Glycolipid Sulfate Research Articles

Biological roles of sulfoglycolipids and pathophysiology of their deficiency.

Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is expressed on the spermatogenic cells. Cerebroside sulfotransferase (CST)-deficient mice generated by gene targeting completely lack sulfatide and seminolipid all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis, indicating that sulfation of glycolipids is essential for myelin formation and spermatogenesis. Moreover, CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction, an experimental model of renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms by which sulfoglycolipids participate in these biological processes are ongoing.

Composition Analysis of Polar Lipids in Halobacteria with Mass Spectrometry

The structures of the polar lipids in halobacteria including their mutants were determined with ESI-MS and FAB-MS. The composition-analyses with ESI-MS gave the results fairly close to these with TLC analyses. The percentages of the composite polar lipids by the mass spectrometric analyses of Halobacterium salinarum S-9 were 20, 77, and 5% for glycolipid sulfate (S-TGD), methyl ester of phosphatidyl glycerophosphate (PGP-OMe), and phosphatidylglycerol (PG), respectively. The MS/MS analysis in the FAB-MS provided an analyzing tool for the structural elucidation of the functional groups in the lipids. Newly proposed methylation in the phosphate was investigated with stable isotope experiments. We found that the Australian halobacteria, Halorubrum sp. aus-1, mainly consist of an isolated glycolipid sulfate with two sugar parts of glucopyranoses. In relationship with the lipid-composition analysis, the chemotaxonomy of halobacteria was discussed. The methylation of the lipids was proved to be essentially independent of that in the photosensory protein of halobacteria on the basis of the wide distribution of the methylated lipids.

Cerebroside Sulfotransferase Deficiency Ameliorates L-selectin-dependent Monocyte Infiltration in the Kidney after Ureteral Obstruction

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Lipid-protein stoichiometries in a crystalline biological membrane: NMR quantitative analysis of the lipid extract of the purple membrane

The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.

Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus membrane: where next?

Recently the UDP-GlcNAc, CMP-sialic acid and GDPmannose transporters from the Golgi apparatus membrane were cloned (Abeijon et al., 1996; Eckhardt et al., 1996; Ma et al., 1997). These transporters, as well as those for other nucleotide sugars, PAPS and ATP, are required for these nucleotide derivatives to reach to Golgi lumen from the cytosol and serve as substrates in glycosylation, sulfation, and phosphorylation of glycoproteins, proteoglycans, and glycolipids. Previously, biochemical and genetic evidence demonstrated that these transporters are highly specific for solute transport (Hirschberg, 1996; Hirschberg and Snider, 1987), are often organelle specific (Hirschberg, 1996), appear to play a regulatory role in the biosynthesis of Golgi lumenal macromolecules (Abeijon et al., 1993; Toma et al., 1996), and use antiporters with the corresponding nucleoside phosphate as the mechanism for concentration of solutes in the Golgi lumen (Hirschberg and Snider, 1987; Milla and Hirschberg, 1989; Waldman and Rudnick, 1990; Abeijon et al., 1993; Berninsone et al., 1994). The aim of this article is not to provide a comprehensive review of this topic, which has been done recently (Hirschberg, 1996), but to highlight some important unanswered questions and new avenues of experimentation of this topic.

Chemical and functional studies on the importance of purple membrane lipids in bacteriorhodopsin photocycle behavior

In native purple membrane (PM), there are approximately 1 squalene, 2 glycolipid sulfate (GLS), and 6 phospholipid (PL) molecules per bacteriorhodopsin (BR) monomer [10]. Brief (∼ 2 min) exposure to 0.1% Triton X-100 removes about 25%, 20%, and 6% of squalenes, GLS, and PL, respectively (this paper) while causing profound changes in the BR photocycle, including the loss of ‘photocooperativity’ [1]. The BR photocycle in Triton-treated PM can be restored to near normal behavior by reconstitution with native PM lipids. Isolated squalenes are not effective whereas PL alone partially restores normal photocycle characteristics.

Sulfation of glycolipids by human gastric mucosa in disease.

The activity levels of sulfotransferase enzymes involved in the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to mucosal membrane and mucus gel glycolipids were studied in fundic and antral mucosal biopsies of patients with severe and chronic gastritis, gastric atrophy, gastric ulcer, and gastric cancer. With sulfotransferase which catalyzes the sulfation of mucus triglucosyl glyceroglucolipid increase in enzyme activity over the control was observed in patients with chronic and severe gastritis, and gastric atrophy, while a decrease in activity was noted in patients with gastric ulcer and gastric cancer. The differences were significant at p less than 0.001 for severe gastritis, gastric ulcer and gastric cancer. The increase in activity of sulfotransferase enzyme involved in the sulfation of membrane galactosylceramide over the control was observed in antral and fundic mucosa of all patients. Significant (p less than 0.001) differences were found in patients with severe gastritis, gastric atrophy and gastric ulcer. The results indicate that considerable changes in the activities of the mucosal sulfotransferase enzymes involved in the synthesis of membrane and secretory sulfolipids occur in gastric disease.

