained within few minutes and a limit of detection (LOD) of 0.5 pmol/μl was obtained. Thanks to the introduction of galactosamine residues on B–CNW, we were able to selectively detect ricin B chain protein in complex media such as serum and soft drinks with enhanced signal intensity. B-CNWs are not toxic and are adaptable to any commercial MALDI-TOF mass spectrometer, showing their great potential as SALDI based materials.

The influences of cycle stage and pregnancy upon cell glycosylation in the endometrium of the mare

From Day 6.5–7 post-conception until its loss around Day 22, the equine embryo is enclosed in a mucinous capsule that prevents direct intercellular interaction between the trophectoderm and uterine epithelium. The embryo is, however, bathed in glycoprotein-rich secretions. In this study, lectin histochemistry was used to characterise the distribution and glycan composition of uterine glycoproteins destined for secretion, and to ascertain the local effect of an embryo on glycosylation in the endometrium. Endometrial biopsies were taken from mares in estrus, on Days 5, 8, 12 and 15 of diestrus, and on Days 12 and 15 of pregnancy and processed for lectin histochemistry. During estrus, lumenal epithelial cells were as truncated pyramids and mainly non-ciliated with glycosylated granules in the cytoplasm. Occasional ciliated cells contained few granules. Five days post-ovulation, non-ciliated cells of the lumenal epithelium were taller, and had accumulated many highly glycosylated apical granules. By Days 12 and 15 post-ovulation these cells were more cuboidal and some showed fewer secretory granules. In marked contrast, by Days 12 and 15 of pregnancy, the ciliated cells were distended, with numerous granules but non-ciliated cells had only a few in the apical cytoplasm. Glycosylation changed dramatically in pregnancy in the luminal and superficial gland epithelium, with fewer fucosylated termini, more N-acetyl galactosamine residues, together with an overall reduction in sialic acid and several other sugar structures. Glycosylation in ciliated cells on Days 12 and 15 of pregnancy showed a striking similarity to that of the blastocyst capsule. The data strongly suggests that glycoprotein production by luminal epithelial cells is influenced by the presence of a conceptus. We speculate that, as well as providing nourishment for the developing embryo, epithelial secretory glycoproteins may contribute components to the capsule, which develops only partially in embryos cultured in vitro.

Definition of the Anti-inflammatory Oligosaccharides Derived From the Galactosaminogalactan (GAG) From Aspergillus fumigatus.

Galactosaminogalactan (GAG) is an insoluble aminosugar polymer produced by Aspergillus fumigatus and has anti-inflammatory properties. Here, the minimum glycosidic sequences required for the induction of IL-1Ra by peripheral blood mononuclear cells (PBMCs) was investigated. Using chemical degradation of native GAG to isolate soluble oligomers, we have found that the de-N-acetylation of galactosamine residues and the size of oligomer are critical for the in vitro immune response. A minimal oligomer size of 20 galactosamine residues is required for the anti-inflammatory response but the presence of galactose residues is not necessary. In a Dextran sulfate induced colitis mouse model, a fraction of de-N-acetylated oligomers of 13 < dp < 20 rescue inflammatory damage like the native GAG polymer in an IL-1Ra dependent pathway. Our results demonstrate the therapeutic suitability of water-soluble GAG oligosaccharides in IL-1 mediated hyper-inflammatory diseases and suggest that α-1,4-galactosamine oligomers chemically synthesized could represent new anti-inflammatory glycodrugs.

Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans.

Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS.

Holothurian fucosylated chondroitin sulfate.

Fucosylated chondroitin sulfate (FucCS) is a structurally distinct glycosaminoglycan found in sea cucumber species. It has the same backbone composition of alternating 4-linked glucuronic acid and 3-linked N-acetyl galactosamine residues within disaccharide repeating units as regularly found in mammalian chondroitin sulfates. However, FucCS has also sulfated fucosyl branching units 3-O-linked to the acid residues. The sulfation patterns of these branches vary accordingly with holothurian species and account for different biological actions and responses. FucCSs may exhibit anticoagulant, antithrombotic, anti-inflammatory, anticancer, antiviral, and pro-angiogenic activities, besides its beneficial effects in hemodialysis, cellular growth modulation, fibrosis and hyperglycemia. Through an historical overview, this document covers most of the science regarding the holothurian FucCS. Both structural and medical properties of this unique GAG, investigated during the last 25 years, are systematically discussed herein.

