Sulfoquinovosyldiacylglycerol Research Articles

BIOSYNTHESIS AND FUNCTION OF THE SULFOLIPID SULFOQUINOVOSYL DIACYLGLYCEROL

The sulfolipid sulfoquinovosyl diacylglycerol is an abundant sulfur-containing nonphosphorous glycerolipid that is specifically associated with photosynthetic membranes of higher plants, mosses, ferns, algae, and most photosynthetic bacteria. The characteristic structural feature of sulfoquinovosyl diacylglycerol is the unique head group constituent sulfoquinovose, a derivative of glucose in which the 6-hydroxyl is replaced by a sulfonate group. While there is growing evidence for the final assembly of the sulfolipid by the transfer of the sulfoquinovosyl moiety from UDP-sulfoquinovose to the sn-3 position of diacylglycerol, very little is known about the biosynthesis of the precursor UDP-sulfoquinovose. Recently, a number of mutants deficient in sulfolipid biosynthesis and the corresponding sqd genes have become available from different organisms. These provide novel tools to analyze sulfolipid biosynthesis by a combination of molecular and biochemical approaches. Furthermore, the analysis of sulfolipid-deficient mutants has provided novel insights into the function of sulfoquinovosyl diacylglycerol in photosynthetic membranes.

Trehalose Increases Freeze–Thaw Damage in Liposomes Containing Chloroplast Glycolipids

Chloroplast thylakoids contain three classes of glycolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). We have investigated the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC) and different chloroplast glycolipids during freezing to −18°C, as a function of the presence of three sugars: glucose, sucrose, or trehalose. Contrary to the situation in thylakoids, where cryoprotection increases from glucose < sucrose < trehalose, liposomes containing 50% DGDG showed the opposite behavior. In fact, carboxyfluorescein leakage increased over the control values (freezing in the absence of sugar) in the presence of trehalose. This effect was not seen in vesicles made from pure EPC, or a mixture of EPC and MGDG, or EPC and SQDG. Liposomes made from mixtures of all three glycolipids, however, showed even more leakage in the presence of trehalose than liposomes containing only DGDG and EPC.

Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana.

Photosynthetic membranes of higher plants contain specific nonphosphorous lipids like the sulfolipid sulfoquinovosyl diacylglycerol in addition to the ubiquitous phospholipid phosphatidylglycerol. In bacteria, an environmental factor that drastically affects thylakoid lipid composition appears to be the availability of phosphate. Accordingly, we discovered an increase in the relative amount of sulfolipid and a concomitant decrease in phosphatidylglycerol in Arabidopsis thaliana grown on medium with reduced amounts of phosphate, as well as in the pho1 mutant of A. thaliana deficient in phosphate transport. To investigate the molecular basis of the observed change in lipid composition, we isolated a cDNA of A. thaliana, designated SQD1, that encodes a protein involved in sulfolipid biosynthesis as suggested by three lines of evidence. First, the cDNA shows high sequence similarity to bacterial sqdB genes known to be essential for sulfolipid biosynthesis; second, the SQD1 gene product is imported into chloroplasts where sulfolipid biosynthesis takes place; and third, transgenic plants expressing SQD1 in antisense orientation show a reduction in sulfolipid content. In the pho1 mutant as well as in wild-type plants grown under reduced phosphate availability, increased amounts of SQD1 mRNA and SQD1 protein are detected, suggesting that the increase in sulfolipid content under phosphate limitation is the result of an increased expression of at least one gene required for sulfolipid biosynthesis in A. thaliana. It is suggested that a certain amount of anionic thylakoid lipid is maintained by substituting sulfolipid for phosphatidylglycerol under reduced phosphate availability.