Uniform preparations of large unilamellar vesicles containing anionic lipids.

A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed. The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55). The procedure takes less than 30 min. The resulting unilamellar vesicles are of a single size with a rather low standard deviation. The sizes of these preparations range between 150 and 1000 nm in diameter. Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy. Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure. Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E. coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid. The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform. Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.

Purification and characterization of galactocerebroside sulfotransferase from rat kidney

Galactocerebroside sulfotransferase (EC 2.8.2.11) was purified to apparent homogeneity from rat kidneys. The purified protein is stable at −20 °C, and has an estimated molecular weight of 64,000 and a pI of 5.1. In contrast to other known sulfotransferases, the enzyme appears not to require divalent metal ions for activity. The K m for the donor, 3′-phosphoadenosine 5′-phosphosulfate, is 5.2 μ m. Structural studies on this “active” sulfate donor show the requirement of a phosphate group at the 3′ position of the ribose moiety. Modification of the amino group at either the 6 or 8 position on the purine ring renders the corresponding compounds poor substrates. Both galactosylceramide and lactosylceramide are effective acceptors for this enzyme, while galactosylsphingosine and galactosylglycerolipids are sulfated only poorly, suggesting that the in vivo sulfation of these glycolipids is carried out by different sulfotransferases. The active site of the enzyme contains arginine residues which appear to be important in binding the sulfate donor. The enzyme protein is hydrophobic and binds 0.17 mg [ 3H]Triton X-100/mg protein. The purified enzyme contains bound lipids, consisting primarily of cholesterol and phosphatidylcholine. The lipid environment affects the activity of the enzyme which, in turn, regulates the sulfation of glycolipids.

Synthesis of two purple-membrane glycolipids and the glycolipid sulfate O-(β- d-glucopyranosyl 3-sulfate)-(1→6)- O-α- d-mannopyranosyl-(1→2)- O-α- d-glucopyranosyl-(1→1)-2, 3-di- O-phytanyl- sn-glycerol

The two purple-membrane glycolipids O-β- d-glucopyranosyl- and O-β- d-galactopyranosyl-(1→6)- O-α- d-mannopyranosyl-(1→2)- O-α- d-glucopyranosyl-(1→1)-2, 3-di- O-phytanyl- sn-glycerol were prepared by coupling O-(2,3,4-tri- O-acetyl-α- d-mannopyranosyl)-(1→2)- O-(3,4,6-tri- O-acetyl-α- d-glucopyranosyl)-(1→1)-2, 3-di- O-phytanyl- sn-glycerol ( 9) with 2,3,4,6-tetra- O-acetyl-α- d-glucopyranosyl bromide or 2,3,4,6-tetra- O-acetyl-α- d-mannopyranosyl bromide, respectively, followed by deacetylation. The glycolipid sulfate O-(β- d-glucopyranosyl 3-sulfate)-(1→6)- O-α- d-mannopyranosyl-(1→2)- O-α- d-glucopyranosyl-(1→1)-2,3-di- O-phytanyl- sn-glycerol was prepared by coupling of 9 with 2,4,6-tri- O-acetyl-3- O-trichloroethyloxycarbonyl-α- d-glucopyranosyl bromide in the presence of Hg(CN) 2/HgBr 2 followed by selective removal of the 3‴-trichloroethyloxycarbonyl group, sulfation of HO-3‴, and deacetylation. The suitably protected key-intermediate 9 could be prepared by two distinct approaches.

Halobacterium mediterranei spec, nov., a New Carbohydrate-Utilizing Extreme Halophile

The extremely halophilic rod-shaped bacterium, strain R-4, has features very different from other known Halobacterium species. As do all other extreme halophiles, the organism possesses a high excess of acidic over basic amino acids in its proteins, a cell wall lacking peptidoglycan, and ether-linked diphytanyl lipids. It differs from the "classic" halobacteria in several important ways: it can use many different compounds as sole sources of carbon and energy. It is strongly amylolytic and lipolytic (against different Tweens), and does not produce H(2)S. The cell envelope of R-4 is much thicker than that of other halobacteria. The proportions of polar lipids in the envelope are different, and a new glycolipid sulfate is present in this strain. The envelope has a relatively low protein content, although a glycoprotein similar to the one of Halobacterium salinarium was detected. The G + C content (60%) is lower than that of other halobacteria. It is suggested that R-4 be designated a new species, Halobacterium mediterranei.

