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A variety of procedures have been developed for determining the sulfate ester content of various biomolecules. Ion chromatography (IC), that is, quantitation of ionic substances by ion conductimetry after separation by anion-exchange chromatography, has been increasingly utilized for the determination of inorganic sulfate in clinical and environmental samples. We adopted suppressed-mode IC to the determination of lipid- or glycolipid-bound sulfate released by acid hydrolysis and found that it has the advantage of increased precision for wide concentration ranges (30 pmol to approximately micromol) and lack of interference from other lipids. To minimize deterioration of the separation column, the lipophilic constituents in the acid hydrolysate were removed by a two-phase partition system of chloroform-methanol-water. The inorganic sulfate was quantitatively extracted into the aqueous phase by replacing water with an alkaline buffer. By this method, the concentration of sulfolipids was determined in the kidney of mammals with various body mass. Sulfolipids were more concentrated in the kidney of smaller animals, which have higher maximum urine concentrating activity per gram of body mass, supporting the hypothesis of the function of sulfolipids as an ion barrier on the luminal surface of renal tubules.

UDP-galactose:ceramide galactosyltransferase (CGT) catalyzes the final step in the synthesis of galactosylceramide (GalCer). It has previously been shown that CGT-deficient mice do not synthesize GalCer and its sulfated derivative GalCer I(3)-sulfate (galactosylsulfatide, SM4s) but form myelin containing glucosylceramide (GlcCer) and sphingomyelin with 2-hydroxy fatty acids. Because relatively high concentrations of GalCer and SM4s are present also in mammalian kidney, we analyzed the composition of lipids in the kidney of Cgt(-/-) and, as a control, Cgt(+/-) and wild-type mice. The homozygous mutant mice lacked GalCer, galabiaosylceramide (Ga(2)Cer), and SM4s. Yet, they did not show any major morphological or functional defects in the kidney. A slight increase in GlcCer containing 4-hydroxysphinganine was evident among neutral glycolipids. Intriguingly, more polar sulfoglycolipids, that is, lactosylceramide II(3)-sulfate (SM3) and gangliotetraosylceramide II(3),IV(3)-bis-sulfate (SB1a), were expressed at 2 to 3 times the normal levels in Cgt(-/-) mice, indicating upregulation of biosynthesis of SB1a from GlcCer via SM3. Given that SM4s is a major polar glycolipid constituting renal tubular membrane, the increase in SM3 and SB1a in the mice deficient in CGT and thus SM4s appears to be a compensatory process, which could partly restore kidney function in the knockout mice.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3′-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.

Specific sugar ligands which bind to rat L-selectin (LECAM-1, lymphocyte homing receptor)-IgG chimera were investigated by thin-layer chromatography-binding assay and ELISA. Rat L-selectin-IgG bound to native sulfated glycolipids such as sulfatide (I3SO3-GalCer), seminolipid, SM3(II3SO3-LacCer), SB2(III3SO3,II3SO3-Gg3Cer), SB1a (IV3SO3,II3SO3-Gg4Cer). The binding activity of the SM2 (II3SO3-Gg3Cer) carrying the internal sulfated Gal was very low, indicating that non-reducing terminal SO3H-3Galβ1- structure is essential for the binding. The reactivity of sulfatide was higher than that of sialyl Lex[Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-Ceramide] either in the presence of 1 mM Ca2+or 1 mM Ca2+and 5 mM EGTA. All the non-fucosylatedgangliosides including ganglio-series andlacto-series type I and II chains tested gave a negative reaction. Neutral glycosphingolipids tested were all negative. Binding assay using synthetic sulfatide analogs indicated that the L-selectin-binding to its sulfated sugar ligands is position specific and depends on the number of the sulfate group, i.e., (SO3)3-3,4,6Gal- > (SO3)2-3,6Gal- > SO3-3Gal- > SO3-3Glc- > SO3-6Gal- > SO3-2Gal-. Lower reactivity of the chimera to SO3-3Glc- rather than SO3-3Gal- indicates that the chimera recognizes the configuration of hydroxyl group linked to the 4 carbon atom of thepyranose ring in galactose.

A cell line (SMKT-R3) established from human renal cell carcinoma was characterized for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with 35S-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.

