Chemical and Immunological Characterization of Galactosyl-β1-3-Globoside in Bovine, Human, and Rat Brain

Abstract: Our studies of bovine brain neutral glycosphingolipids (Ngsls) have revealed the presence of several short-chain (containing -CHO 1–4) and previously uncharacterized long-chain (−CHO > 4–5) Ngsls. We reported the structural characterization of brain GgOse₄Cer (GA1) and have now purified another brain Ngsl to homogeneity. The purified Ngsl migrated close to standard GgOse₄Cer and nLcOse₅Cer on a TLC plate employing two different solvent systems. The carbohydrate molar composition indicated the presence of Gal/Glc/GalNAc in a ratio of 2.8:1.0:0.9. Five permethylated alditol acetate peaks were characterized as 2,3,4,6-OMe₄Gal, 2,4,6-OMe₃Gal, 2,3,6-OMe₃Gal, 2,3,6-OMe₃Glc, and 4,6-OMe₂GalNAcMe by gas chromatography-mass spectrometry. The anomeric carbohydrate sequence has been determined by specific exoglycosidase digestion. Six-hundred megahertz ¹H NMR spectroscopy of the oligosaccharide released by ceramide glycanase hydrolysis confirmed the structure of the Ngsl as Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc\1-1Cer or IV³GalGbOse₄Cer. Using the immunooverlay technique with anti-stage-specific embryonic antigen 3 antibody, it was found in bovine, rat, and normal adult human brain and bovine myelin, but not in human or rat myelin.     

Myelin Gangliosides of Human Peripheral Nervous System: An Enrichment of GM1 in the Motor Nerve Myelin Isolated from Cauda Equina

Myelins of the PNS were isolated from human motor and sensory nerves of cauda equina, and their ganglioside compositions were compared. The predominant ganglioside in the human PNS myelins, both from motor and sensory nerves, was LM1 (sialosylneolactotetraosylceramide). Sialosyl-nLc6Cer and disialosyl-nLc4Cer, GD3, GM3, and GD1b were detected as common components of the two nerve myelins. Furthermore, it was revealed that the motor nerve myelin contained GM1 (about 15% of total gangliosides), whereas sensory nerve myelin contained only a trace amount of GM1 (less than 5%), by TLC analyses together with TLC immunostaining using anti-GM1 antibody. As for the disialoganglioside fraction, the content of GD1a, as well as that of GM1, differed in motor and sensory nerves. Thus, the different contents of the ganglioseries gangliosides in human motor and sensory nerve myelins were demonstrated.     

Gangliosides and Glycosphingolipids of Peripheral Nervous System Myelins—a Minireview

A summary is provided of the available data on the composition of gangliosides and glycosphingolipids in the peripheral nervous system (PNS) including myelins and their antigenic properties. The composition of gangliosides and glycosphingolipids in the PNS is very different from that in the central nervous system (CNS), both quantitatively and qualitatively. One major difference is the abundance of neolacto-series gangliosides in the PNS, with the backbone structure Gal 1-4GlcNAcl-3Gal l-4Glcl-lCer. Their abundance contrasts with the abundance of ganglio-series gangliosides in the CNS. The neolacto-series gangliosides are localized mainly in the myelins of the PNS. In addition to gangliosides, other acidic and neutral glycosphingolipids in the neolacto-series are also characteristic of the myelins of the PNS. The ceramide (fatty acid and sphingosine base) compositions of gangliosides in the PNS are different from those in the CNS gangliosides, having greater percentages of long-chain fatty acids and dehydrosphingosines than found in the CNS gangliosides.     

Chemokines bind to sulfatides as revealed by surface plasmon resonance

Chemokines bind to sulfated cell surface glycosaminoglycans and thereby modulate signaling mediated by G-protein-coupled seven-transmembrane domain chemokine receptors. Similar to glycosaminoglycans, sulfated oligosaccharides are also exposed on the cell surface by sulfatides, a class of glycosphingolipids. We have now identified sulfated glycosphingolipids (sulfatides) as novel binding partners for chemokines. Using surface plasmon resonance (SPR), the binding of proinflammatory and homeostatic chemokines to glycosphingolipids, in particular sulfatides, was investigated. Chemokines were immobilized while glycosphingolipids or additional phospholipids incorporated into liposomes were applied as soluble analytes. A specific affinity of the chemokines MCP-1/CCL2, IL-8/CXCL8, SDF-1alpha/CXCL12, MIP-1alpha/CCL3 and MIP-1beta/CCL4 to the sulfatides SM4s, SM3, SM2a and SB2, SB1a was detected. No significant interactions with the chemokines were observed for gangliosides, neutral glycosphingolipids or phospholipids. Chemokine receptors have been associated with the detergent-insoluble fraction supposed to contain 'rafts', i.e., glycosphingolipid enriched microdomains of the cell surface. Accordingly, the data suggest that early chemokine receptor signaling may take place in the vicinity of sulfated glycosphingolipids on the cell surface, whereby these sulfatides could modulate the chemokine receptor-mediated cell activation signal.     

Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS

By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains.     

GENERAL PATTERN, 35SO4-INCORPORATION AND INTRACELLULAR LOCALIZATION OF GLYCANS IN DEVELOPING SEA URCHIN (ANTHOCIDARIS) EMBRYOS

Glycosaminoglycan (GAG) prepared from sea urchin embryos ( Anthocidaris crassispina ) at various stages with or without pulse ³⁵SO₄-labelling was separated into various fractions by chromatography on DEAE-cellulose with a linear NaCl concentration gradient: fraction "P" (nonacidic) and fractions "A" through "F" (of increasing acidities). The ³⁵SO₄-radioactivity was negligible in "P" and "A", largest in "B" and "C", and decreased in the other fractions three alphabetical order. During development (hatched blastulae to gastrulae) the glycans in fractions "P" and "A" decreased in amount, whereas those in "E" and "F" increased. "E" contained heparin-like (AMPS-1) and dermatanpolysulfate-like (AMPS-2) GAG in addition to a sulfated fucogalactan-like (E₁) glycan. Another sulfated fucogalactan-like (F₁) glycan was found in "F". A sulfated polysialic acid-like (S₁) glycan was found in "C". An EDTA-extract of gastrulae gave AMPS-2, E₁ and F₁. The mitochondria-rich fraction gave AMPS-1, whereas the yolk granule-rich fraction gave S₁. Most of the other still unidentified components in "B", "C", and "D" appeared to be derived from glycoproteins and were mainly located in the crude yolk-mitochondrial and cytosol fractions.     

The Protein Activator Specific for the Enzymic Hydrolysis of GM2 Ganglioside in Normal Human Brain and Brains of Three Types of GM2 Gangliosidosis

Abstract: In order to understand the etiology of Type AB GM2 gangliosidosis, we have purified and characterized the activator protein (GM2 activator) specific for the enzymic hydrolysis of GM₂ ganglioside from normal human brain. The purified activator from human brain moved as one major protein band in various electrophoretic systems. We have also prepared the antiserum against this activator. The levels and the nature of GM₂ activator and β-hexosaminidase A were examined in the brains of five cases of GM₂. gangliosidosis—one Type B, two Type O, and two Type AB. We found that the levels of GM₂ activator in both Type B and Type O cases were markedly elevated, and that the two Type AB cases were the results of different causes. One case had a defective β hexosaminidase A and an elevated level of GM₂ activator. Although this defective β-hexosaminidase A could hydrolyze synthetic substrates, it was inactive in the cleavage of natural glycosphingolipids in the presence of the GM₂ activator. It could, however, hydrolyze asialo-GM₂ and GbOse₄Cer in the presence of sodium taurodeoxycholate. The other case had normal β-hexosaminidase A, but had a very low level of GM₂ activator when analyzed by in vitro assay, suggesting the deficiency of this activator. By immunoelectrophoresis, this case was found to be completely devoid of the protein that cross-reacts with the antiserum against the GM₂ activator.     

Sphingolipid composition of human platelets

Total lipid extracts from washed trypsinized human platelets were fractionated into neutral lipids, glycosphingolipids, and phospholipids by silicic acid chromatography. The concentrations and chemical structures of the neutral and acidic glycosphingolipids were then studied in detail. On the basis of sugar molar ratios, studies of permethylation products, and the action of stereospecific glycosidases on the lipids, identifications were made of four neutral glycosphingolipids. Lactosylceramide was the most abundant type and accounted for 64% of the total neutral glycolipid mixture. The major fatty acids of the lactosylceramide were 20:0, 22:0, 24:0, and 24:1; the major long-chain base was 4-sphingenine. The platelets were surprisingly rich in a ceramide fraction, which represented 1.3% of the total platelet lipids. It had a different fatty acid composition than the neutral glycosphingolipid and ganglioside fractions. Hematoside was also isolated from the total lipid fraction of platelets; the neuraminic acid component was N-acetylneuraminic acid. Treatment of platelets with trypsin, chymotrypsin, or thrombin increased the yield of hematoside as compared with a control, while the level of ceramides was not changed. It was concluded that the platelets are similar to leukocytes, liver, and spleen in that lactosylceramide and hematoside are the principal neutral and acidic glycosphingolipids. The presence of a relatively high proportion of ceramide in platelets may be a unique characteristic of this cellular fraction of blood.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.     

Glycosphingolipids of purified human lymphocytes.

