i(3)so3-galactosylceramide and cholesteryl-sulfate

Matrix metalloproteinase (MMP)-7 binds to cell surface cholesterol sulfate (CS) and acts as a membrane-associated protease. We have previously found that CS modulates the substrate preference of MMP-7, thereby regulating its pericellular proteolytic action. MMP-7 potentially associates with the cell surface via sulfatide (SM4) and cardiolipin (CL) when they are overexpressed on the cell surface. Here, we investigated the molecular interaction between these acidic lipids and MMP-7 or its substrates, and their effects on the activity of MMP-7. Studies using MMP-7 variants with low CS-binding ability suggested that these lipids interact with a similar site on MMP-7. The hydroxamate-based MMP inhibitor TAPI-1 markedly reduced the affinity of MMP-7 for CS and CL, whereas that for SM4 was not affected by TAPI-1. These three acidic lipids also had different effects on the hydrolytic activity of MMP-7 towards a small peptide substrate: SM4, CL and CS reduced the activity to 80%, 92%, and 20%, respectively. Nevertheless, SM4 and CS similarly accelerated the MMP-7-catalyzed degradation of fibronectin and laminin-332, whereas CL did not. The increased proteolysis of substrate was observed only when both substrate and enzyme had affinity for the lipid, suggesting that the lipids probably bring the reactants into closer proximity. Furthermore, MMP-7 bound to cell surface SM4 or CS cleaved specific cell surface proteins and released similar fragments, whereas the cleavage was not stimulated by cell surface CL-bound MMP-7. This study provides a novel mechanism by which acidic lipids differentially regulate pericellular proteolysis by MMP-7 through allosteric alteration of the substrate-binding site and their inherent affinities for MMP-7 substrates.

Topics: Binding, Competitive; Cardiolipins; Cell Adhesion Molecules; Cell Line, Tumor; Cell Membrane; Cholesterol Esters; Dipeptides; Fibronectins; Humans; Hydroxamic Acids; Kalinin; Kinetics; Matrix Metalloproteinase 7; Matrix Metalloproteinase Inhibitors; Protein Binding; Proteolysis; Sulfoglycosphingolipids

Gastric sulfatide, whose carbohydrate moiety resembles that of the anti-ulcer drug sucralfate, has been shown to play a role in mucosal protection in an experimental ulcer model. To elucidate the functional significance of gastric lipids, precise determination of the lipids in human gastric fluid and epithelium was performed, and the anti-ulcer effects of all lipids in the fluid were measured in mouse ulcer models.. The lipids in human gastric fluid and epithelium were analyzed by thin layer chromatography and immunostaining, and the anti-ulcer effects of gastric lipids were determined using mouse ulcer models.. Human gastric epithelium contained both sulfatide and cholesterol sulfate (CS) as sulfolipids, which were also detected in gastric fluid, showing their stable natures in the gastric fluid. Hemorrhaging in HCl-induced gastric lesions was suppressed in a dose-dependent manner by the administration of sulfolipid-containing liposomes, but suppression of stress ulcers was only accomplished with CS-containing liposomes, ie, not with sulfatide-containing ones, due to the longer retainment of CS than sulfatide in the stomach.. Among the lipids in human gastric fluid, CS was revealed to exhibit a gastroprotective activity, which was more effective than that of sulfatide.

Topics: Adult; Aged; Aged, 80 and over; Animals; Anticarcinogenic Agents; Cholesterol Esters; Chromatography, Thin Layer; Cytoprotection; Disease Models, Animal; Female; Gastric Juice; Gastric Mucosa; Humans; Hydrochloric Acid; Lipid Metabolism; Lipids; Liposomes; Mice; Mice, Hairless; Mice, Inbred BALB C; Middle Aged; Stomach Ulcer; Sulfoglycosphingolipids