A novel sulfated glycosphingolipid containing a sulfated glucosyl residue was isolated from rat kidney and purified to homogeneity by column chromatographies with DEAE-Sephadex and silica beads. By compositional analyses, permethylation studies, one- and two-dimensional proton magnetic resonance spectroscopy, infrared spectroscopy, negative secondary ion mass spectrometry, solvolysis, and immunostaining on thin layer chromatogram, the structure of this glycolipid was proposed to be HSO3-3Glc β 1-1Cer (where Cer is ceramide). The ceramide portion consisted of 4-D-hydroxysphinganine as the sole long chain base, and the fatty acid consisted of predominantly tetracosanoic acid, deduced from both composition analysis and negative secondary ion mass spectrometry. The yield of glucosyl sulfatide was about 5 nmol/g of tissue, being about three times as much as that of lactosylceramide sulfate.

A novel bis-sulfated tetraglycosylceramide containing N-acetylgalactosamine was isolated from the lipid extract of rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, alkaline methanolysis, and column chromatographies with DEAE-Sephacel and silica beads. By infrared spectroscopy, proton magnetic resonance spectroscopy, periodate oxidation, solvolysis, chromium trioxide oxidation, and methylation analysis of the native and partially degraded compounds, the structure of this glycolipid is proposed to be (HSO3-3)Gal beta 1-3GalNAc beta 1-4(HSO3-3)Gal beta 1-4Glc beta 1-1Cer. The position of sulfate ester groups and the sequence of carbohydrates were further confirmed by methylation analysis and direct probe mass spectrometry after desulfation-trideuteriomethylation of permethylated glycolipids. The yield of this sulfoglycolipid was 5.5 nmol/g of tissue, which was about 25 and 50 mol %, respectively, of monosulfo-and bis-sulfogangliotriaosylceramide from rat kidney.

A novel sulfated glycosphingolipid that belongs to the “globo-series” was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform/methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative thin layer chromatography. The structure and the properties were studied by infrared spectroscopy, two-dimensional proton magnetic resonance spectroscopy, negative secondary ion mass spectrometry, solvolysis, compositional and methylation analyses, monoclonal antibodies, and sulfatide-binding proteins. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3Galβ1–3GalNAcβ1–3Gala1–4Galβ1–4Glcβ1-lceramide. The proton resonance at 3.93 ppm of the H-3 of the sulfated nonreducing terminal galactose of this lipid was downfield-shifted (Δ0.48 ppm), as compared with H-3 of the internal β-galactose because of the electronegativity of the sulfate ester. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (MC-631) (Kannagi, R., Cochran, N. A., Ishigami, F., Hakomori, S., Andrews, P. W., Knowles, B. B., and Solter, D. (1983) EMBO J.2, 2355–2361), whose epitope is R-3GalNAcβ1–3Galα1–4Galβ1-R′, on thin layer chromatograms and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 0.19 nmol/g of tissue, which was about 0.09 and 0.5 mol % of galactosyl and lactosyl sulfatides in human kidney.

A novel sulfoglycosphingolipid containing two sulfate ester groups was isolated from the lipid extract of rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, alkaline methanolysis, and column chromatographies with DEAE-Sephacel and silica beads. By infrared spectroscopy, proton magnetic resonance spectroscopy, periodate oxidation, solvolysis, chromium trioxide oxidation, and methylation analysis of the native and partially degraded compounds, the structure of this glycolipid is proposed to be (HSO3-3)GalNAc beta 1-4(HSO3-3)Gal beta 1-4Glc beta 1-1Cer. The presence of a unique 3-O-sulfated N-acetylgalactosamine structure was confirmed by gas chromatography-mass spectrometry. The yield of this sulfoglycolipid was 11.2 nmol/g of tissue, which was about half of that of monosulfogangliotriaosylceramide from rat kidney.

Gangliotriaosylceramide 3‘-sulfate (GgOse3Cer-II3-sulfate) contains the sugar sequence similar to that of GM2 ganglioside except that the NeuAc in GM2 is replaced by a sulfate group. Due to this structural similarity, we have studied the in vitro synthesis of GgOse3Cer-II3-sulfate using the system for GM2. Our results showed that GgOse3Cer-II3-sulfate could be synthesized from lactosylceramide 3‘-sulfate and UDP-GalNAc catalyzed by N-acetylgalactosaminyltransferase prepared from rat brain (Dicesare, J. L., and Dain, J. A. (1971) Biochim. Biophys. Acta 231, 385-393). As in the case of GM2, the GgOse3Cer-II3-sulfate biosynthesized in vitro or isolated from rat kidney could also be cleaved by human beta-hexosaminidase A in the presence of GM2-activator (Li, S.-C., Hirabayashi, Y., and Li, Y.-T. (1981) J. Biol. Chem. 256, 6234-6240). The fact that the GM2-activator could stimulate beta-hexosaminidase A to hydrolyze both GM2 and Gg-Ose3Cer-II3-sulfate indicates that these two glycolipids may be catabolyzed by the same mechanism.