Biochemical analysis of the glycosphingolipids (GSLs) of human lymphocytes revealed qualitative and quantitative variations among purified lymphocytes from different tissues. The major neutral GSLs of tonsil lymphocytes are glucosyl ceramide (CMH), lactosyl ceramide (CDH), trihexosyl ceramide (CTH), and globoside. Thymocytes and peripheral blood lymphocytes (PBL) contain only traces of CTH and globoside, and PBL contain more CMH and CDH per cell than tonsil lymphocytes. Thymocytes and PBL contain relatively large amounts of more complex neutral GSLs that are present in only trace amounts in tonsil lymphocytes. Peripheral blood lymphocytes contained three and five times more lipid-bound sialic acid than thymocytes and toncil lymphocytes, respectively. Thymocytes and PBL contained mostly hematoside, whereas tonsil lymphocytes contained more complex gangliosides in addition to hematoside. The observed differences in GSL content among these cells may be related to their content of B cells, which comprise approximately 50% of tonsil lymphocytes, 10% of PBL and 0-2% of thymus cells, and/or the known differences in functional capacities of cells in different lymphoid organs. These findings suggest that cell surface GSLs may serve as markers for identification of functional subpopulations of human lymphocytes.     

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.     

Subcellular Localization of Sulfated Glucuronic Acid-Containing Glycolipids Reacting with Anti-Myelin-Associated Glycoprotein Antibody

Peripheral nerve glycolipids, with which anti-myelin-associated glycoprotein (MAG) antibodies from patients with demyelinating neuropathy and plasma cell dyscrasia cross-react, proved to be novel glycosphingolipids containing a sulfated glucuronyl residue. Consequently, there has been much interest in the immunological role that these sulfated glucuronyl-glycosphingolipids (SGGLs) may play in the pathogenesis of this disorder. For the determination of the distribution of these glycolipids in various nervous tissues and, thereby, the elucidation of their pathogenicity, a quantitative immunostaining-TLC method for their detection has been devised. Using this method, we demonstrated that these glycolipids were distributed in greatly different amounts in the peripheral nerves from human, bovine, chicken, rat, and rabbit. Subcellular localization studies of bovine peripheral nerve also demonstrated that they were enriched in the axolemma-enriched fraction and present in glial-related membranes in lower concentrations. In addition, these glycolipids were present in bovine dura mater and transformed rat Schwann cells. These biochemical results suggest that not only myelin but also axons could be involved as targets of the anti-MAG antibody in macroglobulinemia neuropathy, and it may also be necessary to examine anti-SGGL activity in patients with axonal neuropathy associated with plasma cell dyscrasia.     

Characterization of neutral sphingolipids and gangliosides from chicken liver

The neutral sphingolipids and gangliosides were isolated from 62- and 63-day-old chicken livers and characterized. The total concentration of neutral sphingolipids was 59 nmol/g of liver, and that of gangliosides was 330 nmol/g of liver. The major neutral sphingolipids were free ceramide, galactosylceramide, glucosylceramide, lactosylceramide, galabiosylceramide, and Forssman glycolipid. Galactosylceramide was the most abundant and free ceramide was the second most abundant. The major gangliosides were sialosylgalactosylceramide (GM4) and sialosyllactosylceramide (GM3), each of which contained only N-acetylneuraminic acid as a sialic acid. Sphingosine (d18:1) was a major long-chain base in all the sphingolipids. Considerable amounts of 2-hydroxy fatty acids were present in free ceramide, galactosylceramide, and GM4.     

Phenylalanine 30 plays an important role in receptor binding of verotoxin-1

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb₃). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb₃ and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10⁵ compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galα1-4Galβ1-4Glc trisaccharide portion of Gb₃. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.     

Glycosphingolipid composition of human semen

Glycosphingolipids were extracted from human semen and purified. Based on the fluorometric assay of sphingosine, in spermatozoa a content of 4.4 +/- 0.9 nmol/10(8) cells of gangliosides and 22.1 +/- 1.7 nmol/10(8) cells of neutral glycosphingolipids was determined. Seminal plasma contained 4.1 +/- 0.6 nmol gangliosides and 29.3 +/- 1.5 nmol neutral glycosphingolipids per milliliter. The glycosphingolipid component patterns of human spermatozoa and seminal plasma were determined by thin-layer chromatography. Four neutral glycolipids were isolated and their carbohydrate moieties were characterized. All of these glycolipid components belonged to the globo-series. Gas chromatography, combined gas chromatography/mass fragmentography, and exoglycosidase treatments revealed the following structures for the glycosphingolipids of human semen: Glc1-Cer, Gal beta 1-4Glc1-Cer, Gal alpha 1-4Gal beta 1-4Glc1-Cer, and Gal-NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc1-Cer. In addition, the occurrence of trace amounts of lactoneotetraosyl- and lactoneohexaosylceramide was detected by immunostaining after thin-layer chromatographic separation. Human spermatozoa, as well as seminal plasma, contained the gangliosides Glac1,Glac2, a sialolactoneotetraosylceramide, and a sialolactoneohexaosylceramide. The gangliosides were identified on the basis of their running characteristics by high-performance thin-layer chromatography, exoglycosidase treatment, and immunostaining after thin-layer chromatography. The ceramide composition of the glycolipids in human spermatozoa, as well as in seminal plasma, was dominated by C22:0-behenic acid and the saturated sphingoid d18:0, sphinganine.     

