Glycolipids in human lung carcinoma of histologically different types.

Glycolipids were isolated from primary human lung carcinoma tissue of various histologic types: adenocarcinoma, squamous cell carcinoma, and undifferentiated small cell carcinoma. Each type of carcinoma had a characteristic glycolipid pattern. The major glycolipids isolated were ceramide monohexosides, ceramide dihexosides, ceramide trihexosides, globoside, and hematoside. Squamous cell carcinoma and undifferentiated small cell carcinoma showed marked increases of ceramide monohexosides and dihexosides. Adenocarcinoma had a much higher level of the sulfatide (ceramide 3-sulfate-galactoside) as compared to squamous cell carcinoma, undifferentiated small cell carcinoma, or normal lung tissue. Embryonic tissue had more significant levels of sulfatide than did the other carcinomas. Adenocarcinoma had significantly lower levels of glycolipids due mainly to a decrease in the amount of ceramide monohexosides and dihexosides and hematoside.

Association of elevated sulfatides and sulfotransferase activities with human renal cell carcinoma.

Lactosylceramide sulfate and galactosylceramide sulfate were found to increase markedly in human renal cell carcinoma (adenocarcinoma) as compared to uninvolved tissue. Activities of two sulfotransferases toward galactosylceramide and lactosylceramide as substrates were significantly elevated in the carcinoma compared to the uninvolved tissue resulting in enhanced synthesis of the two sulfatides in the carcinoma. The elevation of the two sulfotransferases was parallel in most tumors, suggesting that the same enzyme is responsible for the enhanced synthesis of two sulfatides. No consistent difference in the activity of arylsulfatase A, which desulfates the two sulfatides, was observed between the carcinoma and uninvolved tissue. Both the present and previous results show that the increased synthesis of the sulfatide(s) due to elevated sulfotransferase activity could be a biochemical characteristic common to adenocarcinomas derived from different tissues.

Lysosomal arylsulfatases of human leukocytes: increment of phosphorylated B variants in chronic myelogenous leukemia.

Lysosomal arylsulfatases A and B of peripheral leukocytes from patients with chronic myelogenous leukemia and from healthy subjects were studied. Two enzyme activities of leukemia cells were significantly higher than those of cells from healthy subjects, irrespective of total and differential counts of leukemic cells. Upon anion-exchange chromatography, the arylsulfatases of chronic myelogenous leukemia cells and normal leukocytes were separated into the basic B enzyme and its anionic variant (B1) and A enzyme. However, the amount of B1 enzyme relative to B enzyme or the activity ratio of B1 enzyme to total arylsulfatase B (B + B1) was higher in chronic myelogenous leukemia cells than in normal cells. The anionic property of the enzyme was found to be due to phosphate groups bound to the carbohydrate moiety of the arylsulfatase, based on the following results. When B1 enzyme was treated with alkaline phosphatase followed by isoelectric focusing, it was changed to a less anionic enzyme with heterogeneous components which are ascribed to phosphodiester groups linked to the heterogeneous carbohydrate moiety of the enzyme; no effect was observed by sialidase treatment. Upon treatment of B1 enzyme with endo-beta-N-acetylglucosaminidase H, which cleaves sugar chains of a high mannose type in glycoproteins, the anionic heterogeneous components were converted to the basic component similar to B enzyme. From our present and previous observations, it can be concluded that the increase of phosphorylated forms of the lysosomal hydrolase represents one characteristic of rapidly proliferating neoplastic cells.

Regulation of activity levels of glycolipid sulfotransferases by transforming growth factor alpha in renal cell carcinoma cells.