Enzymatic sulfation of glycolipids by human gastric mucosa.

A sulfotransferase activity which catalyzes the transfer of sulfate group from 3'-phosphoadenosine-5'-phosphosulfate to galactosylceramide and triglucosyl monoalkylmonoacylglycerol has been demonstrated in antral and fundic mucosa of normal human stomach. With both types of mucosa maximum activity for sulfation of galactosylceramide was obtained at pH 6.8, whereas the pH optimum for sulfation of triglucosyl monoalkylmonoacylglycerol was 7.8. The reactions were stimulated by the addition of Triton X-100, Mg2+ and F1-. The sulfotransferase activity of fundic mucosa for the synthesis of sulfated glyceroglucolipid was about two times higher than that of antral mucosa, while the enzyme activity for sulfation of glycosphingolipid was similar in both areas of the stomach.

Polar lipids of an extremely halophilic bacterial strain (R-4) isolated from salt ponds in Spain

The lipids of an extremely halophilic bacterium, strain R-4, isolated from the salt ponds in Spain at Alicante, were found to contain 93% polar lipids and 7% non-polar lipids. Four major polar lipids were detected, all derivatives of 2,3-di- O-phytanyl- sn-glycerol: (i) a novel glycolipid sulfate, 2,3- di-O- phytanyl-1-O-[α- d- mannopyranosyl-6'- sulfate-(1' → 2')-O-α- d- glucopyranosyl]-sn- glycerol , (ii) phosphatidylglycerol, (iii) phosphatidylglycerophosphate, (iv) a glycolipid, 2,3- di-O- phytanyl-1-O-[α- d-[ mannopyranosyl-(1' a ̊ 2')-O-α- d- glucopyranosyl]-sn- glycerol . Traces of four other phospholipids and one glycolipid were also detected.

Reconstitution of bacteriorhodopsin vesicles with Halobacterium halobium lipids. Effects of variations in lipid composition.

Reconstitution of bacteriorhodopsin into vesicles has been studied using individual and defined mixtures of fractionated Halobacterium halobium polar lipids. The lipids varied greatly in their ability to form vesicles that translocated protons and showed stimulation by valinomycin. Thus, phosphatidylglycerol phosphate gave mainly lipid-protein aggregates, while glycolipid sulfate gave mainly functional vesicles. Addition of glycolipid sulfate to phosphatidylglycerol phosphate promoted vesicle formation. Reconstitutions performed with different combinations of the purified lipids showed: 1) that vesicles with maximal proton pumping are obtained with a mixture of phosphatidylglycerol phosphate and glycolipid sulfate at a ratio close to that found in the purple membrane; 2) increasing the concentration of glycolipid sulfate in vesicles increases the stimulation of proton translocation by valinomycin; 3) phosphatidylglycerol sulfate cannot replace glycolipid sulfate or phosphatidylglycerol phosphate, and 4) vesicles containing sulfated polar lipids only are inferior to those in which liquid combinations containing phosphatidylglycerol phosphate are used.

Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney.

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.

Structural identification of minor glycolipids in Halobacterium cutirubrum

Three minor glycolipids were isolated from the extreme halophile, Halobacterium cutirubrum, and their structures were established by chemical degradation, permethylation analysis and spectrometric procedures as follows: (i) a glycolipid sulfate (GL-1), 2,3-di- O-phytanyl-1- O-[ Galp-(3- SO − 4) β1 → 6Man p(3 → 1 αGalf) α] → 2 Glcpα]- sn-glycerol; (ii) a glycolipid (GL-2), the desulfated product of GL-1, 2,3-di- O-phytanyl- l- O-[Gal pβ1 → 6 Manp(3< ← 1 αGalf) α1 → 2 Glcpα]- sn-glycerol and (iii) a glycolipid (GL-3), 2,3-di- O-phytanyl- l- O[ Galp β1 → 6 Manpαl → 2 Glcpα]- sn-glycerol.

Structural analysis by mass spectrometry and NMR spectroscopy of the glycolipid sulfate from Halobacterium salinarium and a note on its possible function

The major glycolipid sulfate of the extreme halophile Halobacterium salinarium was isolated and characterised mainly by mass spectrometry and NMR spectroscopy. The mass spectrum of the permethylated, desulfated and trimethylsilylated derivative showed the molecule to be a trihexosyl glycerol C 20-diether with the sulfate group on the terminal hexose. A 3-position of the sulfate was indicated by the mass spectrum obtained after acetylation and trimethylsilylation (solvolysis of sulfate and replacement by a trimethylsilyl group). The NMR spectrum of the desulfated permethylated glycolipid gave conclusive evidence for the presence of one β and two α anomeric protons. With the knowledge of degradation data it was possible to assign the β signal to galactose (terminal hexone), and the α signals to glucose and mannose. These data together make it likely that the glycolipid sulfate is identical in structure with the glycolipid from Halobacterium cutirubrum characterised previously (M. Kates and P.W. Deroo, J. Lipid Res., 14 (1973) 438). On the basis of a suggested function of cerebroside sulfate of animal origin (identical polar end with the bacterial glycolipid: β-galactopyranose-3-sulfate) and the present knowledge of ion transport in Halobacteria, it is proposed that the bacterial glycolipid may function as a selective K + receptor for the K + transport from a high-Na + and low-K + outside medium.