Cholesterol sulfate (CS) and sulfatides in the epithelium of the digestive tract were found in the 1000xg supernatants of digestive fluid, particularly in gastric juices containing the duodenal contents and bile acids, there being 14-131 microg of CS and 3-54 microg of sulfatides per mg of protein in the fluid, respectively. CS and sulfatides dissolved in detergents including bile acids inactivated pancreatic trypsin to the same level as by DMSO-solubilized sulfated lipids at 37 degrees C. Similarly, pancreatic DNase I was inhibited by CS solubilized with DMSO or bile acids, but not by sulfatides or other membrane lipids at 37 degrees C. Both the sulfate group and the hydrophobic side chain of CS were indispensable structures for the inhibition of DNase I. Also, the optimum molar ratio of bile acids to CS was important for expression of the inhibitory activity of CS toward DNase I, it being 0.18 of the optimum ratio for sodium taurocholate, and the molar ratio of CS to DNase I for complete inhibition was 342:1. Thus, CS was shown to play a role as an epithelial inhibitor of DNase I in concert with bile acids.

Topics: Adult; Aged; Bile Acids and Salts; Cholesterol Esters; Deoxyribonuclease I; Detergents; Endopeptidases; Enzyme Activation; Female; Gastric Juice; Humans; Lipids; Male; Middle Aged; Pancreas; Serine Proteinase Inhibitors; Sulfates; Sulfoglycosphingolipids; Trypsin

Sulfated lipids, cholesterol sulfate (CS) and I3SO3-GalCer, are commonly present in the epithelia of the digestive tracts of pigs, humans, rabbits, and rats. CS was the only sulfated lipid in the esophageal epithelia of these mammals, and I3SO3-GalCer, together with CS, was detected in the epithelia of the gastrointestinal tracts, at a concentration higher than 0.05 micromol per gram of dry weight. Although no sulfated lipids were present in the pancreatic duct, they were found in relatively high concentrations in the duodenal, jejunal, and ileal epithelia. To elucidate the functional significance of sulfated lipids in the digestive tract, we determined the effect of CS and I3SO3-GalCer on the activities of pancreatic and Pseudomonas aeruginosa elastases and found that both characteristically inhibited the pancreatic elastase but not the P. aeruginosa elastase. Desulfation of CS and I3SO3-GalCer abolished their inhibitory activity, and other membrane constituents including free fatty acids, phospholipids, and gangliosides failed to inhibit pancreatic elastase. In addition, steroid sulfates, such as dehydroepiandrosterone sulfate and pregnenolone sulfate, did not exhibit any inhibitory activity toward pancreatic elastase, indicating that the sulfate group and a suitable hydrophobic side chain are required in the inhibition of elastase. Inhibition of elastase by sulfated lipids occurred in a dose-dependent manner, and the molar ratios of CS and I3SO3-GalCer to elastase at which the enzyme activity was inhibited to 50% of the maximum level were 6:1 and 9:1, respectively. CS-treated elastase had the same Km and a lower Vmax compared with the untreated enzyme, and sulfated lipids were observed to bind tightly to the enzyme, suggesting irreversible inhibition. Thus, CS and I3SO3-GalCer in the digestive tracts of mammals were shown to function as epithelial inhibitors of pancreatic elastase.

Topics: Animals; Bacterial Proteins; Cholesterol Esters; Galactosylceramides; Humans; Intestinal Mucosa; Lipid Metabolism; Metalloendopeptidases; Pancreatic Elastase; Rabbits; Rats; Sulfates; Sulfoglycosphingolipids; Swine

Cholesterol sulfate (CS) recently has been shown to be involved in signal transduction pathway. To evaluate its functional significance, we determined the concentration of CS, and the specific activities of cholesterol sulfotransferase and CS sulfatase in various tissues of rabbit, and compared them with the concentration of sulfoglycolipids in rabbit tissues. CS was present in the epithelia and mucosa, but not in the tunica muscularis, of the digestive tract, trachea, uterine endometrium and uterine cervix. It was also present in lung, spleen, kidney, prostate, skin, hair, and nail at relatively high concentrations. Its concentration in the uterine endometrium was nine times higher in pseudopregnant rabbits than in nonpregnant rabbits because of activation of cholesterol sulfotransferase and inhibition of CS sulfatase in the pseudopregnant rabbits. Sulfoglycolipids were not detected in the uterine endometria of either non-pregnant- or pseudopregnant rabbits. However, sulfoglycolipids were detected at relatively high concentrations in the cerebrum, cerebellum, stomach, duodenum, jejunum, testis, and kidney of rabbits and thus the tissues in which both sulfolipids were detected were the gastrointestinal tract and kidney. In the digestive tract, the concentration of CS decreased in the order esophagus, stomach, duodenum, and jejunum, but that of sulfatide increased in the same order, indicating distribution of CS in the squamous epithelium. In addition, both CS and sulfatide were detected in the serum. On the other hand, CS sulfatase activity was detected in all tissues examined, even in hair, from which the enzyme was liberated by brief sonication, and its highest specific activity was detected in the liver. The specific activity of cholesterol sulfotransferase varied among the tissues examined and was found to be significantly high in the esophageal epithelium and the uterine endometrium of pseudopregnant rabbit, indicating involvement of cholesterol sulfation in the formation of epithelium.

