The inhibitory action of SQDG (sulfoquinovosyl diacylglycerol) from spinach on Cdt1-geminin interaction

A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a glycolipid, sulfoquinovosyl diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major glycolipids, such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the geminin interaction interface on Cdt1, and the sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions.     

Inhibitory effects of glycolipids fraction from spinach on mammalian DNA polymerase activity and human cancer cell proliferation

We succeeded in purifying the fraction containing the major glycolipids in monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol (SQDG) from dried vegetables. This glycolipids fraction was an inhibitor of DNA polymerase alpha (pol alpha) in vitro and also the proliferation of human cancer cells. In this study, eight common vegetables were investigated in terms of the glycolipids fraction, the amounts of major glycolipids, mammalian DNA polymerase inhibitory activity and antiproliferative activity toward human cancer cells. Green tea possessed the largest amount of glycolipids overall. Spinach contained the largest amount of SQDG, followed by parsley, green onion, chive, sweet pepper, green tea, carrot and garlic. Spinach had the strongest inhibitory effect on pol alpha activity and human cancer cell proliferation. A significant correlation was found between SQDG content and inhibition of DNA polymerase. Therefore, the inhibition of pol alpha activity by SQDG may lead to cell growth suppression. Of the six subspecies of spinach (Spinacia oleracea) tested, "Anna" had the largest amount of SQDG, strongest inhibitory activity toward DNA polymerase and greatest effect on human cancer cell proliferation. Based on these results, the glycolipids fraction from spinach is potentially a source of food material for a novel anticancer activity.     

硫代异鼠李糖甘油二酯(SQDG)的生物合成与功能

硫代异鼠李糖甘油二酯(SQDG)是一种含硫的糖脂,分布于高等植物,藓类植物,蕨类植物,藻类植物以及大多数光合细菌的光合膜中。SQDG的含量与生物种类有关。在高等植物中含量一般为总脂的4%,而在藻类中其含量变化较大,一般为总脂含量的10%～70%。SQDG的合成是 在叶绿体内被膜上完成的,催化SQDG合成的酶是UDP-SQ: DAG硫代异鼠李糖基转移酶。SQDG 存在于纯化的叶绿体CF0-CF1 ATPase、LHCⅡ辅基蛋白以及D1/D2异二聚体蛋白中,说明SQDG 可能与膜蛋白复合物的结构和功能有关。SQDG还与植物的抗逆性有关。在磷缺乏时,SQDG能 弥补PG含量的下降,使体内阴离子脂的含量维持在一个稳定的水平。近年来还发现SQDG能有效抑制真核生物DNA聚合酶和HIV反转录酶的活性。     

Studies on a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerols: inhibitory action on cell proliferation

Some chemically synthesized sulfoquinovosylmonoacylglycerols (SQMG)-sulfoquinovosyldiacylglycerols (SQDG) have been reported to selectively and strongly inhibit the activities of mammalian DNA polymerases alpha and beta in vitro. In this study, using human cancer cell lines, we investigated the effects of SQMG-SQDG on the DNA polymerase in the cells. In the presence of n-decane, the IC(50) values on cell growth were approximately 1-5 microM for SQMG and about 0.3-1 microM for SQDG. The values were almost the same as the in vitro enzyme inhibitory levels. The cell lines were arrested in early S-phase by SQMG-SQDG at the concentrations of 0.1-4.7 microM in a manner dependent on incubation time, suggesting that SQMG-SQDG blocked the primary step of DNA replication by inhibiting DNA polymerase, possibly alpha-type. We also demonstrated the localization of SQMG in the cell using the fluorescent SQMG analog, SQMGalpha-NBDD, which was synthesized in our laboratory. SQMGalpha-NBDD was localized in the nucleus and on the nuclear surface, but the binding site seemed not to be the DNA/chromatin, suggesting that the SQMG-SQDG might interact with molecules located close to the DNA/chromatin and on the nuclear surface. These results suggested a correlation between the in vitro biochemical action of the SQMG-SQDGs and their intracellular mode of action.     

Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian DNA polymerase inhibitors

We reported previously that sulfo-glycolipids such as sulfoquinovosyl-diacylglycerol (SQDG) and sulfoquinovosyl-monoacylglycerol (SQMG) are potent inhibitors of DNA polymerase alpha and beta and antineoplastic agents. Then, we succeeded in synthesizing SQDG and SQMG chemically, including their stereoisomers, glucopyranosyl-diacylglycerol (GDG) and glucopyranosyl-monoacylglycerol (GMG). In this study, we demonstrated the structure-function relationship of the synthetic sulfo-glycolipids to DNA polymerase alpha and beta and their relationship to the cytotoxic activity. Both SQDG and SQMG inhibited the activity of mammalian DNA polymerase alpha with IC(50) values of 3-5 microM, but GMG only moderately inhibited it. GDG, diacylglycerol (DG), and monoacylglycerol (MG) did not influence any of the DNA polymerase activities. The sulfate moiety in the quinovose was important in inhibiting the enzyme activity. The one-fatty-acid-sulfo-glycolipids, SQMG, GMG, and MG, prevented the growth of NUGC-3 human gastric cancer cells and induced apoptotic cell death, but the two-fatty-acid-sulfo-glycolipids, SQDG, GDG, and DG, did not. SQMG and GMG could halt the cell cycle at the G1 phase, but the cell cycle was not changed by MG. The relationship between the DNA polymerase inhibition and the cell growth effect by these compounds are discussed.     

The biosynthesis of sulfoquinovosyldiacylglycerol: studies with groundnut (Arachis hypogaea) leaves

The biosynthetic pathway of sulfoquinovosyldiacylglycerol (SQDG) was investigated using groundnut (Arachis hypogaea) leaf discs and 35S-labeled precursors. [35S]SO4(2-) was actively taken up by the leaf discs and rapidly incorporated into SQDG. After 2 h, 1.5% of the [35S]SO4(2-) added to the incubation medium was taken up, of which 28% was incorporated into SQDG. The methanol-water phases of the lipid extracts of the leaf discs were analyzed for the 35S-labeled intermediates. Up to 2 h of incubation, cysteic acid, 3-sulfopyruvate, 3-sulfolactate, 3-sulfolactaldehyde, and sulfoquinovose (SQ) which have been proposed as intermediates [Davies et al. (1966) Biochem. J. 98, 369-373] were not labeled. Only a negligible amount of radioactivity was observed in these compounds after incubation for 4 h and more. Addition of sodium molybdate inhibited the uptake of [35S]SO4(2-) as well as its incorporation into SQDG by the leaf discs, suggesting that 3'-phosphoadenosine-5'-phosphosulfate may be involved in the biosynthesis of SQDG. Addition of unlabeled cysteic acid to the incubation medium enhanced the uptake of [35S]SO4(2-) but did not affect its incorporation into SQDG. 35S-labeled cysteic acid was taken up by the leaf discs and metabolized to sulfoacetic acid but not incorporated into SQ or SQDG. These results show that cysteic acid is not an intermediate in SQDG biosynthesis. [35S]SQ was taken up by the leaf discs and incorporated into SQDG in a time-dependent manner. [35S]Sulfoquinovosylglycerol was also taken up by the leaf discs but not incorporated into SQDG. It is concluded that SQDG is not biosynthesized by the proposed sulfoglycolytic pathway in higher plants. Though [35S]SQ was converted to SQDG, the rates are much lower compared to [35S]SO4(2-) incorporation, which suggests that a more direct pathway involving sulfonation of a lipid precursor may exist in higher plants.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.     

Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii.

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf-2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by approximately 40% and also a lower growth rate than those of the wild-type. Results of genetical analysis of hf-2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf-2 cells restored the lowered PSII activity to the same level as that of wild-type cells, and also enabled the mutant to grow even in the presence of 135 nm 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf-2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.     

Growth inhibition and induction of differentiation and apoptosis mediated by sodium butyrate in caco-2 cells with algal glycolipids

Summary Glycolipids should have potential effects as antitumor agents. However, very few studies have examined this property of digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) on colon cancer cells. Cell viability was determined every 24 h with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay up to 72 h. Alkaline phosphatase activity was measured for assessing cell differentiation. Apoptosis was tested with enzyme-linked immunosorbent assay analysis. Growth of Caco-2 cells was inhibited apparently at 48 h after addition of SQDG and at 72 h with DGDG. Alkaline phosphatase activity of Caco-2 cells obviously increased in combination with DGDG or SQDG and sodium butyrate (NaBT) at 72 h, indicating that DGDG and SQDG enhanced cell differentiation induced with NaBT. An increased enrichment factor was found when the cell was treated in combination with DGDG or SQDG and NaBT. These results strongly suggest that DGDG and SQDG should be considered as the leading compounds of potentially useful colon cancer chemotherapy agents when NaBT is combined.     