The identification of glycerol-3-yl 6-deoxy-6- C-sulfo- α- d-glucopyranoside (glyceryl α-sulfoquinovoside) as a metabolite in Rhizobium, a non-photosynthetic organism

We have isolated and characterized glycerol-3-yl 6-deoxy-6- C-sulfo- α- d-glucopyranoside (glyceryl α-sulfoquinovoside), a key metabolite involved in sulfoquinovosyl diacylglycerol (SQDG) biosynthesis in photosynthetic organisms, from the cellular metabolic pools of Rhizobium. The compound has not been found in any other non-photosynthetic organisms. This result suggests some commonalities between the biosynthesis of SQDG in Rhizobium and in plant organelles and photosynthetic organisms and further adds to the evolving picture that there is considerable gene overlap between Rhizobium and photosynthetic systems, including plants.

The effects of chloroplast lipids on the stability of liposomes during freezing and drying

Chloroplast thylakoids contain four classes of lipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (cpPG). We have investigated the effects of these lipids on the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC), by substitution of different fractions of EPC in the membranes by the various chloroplast lipids. Damage to liposomes after freezing to −18°C was measured as carboxyfluorescein leakage or fusion between vesicles. The presence of all chloroplast lipids increased leakage. However, the maximum amount of leakage and the concentration dependence were dramatically different between the different lipids. Only SQDG induced vesicle fusion, while the non-bilayer lipid MGDG did not. The presence of MGDG in the membranes led to more leakage than the presence of another non-bilayer lipid, egg phosphatidylethanolamine (EPE). In EPE-containing liposomes, leakage was strongly associated with fusion. Combinations of different chloroplast lipids had an additive effect on leakage induced by freezing. Most of the leakage from galactolipid-containing vesicles occurred during the first 15 min of freezing at −18°C. After a 3 h incubation period, most leakage occurred between 0°C and −10°C. Lowering the temperature to −22°C had only a small additional effect. Incubation of liposomes at −10°C in the presence of 2.5 M NaCl without ice crystallization, approximately the same concentration obtained by freezing to −10°C, resulted in very little leakage. Air drying of liposomes to low water contents resulted in massive leakage, both from pure EPC vesicles and from vesicles containing galactolipids. The latter vesicles showed more leakage at any given water content than EPC vesicles.

Sulfoquinovosyl diacylglycerols from the alga Heterosigma carterae.

An extract of the chloromonad Heterosigma carterae (Raphidophyceae), cultivated in natural seawater, contained a complex mixture of sulfoquinovosyl diacylglycerols. Palmitoyl (16:0), three isomers of hexadecenoyl (16:1 cis delta 9, delta 11, delta 13), and eicosapentenoyl (20:5) were found to be the main fatty acyl substituents. Exact double-bond sites were determined by mass spectrometry analysis of the corresponding nicotinyl derivatives. Four major sulfoquinovosyl diacylglycerol components were partially purified and identified as 1-4 by interpretation of their nuclear magnetic resonance and mass spectral data. In addition, complete analysis of the H. carterae sulfoquinovosyl diacylglycerols was performed using high-performance liquid chromatography combined with electrospray tandem mass spectrometry.

Structural characterization of sulfoquinovosyl, monogalactosyl and digalactosyl diacylglycerols by FAB-CID-MS/MS

The glycolipids such as sulfoquinovosyl diacylglycerol (SQDG), isolated from wild-type cyanobacterium Synecho-cystis sp. PCC 6803, and monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), ob-tained from whole wheat flour, have been investigated by using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). Collision-induced dissociation (CID) of sodium-adduct molecular ions [M-H+2Na]+ from SQDG and [M+Na]+ from MGDG and DGDG generates ions which are characteristic of the structure of the polar head group as well as the fatty acid composition. In particular, the charge-remote fragmentation along the fatty acid chains observed in the high-mass range was useful to locate the double-bond positions. The major component ion ([M-H+2Na]+ at m/z 865) of SQDG contains oleic and palmitic acids at the sn-1 and sn-2 positions, respectively. The predominant molecular ion ([M+Na]+ at m/z 801) of MGDG contains two linoleic acids. One of the two major species of DGDG contains palmitic and linoleic acids ([M+Na]+ at m/z 939) at the sn-1 and sn-2 positions, while the other contains two linoleic acids ([M+Na]+ at m/z 963). The utility of FAB-MS/MS in the structural determination of glycolipids in mixtures of biological origin has been demonstrated. © 1997 John Wiley & Sons, Ltd.