The detailed substrate specificity of the UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase to form the Sd(a+) blood group active carbohydrate determinant GalNAc beta 1-4(NeuAc alpha 2-3)Gal was studied using a membrane fraction prepared from human gastric fundic mucosa. Various sialosylated oligosaccharides and gangliosides were examined as acceptor substrates. Oligosaccharide substrates were fluorescence-labelled with 2-aminopyridine, and the transferase activity was quantified by h.p.l.c. using a reversed-phase column. The structures of the products were determined by glycosidase degradation and proton n.m.r. 3′-Sialyl-lactose (II3NeuAcLac), 3′-sialyl-lactotetraose (IV3NeuAcLc4), and 3′-sialyl-lactoneotetraose (IV3NeuAcnLc4) were good substrates for the beta 1-4GalNAc transferase in gastric fundic mucosa, but 6′-sialyl-lactoneotetraose (IV6NeuAcnLc4) or 6′-sialyl-lactose (II6NeuAcLac) were not. Gangliosides with a terminal NeuAc alpha 2-3Gal residue such as GM3, sialylparagloboside, GM1b and GD1a were also studied. The activity of beta 1-4GalNAc transfer to sialylparagloboside was much higher than that to GM2, GM1b or GD1a in spite of them having the same terminal residue. Measurement of the activity of the beta 1-4GalNAc transferase in biopsy specimens demonstrated that the activity was localized in gastric fundic mucosa and was absent in pyloric mucosa, intestinal metaplasia and gastric cancer tissue. Thus the beta 1-4GalNAc transferase present specifically in fundic mucosa required a NeuAc alpha 2-3Gal residue connected to either type-1-chain or type-2-chain oligosaccharides. In glycolipids, the acceptor specificity was restricted to NeuAc alpha 2-3Gal beta 1-4GlcNAc because the NeuAc alpha 2-3Gal beta 1-3GalNAc structure in ganglio-series glycolipids was not a good acceptor substrate.

A Serratia rubidaea isolate with wetting activity when grown at 30 but not 37 degrees C was examined for the production of specific lipids. Two novel lipids (rubiwettins R1 and RG1) were isolated and shown to be able to lower the surface tension of saline to 26 mN/m. These lipids were located in extracellular vesicles found in a 30 degrees C culture of S. rubidaea. Chemical structures of these biosurfactants were determined by degradation product analyses, infrared spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Rubiwettin R1 was proposed to be a mixture of 3-(3'-hydroxytetradecanoyloxy)decanoate, 3-(3'-hydroxyhexadecenoyloxy)decanoate, and minor molecular isomers. The structure of rubiwettin RG1 was proposed to be beta-D-glucopyranosyl 3-(3'-hydroxytetradecanoyloxy)decanoate. The importance of such surface-active exolipids in bacterial occupancy on surfaces was suggested.

A novel class of sulfoglycosphingolipid, a sulfate analog of ganglioside, was isolated from mammals for the first time. This sulfated ganglioside was purified from rat kidney by column chromatographies on anion exchangers and silica beads. One-dimensional 1H NMR, compositional and permethylation analyses showed that this glycolipid has a Gg4Cer core with 1 mol each of sulfate ester and N- glycolylneuraminic acid (NeuGc) at C-3 of galactose. The major ceramide consisted of nonhydroxy fatty acids (24:0 and 22:0) and 4-hydroxysphinganine (t18:0), deduced from the compositional analysis and negative liquid secondary ion mass spectrometry (LSIMS). Mild acid hydrolysis and solvolysis produced compounds which correspond to Gg4Cer IV3-sulfate (SM1b) and II3NeuGcalpha-Gg4Cer (GM1a (NeuGc)), respectively. The abundant ions characteristic for sulfated mono- and disaccharides in high-energy collision-induced dissociation (CID) spectra were consistent with the structure at the non-reducing terminus, HSO3 -O- Hex -O- HexNAc- rather than the alternative structure, NeuGc -O- Hex -O- HexNAc-. The two-dimensional 1H NMR further evidenced the presence of a 3 -O- sulfated galactose in the molecule. From these results the complete structure was proposed to be HSO3-3Galbeta-3GalNAcbeta-4(NeuGcalpha-3)Galb eta-4Glcbeta-1Cer (II3NeuGcalpha-Gg4Cer IV3-sulfate).