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants : use of adenosine-5'-phosphosulfate and adenosine-3'-phosphate 5'-phosphosulfate as precursors

Adenosine-5′-phosphosulfate (APS) and adenosine-3′-phosphate 5′-phosphosulfate (PAPS) have been used as precursors of sulfoquinovosyldiacylglycerol (SQDG) in intact chloroplasts incubated in the dark. Competition studies demonstrated APS was preferred over PAPS and SO₄²⁻. Rates of SQDG synthesis up to 3 nanomoles per milligram of chlorophyll per hour were observed when [³⁵S]APS and appropriate cofactors were supplied to chloroplasts incubated in the dark. The pH optimum for utilization of APS was 7.0. The incorporation was linear for at least 30 minutes. ATP and UTP stimulated the incorporation of sulfur from APS into SQDG, but the most stimulatory additions were DHAP and glycerol-3-P. The concentration curve for APS showed a maximum at 20 micromolar in the absence of DHAP and 30 micromolar in the presence of DHAP. The optimum concentration of DHAP for conversion of APS into SQDG was 2 millimolar. Rates of synthesis up to 4 nanomoles per milligram of chlorophyll per hour were observed when [³⁵S]PAPS was the sulfur donor and appropriate cofactors were supplied to chloroplasts. Optimal rates for conversion of sulfur from PAPS into SQDG occurred with concentrations of DHAP between 5 and 10 millimolar. DHAP was by far the most effective cofactor, although ATP and UTP also stimulated the utilization of PAPS for SQDG biosynthesis. In general, triose phosphates, including glycerol-3-P were not effective cofactors for SQDG biosynthesis.     

Sulfoquinovosyldiacylglycerol and phosphatidylglycerol bilayers share biophysical properties and are good mutual substitutes in photosynthetic membranes

From cyanobacteria to higher plants, photosynthetic membranes are composed of two galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), and two negatively charged lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). In many environments, plants and algae grow in a shortage of nutrients, leading to the development of nutrient-saving mechanisms. For example, at the cellular level, in phosphate starvation, these mechanisms include conversion of phospholipids into phosphorus-free lipids. In photosynthetic membranes, PG is supposed to be replaced by SQDG in phosphate starvation whereas the opposite occurs in sulfur deprivation. All biological data confirm a complementary relationship between SQDG and PG and suggest the importance of maintaining the total amount of anionic lipids in photosynthetic membranes. Using neutron diffraction on reconstituted SQDG or PG lipid membranes, we demonstrate that, despite chemically different headgroups, PG and SQDG have similar physicochemical properties. With an equivalent diacylglycerol backbone, PG and SQDG membranes have a similar bilayer thickness and bending rigidity. They also have essentially the same response to hydration in terms of repulsion and interaction forces. The results presented here establish that SQDG and PG are good substitutes to each other in nutrient starvation conditions to maintain the chloroplast functional organization and its photosynthesis activity.     

Glyco- and sphingophosphonolipids from the medusa Phyllorhiza punctata: NMR and ESI-MS/MS fingerprints

The medusa Phyllorhiza punctata has been found in Brazilian waters where it is an exotic species, having arrived in ballasts from the Indo-Pacific Ocean in the general region of North Australia and Indonesia. Fatty acids of the intact animal and its component umbrella, oral arms, and mucus were identified. Two different groups of glycolipids and a sphingolipid were isolated by silica-gel column chromatography and characterized using GC-MS, ESI-MS, 1D, 2D (13)C, (1)H and (31)P NMR spectroscopy. They were sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and ceramide aminoethylphosphonate (CAEP). The CAEP long chain base (LCB) and its polar head group (PHG) formed by partial hydrolysis, were analyzed by ESI-MS/MS. The probable origin of MGDG and SQDG in the jellyfish is the result of an endosymbiotic association with a microalga of the Dinoflagellate group, since these lipids are commonly found in photosynthetic membranes.     