Topics: Animals; Antibodies, Monoclonal; Arylsulfatases; Carbohydrate Sequence; Cholesterol Esters; Chromatography, Thin Layer; Densitometry; Epitopes; Female; Glycolipids; Humans; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Rabbits; Steryl-Sulfatase; Sulfoglycosphingolipids; Sulfotransferases; Tissue Distribution

In this study, we determined the concentrations of acidic lipids, including cholesterol sulfate (CS), sulfatide and GM3 ganglioside, in human sera of non-pregnant state and during the course of pregnancy. In human sera of non-pregnant women, GM3 was present at a concentration of 8 nmol/ml and the concentrations of CS and sulfatides were less than 20% of that of GM3. The concentration of sulfatides in sera at the second trimester of gestation was decreased, but CS gradually increased from the first to the third trimester of gestation with a correlation coefficient of 0.66, and a correlation between the concentration of CS and weeks of gestation (p<0.01). CS was also contained in the placental villi, and its concentration increased from the first to the third trimester of gestation, suggesting that placental CS is one of the source of CS in the blood by shedding.

Topics: Cholesterol Esters; Chorionic Villi; Chromatography, Thin Layer; Female; G(M3) Ganglioside; Humans; Lipids; Pregnancy; Spectrometry, Mass, Fast Atom Bombardment; Sulfoglycosphingolipids; Time Factors

The substrate specificity of the purified, mammalian phosphatidylinositol 3-kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed.

Topics: Adenosine Triphosphate; Animals; Catalysis; Cell Line; Cholesterol; Cholesterol Esters; Detergents; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Saccharomyces cerevisiae; Spodoptera; Substrate Specificity; Sulfoglycosphingolipids

Region II of the malaria circumsporozoite (CS) protein is highly conserved between the CS proteins of different species of malaria. Amino acid sequences homologous to that of region II are found in thrombospondin, properdin, von Willebrand factor and a few other proteins. We show here that the native CS protein from the rodent parasite Plasmodium berghei, and recombinant Plasmodium vivax and Plasmodium falciparum CS proteins containing region II, but not recombinant proteins lacking region II, specifically bind to sulfatides and cholesterol-3-sulfate. The binding is abolished following reduction and alkylation of the proteins. Region II contains 2 cysteines separated by only 3 amino acids, S(N), V, T, and these are the only cysteines present in our recombinant proteins. Therefore, our findings strongly suggest that the region II cysteines are linked by a disulfide bond forming a small peptide loop. We also present evidence that the recognition of sulfatides, cholesterol-3-sulfate, or other cross-reactive sulfated macromolecules by region II may be required during sporozoite invasion of liver cells. Antibodies to a peptide representing region II react with live sporozoites and with sporozoites fixed with glutaraldehyde, indicating that this region is exposed on the surface of the parasites. Furthermore, we have found that the sulfatide and cholesterol-3-sulfate recognition by the CS proteins, and the invasion of hepatocytes by P. berghei sporozoites, are specifically inhibited by dextran sulfate.