Characterization and composition of the purple and red membrane from Halobacterium cutirubrum;.

The purple membrance (bacteriorhodopsin) isolated from cells of Halobacterium cutirubrum grown anaerobically in the light was shown to contain 77% protein and 20% lipils by weight. The protein component consisted of a single protein moeity, having a molecular weight of (19.6 plus or minus 0.8) times 10-3, complexed with retinal in mole ratio of 2:1, respectively. The protein moeity is not glycosylated but may be phosphorylated (ca. 2 mol of phosphate per mole of protein). The red membrance contains 56%protein and 38% lipids, including bacterioruberins. Several polypeptide components are present including some which may be glycosylated and/or phosphorylated. The lipids of both membranes contained phosphatidyl glycerophosphate (52%) and phosphatidyl glycerol (3-4%) but the sulfated lipid components, glycolipid sulfate and phosphatidyl glycerosulfate, were present exclusively in the purple membrane, the red membrane containing instead two unidentified glycolipids. Neutral lipids (squalenes, vitamin 75-8, etc.) were present in both membranes to the extent of 7-9%.

Osmometric and microscopic studies on bilayers of polar lipids from the extreme halophile, halobacterium cutirubrum

The major membrane polar lipid components in Halobacterium cutirubrum are the diphytanyl ether analogues of phosphatidylglycerol phosphate, glycolipid sulfate, phosphatidylglycerol and phosphatidylglycerol sulfate. Dispersions of total polar lipids in water formed large birefringent liposomes showing concentric lipid bilayers in the elctron microscope; they behaved as ideal osmometers in KCl or NaCl solutions in the concentration range 0.005–0.2 M. At concentrations above 0.2 M KCl the liposomes shrank to spherical particles which were much less birefringent, showed no distinct bilayer structures by electron microscopy, and no longer behaved as ideal osmometers. Dispersions of phosphatidylglycerol phosphate, phosphatidylglycerol or phosphatidylglycerol sulfate alone did not behave as osmometers at any concentration of KCl or NaCl, but glycolipid sulfate alone or mixed with phosphatidylglycerol phosphate or phosphatidylglycerol phosphate + phosphatidylglycerol sulfate showed ideal osmometer behavior in 0.005–0.2 M KCl or NaCl. The highly negatively charged total polar lipids of H. cutirubrum thus can form stable lipid bilayers only at low ionic concentrations (0.005–0.2 M), much lower than the salt concentration (4 M) of the growth medium, and the presence of glycolipid sulfate is essential. Stability of the membrane in 4 M salt appears to require direct participation of the protein components.

Structure determination of the glycolipid sulfate from the extreme halophile Halobacterium cutirubrum

A sulfur-containing glycolipid, accounting for ca. 25% of the total polar lipids, has been isolated from the extreme halophile Halobacterium cutirubrum. The ammonium salt of the lipid was found to have the molecular formula C(61)H(117)O(21)S.NH(4), and on strong acid hydrolysis it yielded 2,3-di-O-phytanyl-sn-glycerol, glucose, mannose, galactose, and sulfate in equimolar proportions. Infrared and NMR spectra indicated the presence of a secondary sulfate group. Solvolysis of the lipid in 0.004 m HCl in tetrahydrofuran resulted in rapid release of inorganic sulfate and formation of galactosyl-mannosyl-glucosyl diphytanyl glycerol ether. With higher acid concentration (0.25 m methanolic HCl), stepwise hydrolysis of monosaccharide units occurred, giving mannosyl-glucosyl glycerol diphytanyl ether and monoglucosyl glycerol diphytanyl ether. The position of attachment of the sugars and of the sulfate group was determined by methylation of the free acid form of the glycolipid sulfate, followed by acid hydrolysis and gas-liquid chromatographic analysis of the partially methylated sugars as the alditol acetates. The configuration of the glycosidic linkages was established both by optical rotation measurements and by specific enzymatic hydrolysis. The results obtained established the structure as 2,3-di-O-phytanyl-1-O-[beta-d-galactopyranosyl-3'-sulfate-(1' -->6')-O-alpha-d-mannopyranosyl-(1' --> 2')-O-alpha-d-glucopyranosyl]-sn-glycerol.