The Galactosamine Residue in Mycobacterial Arabinogalactan Is α-Linked

Previous studies have demonstrated that cell wall arabinogalactan from mycobacteria possesses a single galactosamine (GalN) residue. This moiety, which is one of the rare natural occurrences of galactosamine lacking an acetyl group on the nitrogen, has been identified as a pendant substituent attached to a highly branched arabinofuranose residue in the arabinan core. However, the stereochemistry by which the GalN residue is linked to the polysaccharide remains unknown. We report here the synthesis of two tetrasaccharides, 1 and 2, consisting of GalN attached through either an α- or β-linkage to a trisaccharide fragment of mycobacterial arabinan. These molecules represent the first synthetic GalN-containing oligosaccharides, and the preparation of both targets was achieved from a single donor species by modulation of the reaction solvent. Comparison of the NMR spectra of 1 and 2 with those obtained from a sample derived from the natural glycan revealed that the GalN residue in the polysaccharide is attached via an α-linkage.

Synthesis of the repeating unit of the lipoteichoic acid of Streptococcus pneumoniae

The lipoteichoic acid repeating unit of Streptococcus pneumoniae is a complex pseudopentasaccharide ( 3). It consists of one ribitol-phosphate, one 2-acetamino-4-amino-2,4,6-trideoxy-galactose, one glucose and two galactosamine residues each differently linked, but both carrying one phosphocholine substituent, at position 6. Suitable building blocks ( 6– 10) for efficient and diastereocontrolled ligations were designed, thus providing, after complete deprotection, the target molecule in high purity. Biological tests revealed that repeating unit 3, lacking the lipid moiety, did not stimulate a pro-inflammatory response in human monocytes (hMNCs).

Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase

A typical filamentous bacterium, Sphaerotilus natans, secretes a thiolic glycoconjugate which is assembled into a microtube, so called sheath. The glycoconjugate is known to consist of a pentasaccharide-dipeptide repeating unit, but its chemical structure has not been completely elucidated. In order to determine its chemical structure, the sheath was broken down by performic acid oxidation. The released sulfonated derivative was water soluble which was suitable for detailed NMR analysis. The data exhibited the presence of two stoichiometric and one substoichiometric (relative abundance was about 0.5) acetylations, suggesting that the glycoconjugate is composed of two equimolar pentasaccharide-dipeptide repeating units each having either two or three acetyl groups. However, the position of substoichiometric acetylation could not be defined. To determine the position, the sheath was derivatized with a thiol selective fluorescent reagent followed by digestion with a specific polysaccharide lyase prepared from a sheath-degrading bacterium, Paenibacillus koleovorans. As expected, two fluorescent digests were recovered by reverse-phase HPLC and were subjected to NMR analysis. The data revealed that both digests are pentasaccharide-dipeptides which have unsaturated glucuronic acid and galactosamine residues at their reducing and non-reducing ends, respectively. It was also confirmed that one digest has 3- O-acetylated glucose residue while the other has non-derivatized glucose residue. The substoichiometric acetylation was thus identified with the 3- O-acetylation, and structural determination of the thiolic glycoconjugate was completed. By virtue of the clarification of the two digests’ structures, the cleavage site was specified as (1 → 4)-α-galactosaminic bond to glucuronic acid. Based on the present and earlier findings, we propose a novel glycoconjugate category named thiopeptidoglycan and a novel polysaccharide lyase named thiopeptidoglycan lyase.

POLAR LIPIDS OF RUANIA ALBIDIFLAVA, A NOVEL MEMBER OF THE SUBORDER MICROCOCCINEAE

Ruania albidiflava represents a novel species and new genus within the suborder Micrococcineae in Actinobacteria classis. The aim of the studies was to establish a polar lipid profile of Ruania and to compare it with other representatives of actinobacteria. Additionally, isolation, purification and chemical characteristics of major lipid compounds were elaborated. Major lipids were isolated using column adsorption chromatography and purified by TLC and high performance liquid chromatography. The polar lipids of Ruania were phosphatidylglycerol, diphosphatidylglycerol and two unknown lipids containing sugar and phosphorus. GLC-MS and methylation analysis of sugar part of major glycolipids revealed the presence of glycerol, inositol; and terminal residues of mannose and galactosamine. Fatty acids were mainly iso and anteiso C15:0 branched. Comparative TLC showed its glycolipid profile of Ruania different from previously reported for Rothia, Arthrobacter, Micrococcus and Propionibacterium, but was similar to Oerskovia which belongs to Micrococcineae suborder.

Oligosaccharide composition is similar in drusen and dense deposits in membranoproliferative glomerulonephritis type II

Drusen are a feature of age-related macular degeneration (AMD). Lesions similar in appearance to drusen are also found in the fundi of patients with membranoproliferative glomerulonephritis type II (dense deposit disease, DDD). The lamina densa of the glomerular basement membrane, in DDD, is transformed into an electron-dense structure by deposition of microscopically homogeneous material. Our study sought to compare the saccharide composition of drusen and dense deposits in the formalin-fixed, paraffin-embedded tissue from the eye and kidney. Six eye specimens were obtained from patients diagnosed with AMD but another eye was obtained from a patient with partial lipodystrophy, who died after renal failure presumably because of DDD. The kidney specimens were from three biopsy-proven cases of DDD. Glycosylation patterns were measured by the binding of 19 biotinylated lectins before and after neuraminidase pre-treatment. High mannose, bi/tri-antennary non-bisected and bisected complex N-glycan, N-acetyl glucosamine, galactose, and sialic acid residues were found in both drusen and dense deposits. Treatment with neuraminidase exposed subterminal galactose in both sites and sparse N-acetyl galactosamine residues in drusen alone. Our study found similar pathologic oligosaccharide structures in the eye and kidney, suggesting that drusen may be a common end result of retinal and glomerular disease.