Structural characterization of sulfoquinovosyl, monogalactosyl and digalactosyl diacylglycerols by FAB‐CID‐MS/MS

Structural characterization of sulfoquinovosyl, monogalactosyl and digalactosyl diacylglycerols by FAB‐CID‐MS/MS

Accumulation of Sulfoquinovosyl-1-O-dihydroxyacetone in a Sulfolipid-Deficient Mutant ofRhodobacter sphaeroidesInactivated insqdC

The biosynthesis of the sulfolipid sulfoquinovosyl diacylglycerol in the purple bacteriumRhodobacter sphaeroidesrequires at least four genes:sqdA, sqdB, sqdC,andsqdD.As part of our strategy aimed at the elucidation of the function of the differentsqdgene products, we insertionally inactivatedsqdCofR. sphaeroides.The resultingsqdCnull mutant showed only a 90% reduction in sulfolipid content. Apparently, thesqdCgene product is required for optimal sulfolipid biosynthesis, but either catalyzes no essential reaction in the pathway or can be functionally replaced to a certain extent by a different protein. The mutant accumulated a35S-labeled compound that was purified to homogeneity from cell extracts. Matrix-assisted laser desorption mass spectrometry and nuclear magnetic resonance spectroscopy provided conclusive structural evidence to identify the compound as α-d-sulfoquinovosyl-1-O-dihydroxyacetone that exists in two interconvertible, keto and hemiacetal forms. Incubation of wild-type protein extracts with the labeled compound did not result in the incorporation into sulfolipid as would be expected for an intermediate of the pathway. Based on our results we propose that thesqdCgene product mediates the substrate specificity of the UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase that is encoded bysqdDand that catalyzes the final reaction of sulfolipid biosynthesis.

Capacitance measurements of photovoltaic cells based on mixed monolayers of chlorophyll a and sulfoquinovosyldiacylglycerol

The capacitance measurements of Al/Chlorophyll a/Ag and Al/Chlorophyll a-Sulfoquinovosyldiacylglycerol/Ag sandwich cells have been carried out at different frequencies in dark and under illumination. The results show that while a voltage-dependent capacitance and a linear 1 C 2 versus V a plot is obtained for chlorophyll a (Chl a) at low frequencies, the addition of sulfoquinovosyldiacylglycerol (SQDG) in proportions ∼ 0.25 results in totally voltage-invariant capacitance. The capacitance characteristic of Chi a-SQDG (0.25) cells resemble more those for an insulator than those for a Schottky barrier. These results are explained in terms of negatively charged SQDG playing a role of traps which immobilize the holes. Alternatively, the interaction of negative polar head group of SQDG with Mg 2+ of Chl a can also be invoked as a possible reason for the observed results. The bound Chl a- SQDG species possibly does not form a Schottky barrier or forms a weak barrier with either Al or Ag electrodes, resulting in insulator-like capacitance characteristics of Al/Chl a-SQDG(0.25)/Ag cells.

Lipids of Ectocarpus fasciculatus (Phaeophyceae). Incorporation of [l-14C]Oleate and the Role of TAG and MGDG in Lipid Metabolism