Involvement of sulfoquinovosyl diacylglycerol in the structural integrity and heat-tolerance of photosystem II

To examine the role of sulfoquinovosyl diacylglycerol (SQDG) in thylakoid membranes, we compared the structural and functional properties of photosystem II (PSII) between a mutant of Chlamydomonas reinhardtii defective in SQDG ( hf-2) and the wild type. The PSII core complex of hf-2, as compared with that of the wild type, showed structural fragility when solubilized with a detergent, dodecyl beta- d-maltoside, suggesting that the physical properties of the PSII complex were altered by the loss of SQDG. On the other hand, exposure of the cells to 41 degrees C for 120 min in the dark decreased the PSII activity to 70% and 50% of the initial levels in the wild type and hf-2, respectively, which implies that the PSII activity, in the absence of SQDG, becomes less stable under heat-stress conditions. PSII inactivated to 60% of the initial level by dark incubation at 41 degrees C was reactivated by following illumination even at 41 degrees C to more than 90% in the wild type, but only to 70% in hf-2. These results suggest that PSII inactivated by heat recovers through some mechanism dependent on light, and that SQDG participates in functioning of the mechanism. The conformational disorder of PSII caused by the defect in SQDG might be correlated with the increased susceptibility of its activity to heat-stress.     

Sulpholipid reflects plant resistance to stress-factor action

The results of studying the behaviour of sulphoquinovosyldiacylglycerol (SQDG) during environmental-factor action are discussed. Low-temperature action caused SQDG accumulation in 1-year-old shoot bark of resistant apple varieties, and was especially striking in the Siberia apple, a variety of extreme hardiness. Another unfavourable factor, high temperature, induced SQDG accumulation in leaves and chloroplasts of drought-resistant plants while in sensitive plants a decrease in its content took place. Water deficit caused the same effect. SQDG increase was also observed in field experiments with artificial irrigation in the drought region of Ukraine, at the stages of stooling and milk ripeness. Besides, SQDG accumulation was observed in wheat plants infected by Puccinia graminis and kidney bean plants infected by tobacco mosaic and potato x viruses. But lead supplied at various concentrations caused an SQDG decrease in wheat seedling leaves and roots. The reasons for these phenomena seemed to be mainly the stabilization of the photosynthetic processes, particularly ATP synthesis and light-harvesting complex II functioning, but signal function also cannot be excluded.     

Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii

Information is limited on sulfur (S)-sources inside plant cells for synthesis of the proteins for acclimation to S-starvation. We found that a green alga, Chlamydomonas reinhardtii, when transferred to S-starved conditions, degrades 85% of a chloroplast membrane lipid, sulfoquinovosyl diacylglycerol (SQDG), to redistribute its S to a large part of protein fraction as early as by 6h. Furthermore, the degradation of SQDG preceded that of proteins such as ribulose bisphosphate carboxylase/oxygenase, the candidates of internal S-sources. SQDG was thus demonstrated to yield a major internal S-source for protein synthesis during the early phase of acclimation process to S-starvation.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(α-d-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase α and β. However, since the α-configuration of SQDG with two stearic acids (α-SQDG-C₁₈) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (α-SQMG-C₁₈) from α-SQDG-C₁₈ by hydrolysis with a pancreatic lipase. α-SQMG-C₁₈ inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. α-SQMG-C₁₈ arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that α-SQMG-C₁₈ induced cell death through a p53-dependent apoptotic pathway.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.     

Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria.

Sulfoquinovosyl diacylglycerol (SQDG) is involved in the maintenance of photosystem II (PSII) activity in Chlamydomonas reinhardtii[Minoda, A., Sato, N., Nozaki, H., Okada, K., Takahashi, H., Sonoike, K. & Tsuzuki, M. et al. (2002) Eur. J. Biochem.269, 2353-2358]. To understand the spread of the taxa in which PSII interacts with SQDG, especially in cyanobacteria, we produced a mutant defective in the putative sqdB gene responsible for SQDG synthesis from two cyanobacteria, Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942. The mutant of PCC6803, designated SD1, lacked SQDG synthetic ability and required SQDG supplementation for its growth. After transfer from SQDG-supplemented to SQDG-free conditions, SD1 showed decreased net photosynthetic and PSII activities on a chlorophyll (Chl) basis with a decrease in the SQDG content. Moreover, the sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine was increased in SD1. However, SD1 maintained normal amounts of cytochrome b559 and D1 protein (the subunits comprising the PSII complex) on a Chl basis, indicating that the PSII complex content changed little, irrespective of a decrease in the SQDG content. These results suggest that the role of SQDG is the conservation of the PSII properties in PCC6803, consistent with the results obtained with C. reinhardtii. In contrast, the SQDG-null mutant of PCC7942 showed the normal level of PSII activity with little effect on its sensitivity to PSII herbicides. Therefore, the difference in the SQDG requirement for PSII is species-specific in cyanobacteria; this could be of use when investigating the molecular evolution of the PSII complex.