Topics: Amino Acid Sequence; Animals; Antigens, Protozoan; Binding Sites; Cholesterol Esters; Cysteine; Dextran Sulfate; Disulfides; Molecular Sequence Data; Plasmodium; Protein Binding; Protozoan Proteins; Recombinant Proteins; Sulfoglycosphingolipids

The effect of galactocerebroside 3'-sulfate (sulfatide) or cholesterol sulfate on syncytium formation induced by bovine immunodeficiency virus (BIV)-infected cells was investigated in vitro. Sulfatide was purified from bovine brain and incorporated in liposomes which were composed of egg phosphatidylcholine (PC), cholesterol (Chol), and dipalmitoylphosphatidic acid (DPPA). Either sulfatide- or cholesterol sulfate-containing liposomes effectively prevented syncytium formation induced by BIV-infected cells, but the inhibitory effect of sulfatide alone on syncytium formation was low. On the other hand, neither liposomes containing galactocerebroside nor liposomes composed of egg PC, Chol, and DPPA had any effect on syncytium formation induced by BIV-infected cells. These results suggest that liposomes containing sulfatide or cholesterol sulfate are an efficient agent to inhibit syncytium formation induced by BIV-infected cells, and that sulfate residue might play an important role in the inhibition of syncytium formation.

Topics: Animals; Cattle; Cholesterol Esters; Cytopathogenic Effect, Viral; Giant Cells; Immunodeficiency Virus, Bovine; Liposomes; Sulfoglycosphingolipids

Cholesterol sulfate was found to display a strong ability to trigger the activation of Factor XII and prekallikrein in the presence of HMW kininogen. Other sulfate ester derivatives of testosterone, estrone, pregnenolone and dehydroepiandrosterone and cholesterol tested did not show any effect on the activation of Factor XII and prekallikrein. The activity of cholesterol acetate and sulfodeoxycholic acid was very weak. Cholesterol sulfate markedly shortened the partial thromboplastin time of normal human plasma, but not plasmas deficient in Factor XII, Factor XI and HMW kininogen. Upon prolonged incubation, the partial thromboplastin time of prekallikrein-deficient plasma was also shortened. Moreover, as well as kaolin and sulfatide, cholesterol sulfate shortened the partial thromboplastin time of plasmas from monkey, dog, rat, guinea pig, sheep, cow, hog and horse, but not from duck and chicken. Since cholesterol sulfate is distributed in erythrocytes, various organs and body fluids, it may play an important role in the activation of the intrinsic blood coagulation system.

Topics: Animals; Blood Coagulation; Cattle; Cholesterol Esters; Factor XII; Humans; Kallikreins; Kaolin; Prekallikrein; Sulfoglycosphingolipids; Time Factors

The lipid composition of the brain, including myelin, was studied in detail in two cases with a variant form of metachromatic leukodystrophy (multiple sulphatase deficiency type). In the white matter, the sulphatide concentration was 3-4 times higher than the normal level in both cases. There was a significant accumulation of cholesterol sulphate in the brain, liver and kidney of both cases. The ganglioside pattern in the grey and white matter was abnormal, with a higher proportion of GM3, GM2 and GD3-gangliosides. Non-lipid hexosamine contents were increased 1.5-2 times in brain, 8-10 times in liver and 2-3 times in kidney. Increased amounts of glucocerobroside, ceramide lactoside and ceramide trihexoside were present in grey and white matter of both cases. Recovery of purified myelin from two patients' brains was much less than from control (1-2% in case 1 and 20-30% in case 2). The lipid composition of myelin was almost normal except for a higher proportion of sulphatide, with a decreased amount of cerebroside. The fatty acid compositions of myelin sulphatide and sphingomyelin were almost normal, while non-hydroxy fatty acids of cerebroside contained less long-chain fatty acids, as characterized by a significant increase of C16:0 and C18:0 fatty acids. The myelin polypeptide pattern by SDS-disc gel electrophoresis showed a relative decrease of basic protein and of proteolipid protein. A possible mechanism of myelin loss in MSD is discussed.

Topics: Antigens, CD; Brain Chemistry; Cerebrosides; Child; Child, Preschool; Cholesterol Esters; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Female; Gangliosides; Glucosylceramides; Glycosphingolipids; Hexosamines; Humans; Kidney; Lactosylceramides; Liver; Male; Myelin Sheath; Sphingolipidoses; Sulfoglycosphingolipids