Lipids and fatty acids of Ectocarpus fasciculatus (Ectocarpales, Phaeophyceae) were analyzed. Major polar lipids are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), diacylglycerylhydroxymethyl-iV.A'j.N-trimethyl/J-alanine (DGTA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI). Diphosphatidylglycerol (OPG), phosphatidic acid (PA) and phosphatidyl-O-[iV-(2-hydroxyethyl)glycine] (PHEG) were also present in small amounts. Nonpolar lipids mainly consist of triacylglycerol (TAG) and diacylglycerol (DAG). Major fatty acids are 16:0,18:1, al8:3, 18:4, 20:4 and 20:5. The positional distribution of fatty acids showed that molecular species of eukaryotic structure account for 99% in MGDG, 98% in DGDG, 62% in PG and 23% in SQDG. On incubation with [1- 14 C]18:1 for 30 min, 33% of the total label was detected in TAG, 16% in PG, 14% in PE, 10% in PC and 8% in MGDG. During 7 days of chase, the label in TAG, PG, PE and PC decreased and simultaneously increased in MGDG up to 41% of the total. In SQDG, labelled fatty acids were found in prokaryotic as well as in eukaryotic molecular species. During the experiment, the label shifted from 18:1 to 18:2, 18:3,18:4 and, to a minor extent, to 20:4 and 20:5 acids indicating 18:1 to be processed by elongation and/or desaturation. These results suggest TAG to act as a major primary acceptor of exogenous oleate and to be involved in the transfer of fatty acids to MGDG and other polar lipids.

Conformation of sulfoquinovosyldiacylglycerol bound to a magnetically oriented membrane system

The conformation of uniformly 13C-labeled sulfoquinovosyldiacylglycerol (SQDG) is studied in both membrane and solution environments using NMR spectroscopy. Analysis in a membrane-like environment is based on the measurement of dipolar interactions between 13C-13C and 1H-13C spin pairs and on the measurement of 13C chemical shift anisotropy offsets, which appear in magnetically oriented phospholipid-based membrane fragments. Potential energy maps for glycosidic torsions, phi, psi and theta 1, are calculated with a membrane interaction energy and are used in the interpretation of experimental data. The membrane-bound description for SQDG is most consistent with a set of low-energy conformations that extends the headgroup of SQDG away from the membrane surface. Analysis of the conformation of SQDG in CD3OD solution is based on measured 3JCH scalar couplings. The description of the solution conformation is modeled as a mixture of low-energy conformers predicted in the absence of a membrane interaction term and involves more extensive motional averaging than the model for SQDG embedded in the lipid matrix.

Membrane lipid composition of the unusual cyanobacterium Gloeobacter violaceus sp. PCC 7421, which lacks sulfoquinovosyl diacylglycerol

Gloeobacter violaceus sp. PCC 7421 is an unusual cyanobacterium with only one cellular membrane, which lacks the thylakoid membranes found in other oxygenic photosynthetic organisms. The cell membrane lipids in G. violaceus sp. PCC 7421 are monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidyl glycerol and phosphatidic acid in the molar proportion of 51, 24, 18 and 4% respectively. This lipid composition resembles that of the cell membrane from other cyanobacteria, but completely lacks sulfoquinovosyl diacylglycerol. This lack of sulfoquinovosyl diacylglycerol is exceptional for a photosynthetic membrane. The membrane lipids are esterified to 14:0, 16:0, 16:1, 18:0, 18:1, 18:2 and α18:3 fatty acids.

Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids.

The pancreatic lipase family contains three subfamilies, the 'classical' lipases and the pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2). Galactolipids are present in membranes of leaves and vegetables and consist of digalactosyldiacylglycerol (DGalDG) monogalactosyldiacylglycerol (MGalDG) and sulfoquinovosyldiacylglycerol (SQDG). These lipids were incubated with PLRP2 from guinea-pig (GPLRP2) and rat (RPLRP2). In the presence of bile salts DGalDG was efficiently hydrolyzed by GPLRP2 and, although less efficiently, by RPLRP2 to digalactosylmonoacylglycerol (DGalMG), free fatty acids and water-soluble galactose-containing compounds. Also, MGalDG and SQDG were hydrolyzed by GPLRP2 and RPLRP2. These data suggest a possible role of PLRP2 in the digestion of dietary galactolipids.