Biosynthesis of Undecaprenyl Phosphate-Galactosamine and Undecaprenyl Phosphate-Glucose in Francisella novicida

Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-beta-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-beta-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-beta-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-alpha-l-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-alpha-l-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.

The Identification and Location of Succinyl Residues and the Characterization of the Interior Arabinan Region Allow for a Model of the Complete Primary Structure of Mycobacterium tuberculosis Mycolyl Arabinogalactan

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-alpha-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear alpha-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

Glycoproteins of drusen and drusen-like lesions

Drusen are a marker of age-related macular degeneration (AMD). Lesions similar to drusen, both in histology and their clinical appearance, are also seen in choroidal tumours, chronic inflammatory and degenerative conditions of the eye, and in mesangiocapillary glomerulonephritis type II (MCGN-II). This study aims to compare the saccharide composition of these drusen-like lesions in the various ocular pathological groups and in MCGN-II. Formalin fixed and paraffin wax embedded tissue from 21 eyes was studied. The histological diagnoses included AMD, retinal detachment, phthisis bulbi following failed retinal detachment surgery, malignant melanoma, long-standing uveitis, glaucoma and MCGN II. Glycosylation was examined using a panel of twenty biotinylated lectins and an avidin-peroxidase DAB-cobalt revealing system, with and without neuraminidase pre-treatment. High mannose, bi/tri-nonbisected and bisected complex N-glycan, N-acetyl glucosaminyl, galactosyl and sialyl residues were found to be expressed by drusen, while treatment with neuraminidase exposed subterminal N-acetyl galactosamine and galactosyl residues. Similar binding patterns were found in the various pathological groups studied. As there was no significant difference in the lectin-binding pattern in drusen in different pathologies, a common pathogenesis or at least a final common pathway for the elaboration of carbohydrate components of drusen is suggested.

Two E.coli ArnC Homologues in Francisella novicida and their Functions in Lipid A Modification

Two F. novicida homologues of E. coli ArnC synthesize substrates required for lipid A glycosylation at 1- and 6′-positions. ArnC1 selectively condenses undecaprenyl phosphate and UDP-glucose to form undecaprenyl phosphate-glucose; ArnC2 catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine, instead of UDP-galactosamine, to form undecaprenyl phosphate-N-acetylgalactosamine. The ArnC1 knockout of F. novicida produces only one free lipid A species, which is modified with a galactosamine residue at the 1-position. The ArnC2 knockout makes two free lipid A species, the major form of which is not glycosylated at either the 1- or the 6′-positions, and the minor form of which contains glucose at the 6′-position. Membranes of the ArnC1 mutant cannot catalyze formation of undecaprenyl phosphate glucose, while membranes of the ArnC2 knockout catalyze this reaction with similar efficiency as wild-type. Membranes of the ArnC2 knockout cannot generate undecaprenyl phosphate-N-acetylgalactosamine, while membranes of the ArnC1 knockout and the wild-type do so at similar rates. We conclude that ArnC1 generates the immediate donor substrate for the modification of F. novicida lipid A with glucose, while ArnC2 makes a precursor of the actual donor substrate for modification of F. novicida lipid A with galactosamine. Supported by NIH grant R37-GM51796 to CRHR.

Identification of the Origin of Chondroitin Sulfate in "Health Foods"

Twelve "health foods" products containing chondroitin sulfate (CS) were purchased from the Japanese market and the origin of the CS was investigated by conducting disaccharide compositional analysis after enzymatic depolymerization and by 1H-NMR spectroscopy. Nine of the 12 products had labels indicating that the origin of the CS was shark cartilage. However, two of them were found to contain mammalian CS. Next, we compared the ratio of the sulfate group to the galactosamine residue after the acid hydrolysis of CS. The results suggest that all of the CS from sharks had a ratio of more than 1.0, while the CS from mammals had a ratio of less than 1.0. Since this comparative analysis does not require expensive purified enzyme, it would be an economical way to identify the origin of CS in "health foods." Being able to determine the origin of the ingredients in natural products is very important for ensuring their quality, safety, and efficacy. Therefore, we think that regulatory requirements for accurately indicating the origin of "health foods" and effective enforcement of these requirements are needed.

Structure and Biosynthesis of Free Lipid A Molecules That Replace Lipopolysaccharide in Francisella tularensis subsp. novicida

Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an alpha-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An alpha-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.