Accumulation of sulfolipids associated with markedly elevated levels of glycolipid sulfotransferase activities was previously demonstrated in human renal cell carcinoma cells. To explore the regulation mechanisms of sulfoglycolipid synthesis in renal cancer, effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated by using a human renal cell carcinoma cell line, SMKT-R3. Among the growth factors tested, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to increase the sulfotransferase activity markedly (about 300%), but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. The augmented effects of TGF-alpha was abolished by cycloheximide. Since TGF-alpha is known to bind to the same receptor as EGF, SMKT-R3 cells were investigated for the EGF receptor by affinity cross-linking with 125I-EGF. A radiolabeled protein with a molecular mass of 175 kDa corresponding to the ligand-receptor complex was immunoprecipitated with a monoclonal anti-EGF receptor antibody. When production of the growth factors was examined immunochemically, the cells were found to secrete TGF-alpha at a low level and retain it in a membrane-bound form, whereas EGF was not detected. These observations suggest that the sulfotransferase activities are regulated through the autocrine, paracrine, and/or juxtacrine modes of intercellular stimulation by TGF-alpha in human renal cancer cells.

Involvement of protein kinase C in the regulation of glycolipid sulfotransferase activity levels in renal cell carcinoma cells.

Accumulation of sulfolipids associated with elevated levels of glycolipid sulfotransferase activities has previously been demonstrated in renal cell carcinoma cells. To investigate the role of protein kinase C in the synthesis of sulfolipids, the effects of 12-O-tetradecanoylphorbol-13-acetate and protein kinase C inhibitors on glycolipid sulfotransferase activity levels were examined in a human renal cell carcinoma cell line, SMKT-R3. Continuous treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate caused a dose- and time-dependent reduction of the sulfotransferase activity levels. Similarly, protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and staurosporine, reduced the enzyme activities in a dose-dependent manner. These observations suggest that the glycolipid sulfotransferase activity levels are regulated by protein kinase C in SMKT-R3 cells. Furthermore, long-term 12-O-tetradecanoylphorbol-13-acetate treatment resulted in a reduction of sulfolipid synthesis and a decrease of the expression of sulfolipids on the cell surface. Taken together, it is suggested that protein kinase C is involved in the synthesis of sulfolipids through the regulation of the glycolipid sulfotransferase activity levels in renal cell carcinoma cells.

Elevated activities and properties of arylsulfatases A and B and B-variant in human lung tumors.

The activities of arylsulfatases A and B were determined in human primary and secondary tumor tissues (total, 53 cases) of various histological types. Significantly higher activities of these sulfatases were found in almost all the primary lung carcinomas as compared to their corresponding uninvolved tissues. No significant correlation was demonstrated between the enzyme activities and histological figures (stroma amounts, etc.). Lung adenocarcinoma and squamous cell carcinoma showed the presence of an additional arylsulfatase component (B1) which was not detected in normal human lung. The tumor arylsulfatase B1 had an isoelectric point (pI) of 6.7 and was clearly distinguished from arylsulfatase A (pI 4.9) and arylsulfatase B (pI 9.1 to 9.2) in normal lung and lung tumor. The tumor B1 enzyme was demonstrated to be most probably an isoenzyme of arylsulfatase B, since this unusual enzyme was indistinguishable from arylsulfatase B in terms of Ag+ inhibition; its kinetic parameters of Km for p-nitrocatechol sulfate, which was 2.9 mM with B1; optimum pH of 6.3 for B1; heat stability; and substrate specificity for three synthetic and two physiological substrates.

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.

Human seminal plasma Zn-alpha 2-glycoprotein: its purification and properties as compared with human plasma Zn-alpha 2-glycoprotein.

On the basis of the datum that the level of Zn-alpha 2-glycoprotein (Zn alpha 2gp) in human seminal plasma was about 6-times higher than that in adult serum, Zn alpha 2gp was purified from fresh human seminal plasma approx. 70-fold with 60% yield over seminal plasma by DEAE-Sephacel, Zn-chelate Sepharose 4B and DEAE-5PW column chromatographies. The molecular weight of seminal plasma Zn alpha 2gp was 50,000 on Superose column chromatography, and 40,500 and 41,500 on SDS-polyacrylamide gel electrophoresis in the absence and presence of beta-mercaptoethanol, respectively. Plasma Zn alpha 2gp is a glycoprotein, while the protein from seminal plasma does not contain carbohydrate. The amino acid sequence of the first 17 residues of seminal plasma Zn alpha 2gp was Glu-Asn-Gln-Asp-Gly-Asn-Tyr-Ser-Leu-Thr-Tyr-Ile-Tyr-Thr-Gly-Leu-Ser. This sequence was completely identical with the amino acid residues from Glu-2 to Ser-18 in the N-terminal amino acid sequence of plasma Zn alpha 2gp. These data suggest that both Zn alpha 2gps in plasma and seminal plasma may be expressed from one gene, but their posttranslational modifications are different.

Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.

Human blood group B-active ganglio-glycosphingolipid in rat glioma.

A human blood group B-active glycosphingolipid, belonging to the ganglio-series, was isolated from rat glioma cell line RG2 subcutaneous isografts. The oligosaccharide structure of the glycosphingolipid was completely characterized as Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1- 1'ceramide by NMR spectrometry, negative fast atom bombardment-mass spectrometry, sequential degradation by glycosidases and methylation analysis. Human blood group B antigenicity and the activity of this glycosphingolipid were confirmed by immunostaining on thin-layer chromatography and the inhibition of hemagglutination, respectively. Although the lipid has been detected in rat granuloma, bone marrow cells, spleen, thymus, ascites hepatoma cells and gastric mucosa, this is the first report of the occurrence of the B-active lipid in glioma.

Effects of exercise stress and cold stress on glutathione and gamma-glutamyltransferase in rat liver.

Effects of acute and chronic stress (exercise and cold) on glutathione and gamma-glutamyltransferase (gamma GT) in the rat liver were investigated. Such stress, except for in the case of acute exercise, had no definite influence on the glutathione level. On the other hand, gamma GT activity in both the extramicrosomal and microsomal fractions varied substantially, suggesting that acute exercise increases the release ability of the microsomal membrane of the rat liver, and that swimming training and long-term cold exposure stabilize the membrane. Immunoreactive gamma GT, however, did not always correlate with the enzyme activity, especially in the extramicrosomal fraction. Cross-adaptation appeared to exist between swimming training and chronic cold exposure.

Proteolytic processing of human lysosomal arylsulfatase A.

Arylsulfatase A purified from human placenta contained an unreported component with an apparent molecular mass of 7 kDa in addition to the two known components with apparent molecular masses of 58 and 50 kDa. The detailed relationship between the 58 kDa component and the 50 kDa component is as yet unknown. The present study was undertaken to define the structure of the subunits of the sulfatase. The N-terminal sequence of the 50 kDa component was identical to that of the 58 kDa component. Furthermore, the peptide maps of the 50 kDa component, which was separately digested with trypsin and Achromobacter proteinase I, were quite similar to those of the 58 kDa one. Through sequence analysis of the incompatible peaks in the peptide maps, the 50 kDa component was found to lack a sequence from Val-445 to the C-terminus. On the other hand, the N-terminal sequence of the 7 kDa component began with Ala-448, though there was a minor sequence commencing with Thr-449. These observations suggest that the 50 and 7 kDa components were produced by limited proteolysis near the C-terminus of the 58 kDa component. Through analysis using unreducing SDS-PAGE, the 58 and the 7 kDa components were found to be linked by disulphide bonds. Arylsulfatase A purified from human liver was also composed of the same subunits as the placental one. This finding suggests that human arylsulfatase A undergoes similar proteolytic processing regardless of the tissue involved.

Differential lipid specificities of the repeated domains of annexin IV.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

Expression, purification and binding to the receptor of human insulin-like growth factor II.

Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase. Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography. The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane. Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II. In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes.

Mechanism of the immunosuppressive effect in vivo of novel immunosuppressive drug beta-SQAG9, which inhibits the response of the CD62L+ T-cell subset.