The use of nicotinates and sulfoquinovosyl monoacylglycerols in the analysis of monounsaturated n-3 fatty acids by mass spectrometry.

Fatty acyl groups (16:1 and 16:0) liberated from purified sulfoquinovosyl diacylglycerols produced by the unicellular marine microalga, Heterosigma carterae (formerly H. akashiwo), were converted to either the corresponding alcohols or methyl esters. Nicotinate derivatives of the alcohols were examined by combined gas chromatography/mass spectrometry, and the methyl esters were examined by nuclear magnetic resonance (NMR) spectroscopy after separation by high-performance liquid chromatography. Three different hexadecenoyl fatty acyl groups were identified, one of which was cis 13-hexadecenoyl (16:1n-3). Both the configuration and the n-3 position of the double bond in the cis 13-hexadecenoyl moiety were unequivocally established by NMR analysis of the corresponding methyl ester. The nicotinate derived from the alcohol of the 16:1n-3 fatty acyl moiety gave a characteristic fragmentation series in the electron impact mass spectrum which, by careful interpretation, was consistent with, but not unambiguous for, the assigned location of the double bond. Tandem mass spectrometry experiments on a sulfoquinovosyl monoacylglycerol containing the cis 13-hexadecenoyl group in the sn-2 position, using negative-ion liquid secondary ion mass spectrometry, also gave a fragmentation pattern which was consistent with the positional assignment of the double bond.

Cyclodextrins: a new tool for the controlled lipid depletion of thylakoid membranes

Cyclodextrins (CDs) have been used in a controlled lipid depletion of thylakoid membranes avoiding the use of either detergents or lipolytic enzymes. Spinach thylakoid membranes were first treated with different CDs under various conditions. After removal of the CDs by washing, the amounts of mono- and digalactosyldiacylglycerol (MGDG and DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), protein, pigment and plastoquinone remaining in the membranes were determined. The main results, obtained with α-CD and heptakis-(2,6-di- O-methyl)- β-CD (DM-β-CD), were as follows. (1) Acyl lipids were removed from thylakoid membranes by both CDs (DM-β-CD being more efficient than α-CD); the extent of removal depended on both CD and chlorophyll concentrations. (2) α-CD presented a higher selectivity towards lipid classes than did DM-β-CD, but in both cases the removal order was SQDG > PG > MGDG > DGDG. (3) α-CD showed a preference for those lipids containing saturated 16-carbon acyl chains whereas DM-β-CD was essentially insensitive to the fatty acid composition of the lipids. (4) The protein, chlorophyll and carotenoid contents of thylakoids were not affected by CD treatments. (5) Plastoquinones were removable but in small amounts only and with a low efficiency (DM-β-CD > α-CD). (6) For all lipid classes, the extent of lipid removal was higher at 0° than at 20°C. (7) The presence of MgCl 2 reduced the removal of PG and SQDG but did not affect galactolipid depletion levels. (8) Stable lipid depletion levels in thylakoid membranes were reached after 5–10 min of CD treatment at 0°C. (9) Of the four CDs tested, only three (α-CD, β-CD, and DM-β-CD) promoted lipid depletion whereas one (hydroxypropyl-β-CD) failed completely to do so. It is concluded that CD-mediated lipid removal provides a valuable and versatile tool to achieve controlled and specific lipid depletions in biological membranes. A few examples of the consequences of a CD-induced lipid depletion on fluorescence and electron transport properties of thylakoids are given to show the usefulness of CDs in the investigation of structure-function relationships in photosynthetic membranes.

Digalactosyldiacylglycerol suppression of inhibition by sulfoquinovosyldiacylglycerol of alpha-glucosidase.

Digalactosyldiacylglycerol (DGDG) suppressed the inhibition by sulfoquinovosyldiacylglycerol (SQDG) of an alpha-glucosidase reaction. Suppressing was considered to be an apparent decrease in inhibitory activity of SQDG ascribed to direct interaction between SQDG and DGDG. This suppression was presumed to be caused by less access of SQDG to the enzyme because SQDG and DGDG formed mixed micelles.