Factors affecting the glycosphingolipid composition of murine tissues

The effects of genetic strain, sex, age, and pathological state on the distribution and concentration of glycosphingolipids in mouse kidneys and livers were studied. The concentrations of glycosphingolipids and phospholipids in the kidneys and livers of different strains were compared. The major glycosphingolipid in the kidneys of male and female BALB/c, C3H/He, C57/BL, A, and C57xA (F(1) hybrid) mice was a sulfatide, monoglycosyl-(3-sulfate) ceramide; monoglycosyl ceramide was the major component in livers. The kidneys of males of all strains contained significant amounts of diglycosyl ceramide, but those of females contained, at most, only traces. Glycosphingolipid concentration in the male kidneys of C57/BL and C57xA (F(1) hybrid) was much higher than in the female and was also much higher than in the male kidneys of C3H/He, BALB/c, and A strains. The kidneys of "old" (36 wk) male and female C3H/He mice contained much higher proportions of monoglycosyl ceramide than 10-12-wk-old adults. The distributions of glycosphingolipids in kidneys of female C3H/He mice with BP8 ascites tumors and male C57xA (F(1) hybrid) mice with EL4 ascites tumors differed from those in the normal mice. An unknown lipid, present in all glycosphingolipid extracts from kidney and liver, was tentatively identified as cholesterol sulfate.     

SPHINGOGLYCOLIPIDS IN THE NERVOUS SYSTEM IN FABRY'S DISEASE

Abstract— Two glycolipids, accumulated in the spinal ganglia of a patient with Fabry's disease were identified as: galactosyl (α1 → 4) galactosyl (β1 → 4) glucosyl(1 → 1) ceramide (CTH) and galactosyl (α1 → 4) galactosyl(1 → 1) ceramide (CDG). Only one glycolipid which had the same structure as the CTH in the spinal ganglia accumulated in the sympathetic ganglia of the patient. In the nervous system, CTH contained behenic acid (C_22:0) as the major fatty acid. In the spinal ganglia, CDG also contained behenic acid as the major fatty acid.     

Acid-stable cytochromes in ferrous ion oxidizing cell-free preparations from Thiobacillus ferrooxidans

Abstract Cell-free preparations from ferrous ion- and sulfur-grown Thiobacillus ferrooxidans prepared under neutral (pH 7.5) or acidic conditions (pH 2.0) were compared. Under neutral conditions the ferrous ion-oxidizing system of T. ferrooxidans was membrane-bound. At acidic conditions, the enzyme system became partially solubilized. In ferrous ion-oxidizing membranes of ferrous ion-grown cells (neutral conditions) a₁-, c- and b-type cytochromes were present. The acidic preparations contained only cytochrome a₁ and c. At least three acid-stable c-type cytochromes (M_r 60 000, 30 000 and 25 000), an acid-stable protein with non-convalently bound heme group (M_r probably rusticyanin, were detected in ferrous ion oxidizing preparations. Membranes of sulfur-grown cells prepared under neutral conditions still oxidized ferrous ions, and contained a₁-, b-, c- and in addition an aa₃-type cytochrome. Cytochrome b and aa₃ were acid-labile.     

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda: Ascaridida)

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(β1-1)ceramide, Man(β1-4)Glc(β1-1)ceramide, GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide and Gal(α1-3)GalNAc(β1-4)GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine. Abbreviations: CDH, ceramide dihexoside; Cer, ceramide; CMH, ceramide monohexoside; CPH, ceramide pentahexoside; CTH, ceramide trihexoside; CTetH, ceramide tetrahexoside; Hex, hexose; HexNAc, N-acetylhexosamine; HPTLC, high-performance thin-layer chromatography; LSIMS, liquid secondary-ion mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; N-, Nz- and A-glyco(sphingo)lipids, neutral, neutralzwitterionic and acidic glyco(sphingo)lipids, respectively This revised version was published online in November 2006 with corrections to the Cover Date.     

Composition of Pneumocystis carinii Neutral Lipids and Identification of Coenzyme Q10 as the Major Ubiquinone Homolog

The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ₁₀ was the predominant homolog with lesser amounts of CoQ₉ present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ₉ and lesser amounts of CoQ₈, but no detectable CoQ₁₀. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ₁₀ (if not both CoQ1₁₀ and CoQ₉) in P. carinii as not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.