INTRODUCTION: We synthesized sulfo-glycolipid, beta-SQAG9 (designate square beta-SQAG9 liposome, because it efficiently forms a liposome structure) that possessed immunosuppressive effects such as inhibition of T-cell responses in human allogeneic MLR and skin allograft survival in rats, and bound to CD62L (L-selectin) in vitro. In this study, we further investigated the immunosuppressive mechanism in vivo by beta-SQAG9 liposome in a skin-allografted rat model. METHODS: ACI rats (RT1(a)) were grafted skin of LEW rats (RT1(1)) treated with PBS or beta-SQAG9 liposome IV once a day for 7 days. Subsequently, we investigated the population of T cells and CD62L(+) T-cell subset in the spleen, axillary lymph nodes (ALNs), and peripheral blood of skin-allografted rats by two-color flow cytometry. RESULTS: Five of 11 (45.5%) rats that were treated with 50 mg/kg beta-SQAG9 liposome showed graft survival and another showed moderate rejection in graft. The CD62L(+) T-cell subset population in ALNs of beta-SQAG9 liposome-treated rats decreased in a dose-dependent manner. No significant difference in the T-cell population was observed between the beta-SQAG9 and control groups. These data suggest that beta-SQAG9 could bind to the CD62L(+) T-cell subset in vivo as well as in vitro and affect T-cell migration, which might lead to T-cell tolerance in vivo.

Human blood group A and H glycolipids in porcine plasma. Evidence for acquisition of the erythrocyte antigens from plasma.

Human blood group A- and H-antigenic glycosphingolipids were isolated from pooled porcine plasma. The structures of the A-active hexa- and H-active pentaglycosylceramides of lactoseries (type 1 sugar chain) were the same as those in porcine erythrocytes. These results endorse biochemically the previous observations that the A and H antigens on porcine erythrocytes are taken up from plasma.

Protein phosphorylation of lysosomal arylsulfatase B in normal and leukemic leukocytes.

An acidic variant form of arylsulfatase B from normal leukocytes and chronic myelogenous leukemia (CML) leukocytes was found to be phosphorylated at its serine and threonine residues through in vivo phosphorylation with 32Pi. However, the predominant phosphorylation site was serine in normal cells, in contrast to threonine in CML cells. A cyclic AMP-dependent protein kinase was responsible for phosphorylation of the sulfatase of CML cells.

Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine.

A novel O-acetylated GM3 containing 3-O-acetyl 4-sphingenine was isolated with one having a non-acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one- and two-dimensional proton nuclear magnetic resonance, and fast atom bombardment-mass spectrometries. In addition, the O-acetyl GM3 showed higher immunological activity toward anti-melanoma antibody in the presence of non-acetylated GM3 in complement-dependent liposome lysis than did non-acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O-acetyl GM3.

Elevated serum level of glycolipid sulfotransferase in patients with hepatocellular carcinoma.

Activity of glycolipid sulfotransferase (cerebroside sulfotransferase) in serum was elevated in 21 (33%) of 63 patients with hepatocellular carcinoma (HCC, mean +/- S.E., 349 +/- 32 pmol/ml per h, n = 63, P less than 0.001) compared to healthy subjects (172 +/- 12, n = 85). Ho significant elevation of the sulfotransferase level was observed in liver cirrhosis (219 +/- 28, n = 10) in which many of biochemical HCC markers increase concomitantly. The elevation of sulfotransferase was independent of the production of alpha-fetoprotein and of aminotransferase levels in HCC, providing complementary value for alpha-fetoprotein-negative HCC cases. However, the sulfotransferase levels (234 +/- 21, n = 32, P less than 0.01) in sera from patients with renal cell carcinoma, in whose involved tissues the enzyme was demonstrated to increase markedly, were less than in HCC.

Distinctive glycolipid patterns in Wilms' tumor and renal cell carcinoma.

Glycolipid patterns were analysed chromatographically in Wilms' tumor and renal cell carcinoma tissues and compared with those of uninvolved tissue. Ganglioside GM3 was found to be increased in both cancer tissues, whereas sulfatides accumulated only in renal cell carcinoma, as reported earlier. Neolactotetraosylceramide was detected in both cancer tissues, but not in the uninvolved kidney tissues. In four cases of Wilms' tumors, only a low level of sulfotransferase towards galactosylceramide was found in one case, while no activity was detected in the three other cases. Present results show that the increased sulfatide(s) in the renal cell carcinoma and the deficiency of the sulfatides in Wilms' tumors appear to be biochemical characteristics of histologically different carcinomas.