Impaired photosystem II in a mutant of Chlamydomonas reinhardtii defective in sulfoquinovosyl diacylglycerol.

The photosynthetic apparatus was characterized in a mutant of Chlamydomonas reinhardtii, hf-2, defective in the synthesis of a chloroplast-specific lipid, sulfoquinovosyl diacylglycerol (SQui-acyl2Gro). hf-2 showed reduced photosystem II (PSII) activity with little effect on photosystem I (PSI) activity, as compared with the parent. PAGE in the presence of dodecyl beta-D-maltoside (DodGlc2) of C. reinhardtii thylakoid membranes was used to isolate chlorophyll-protein complexes without chlorophyll (Chl) release in order to examine lipid species bound to these complexes. The four complexes obtained were shown to be the PSI complex, the PSII core complex and the two groups of the light-harvesting complex of PSII by analyses of 77-K emission spectra of Chl fluorescence and of subunit compositions. Lipid analysis of Chl-protein complexes in the parent revealed the localization of SQui-acyl2Gro in the PSII core complex and the two groups of the light-harvesting complex of PSII, but not in the PSI complex. These results suggest that SQui-acyl2Gro is responsible for PSII activity by associating with the core and light-harvesting complexes of PSII.

Hydrolysis of galactolipids by human pancreatic lipolytic enzymes and duodenal contents.

Monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG) and sulfoquinovosyldiacylglycerols (SQDG) are major lipids in vegetable food. Their digestion and absorption are unknown. This study examines the hydrolysis of galactolipids in vitro with human duodenal contents, pancreatic juice, and purified human pancreatic lipases. Galactolipids were incubated with human duodenal contents, pancreatic juice, pure pancreatic carboxyl ester lipase (CEL), and colipase-dependent lipase with colipase (Lip-Col). Hydrolysis was estimated as release of free fatty acids and by the use of [3H]galactose or [3H]fatty acid-labeled DGDG. Pancreatic juice and duodenal contents hydrolyzed DGDG to fatty acids, digalactosylmonoacylglycerol (DGMG) and water-soluble galactose-containing compounds. The hydrolysis of DGDG was bile salt-dependent and had a pH optimum at 6.5-7.5. Human pancreatic juice released fatty acids from MGDG, DGDG, and SQDG. Purified CEL hydrolyzed all three substrates; the hydrolysis rate was MGDG > SQDG > DGDG. Pure Lip-Col had activity toward MGDG but had little activity against DGDG. Separation of pancreatic juice by Sephadex G100 gel filtration chromatography revealed two peaks with galactolipase activity that coincided with CEL (molecular mass 100 kD) and lipase (molecular mass 50 kD) peaks. In contrast to pure Lip-Col enzymes of the latter peak were as active against DGDG as against MGDG. Thus, DGDG is hydrolyzed both by CEL and by a pancreatic enzyme(s) with a molecular mass of 40-50 kD to fatty acids and lyso DGDG. MGDG, DGDG, and SQDG are all hydrolyzed by human pancreatic juice. Pure CEL hydrolyzed all three substrates.

Isolation and Characterization of Mutants Affected in Lipid Metabolism of Chlamydomonas Reinhardtii

Two mutants affected in lipid metabolism of Chlamydomonas reinhardtii were isolated by treating cells with ultraviolet light. Both mutants showed high chlorophyll fluorescent yields, as compared with parents, and were designated as hf‐2 and hf‐9 (for high fluorescence), hf‐2 was shown to be defective in the synthesis of a chloroplast‐specific lipid, sulfoquinovosyl diacylglycerol. hf‐9 was shown to be defective in desaturation at the ω6 position of fatty acids of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. The mutants exhibited alterations in photosynthetic activity and chloroplast ultrastructure.