Isotopic Combinatomer Analysis Provides in Vivo Evidence of the

Sep 21, 2016 - Isotopic Combinatomer Analysis Provides in Vivo Evidence of the Direct Epimerization of Monoglucosyl Diacylglycerol in Cyanobacteria. N...
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Isotopic Combinatomer Analysis Provides in Vivo Evidence of the Direct Epimerization of Monoglucosyl Diacylglycerol in Cyanobacteria Naoki Sato,*,†,‡ Yozo Okazaki,§ and Kazuki Saito§,∥ †

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan JST, CREST, Tokyo 102-0076, Japan § RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan ∥ Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan ‡

S Supporting Information *

ABSTRACT: Galactolipids constitute the majority of photosynthetic membranes called thylakoid membranes in cyanobacteria and chloroplasts of land plants and algae. The galactolipids, although identical in headgroup structure, are synthesized by significantly different pathways in cyanobacteria and chloroplasts. In the cyanobacterial pathway, monoglucosyl diacylglycerol (GlcDG) is synthesized first and then converted to monogalactosyl diacylglycerol (MGDG). On the basis of circumstantial evidence, the mechanism of conversion was thought to be epimerization at C-4, but no direct evidence has yet been provided, because there is no in vitro enzymatic system of the putative membrane-bound reaction. Labeling studies with 14C and 13C suggested that the labels in the headgroup and the acyl groups were kept at a reasonably constant ratio before and after the conversion. We then provide in vivo evidence of the direct epimerization based on detailed isotopomer analysis of the conversion, named “combinatomer analysis”. The different types of molecules formed by the combination of labeled or unlabeled parts (sn-1 acyl, sn-2 acyl, glycerol, and hexose) are called here “combinatomers”. Combinatomer analysis of the experiments with pulse labeling with 13C and chase in Anabaena sp. PCC 7118 indicated that the composition of combinatomers in the precursor GlcDG was kept unchanged in the product MGDG. Production of combinatomers resulting from exchange of hexose was minimal. This provides solid evidence of the epimerization of the glucose moiety of GlcDG, as well as the direct desaturation of acyl groups at the sn-1 position.

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A breakthrough was achieved by the discovery of the mgdE gene for the conversion of GlcDG to MGDG in the cyanobacterium Synechocystis sp. PCC 6803.13 This clearly identified that the “epimerization” is catalyzed by a single enzyme consisting of a single polypeptide. The gene name mgdE is also suggestive of “epimerization”. The enzyme MgdE contains an oxidoreductase domain that could perform the suspected reversible oxidation and reduction at C-4 of glucose. We have to be cautious about the “epimerization”, however, because currently known epimerization between glucose and galactose (4-epimerase) involves their UDP derivatives.14 Free glucose is known to be a poor substrate, although free monosaccharide is a substrate for other types of epimerization, such as the 3-epimerization of psicose15 and ribulose (such as that in the Calvin−Benson cycle). Examples of epimerization of the sugar moiety within a polysaccharide or an oligosaccharide chain of glycoprotein are quite limited, such as glucuronyl C-5 epimerase16 and cellobiose 2-epimerase.17 On the basis of this situation, we consider the “epimerization” of the glucose within

alactolipids constitute the majority of photosynthetic membranes called thylakoid membranes in cyanobacteria and chloroplasts of land plants and algae. The galactolipids, although identical in headgroup structure, are synthesized by significantly different pathways in cyanobacteria and chloroplasts.1−4 In plants, monogalactosyl diacylglycerol (MGDG) is synthesized by the transfer of galactose from UDP-galactose to diacylglycerol (DAG) by the enzyme MGD1,5 and then MGDG is further galactosylated to digalactosyl diacylglycerol (DGDG) by the enzyme DGD1.6 In the cyanobacterial pathway, monoglucosyl diacylglycerol (GlcDG) is synthesized first by the transfer of glucose from UDP-glucose to DAG by the enzyme MgdA7,8 and then converted to MGDG by an unknown mechanism (for chemical structures, see Figure 1A). DGDG is also formed by galactosylation of MGDG by the enzyme DgdA,9,10 an enzyme distinct from DGD1. On the basis of circumstantial evidence, the mechanism of conversion of GlcDG to MGDG was thought to be epimerization at C411,12 (Figure 1A), and we sometimes use the word “epimerization” for this conversion for the sake of simplicity; however, no direct evidence has yet been provided for the “epimerization” in experiments with membrane fractions in spite of the efforts of many researchers. © 2016 American Chemical Society

Received: July 28, 2016 Revised: September 19, 2016 Published: September 21, 2016 5689

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Figure 1. Conversion of exogenously added [14C]GlcDG to MGDG in Anabaena variabilis M3. (A) Structure of monoglucosyl diacylglycerol (GlcDG) and monogalactosyl diacylglycerol (MGDG). Note that the configuration of the OH group at C-4 is different. (B) Time course of incorporation of labeled GlcDG into the cells. Radioactivity expressed in counts per minutes may be converted to disintegrations per minute by assuming an average counting efficiency of ∼90%, which was normally the case in our experiments. (C) Conversion of GlcDG to MGDG. (D) Distribution of radioactivity in the acyl groups (gray) and the polar groups (hatched) in the input GlcDG as well as the GlcDG and MGDG recovered after incubation for 5 h. Average ± standard deviation of three replicates.

Anabaena sp. PCC 7118 was originally obtained from the Pasteur Culture Collection and grown in BG-11 medium. Labeling with [14C]GlcDG. A 20 mL culture of A. variabilis M3 was labeled with 3.7 MBq of NaH14CO3 under light at 22 °C for 20 min. Total lipids were extracted, and GlcDG (∼37 kBq) was purified by TLC. The purity of this preparation was >95% (major contaminants were MGDG and lyso products) as checked by TLC and autoradiography. GlcDG (3 kBq) was added to a 10 mL culture of A. variabilis M3 in BG-11 medium and incubated under illumination at 32 °C for 5 h. A 2 mL aliquot was withdrawn at 0, 1, 3, and 5 h, mixed with 50 μL of 0.1% Tween 20, and centrifuged at 15000 rpm for 1 min. The cells were washed in a BG-11/Tween 20 mixture and finally suspended in phosphate-buffered saline. The detergent was used to remove unincorporated radioactive lipid adsorbed on the cell surface. Note that Tween 20 is a mild detergent that does not lyse the cells. The cell suspension was transferred to a glass tube, and then lipids were extracted and analyzed by TLC using solvent 3 (chloroform/methanol/ammonia−water, 65:35:5, by volume).12 Labeling and Chase Experiments with NaH13CO3. A 500 mL culture of Anabaena sp. PCC 7118 grown at 30 °C was collected by centrifugation, resuspended in 410 mL of prewarmed BG-11 medium, and transferred to a closed culture vessel equipped with a magnetic stirrer (Corning catalog no. 3153), which was placed in a water bath at 30 °C with lateral illumination (100 μE m −2 s −1 ); 0.40 g of NaH 13 CO 3

a glycolipid must be a new biochemical reaction, and we need to demonstrate it experimentally. We considered that, as an alternative to in vitro experiments, in vivo evidence of the epimerization might be obtained by detailed isotopomer analysis of the conversion from GlcDG to MGDG. That is why we performed labeling experiments with 14 C and 13C in this study. The results of initial labeling analysis suggested that the labels in both the headgroup and the acyl groups were kept at a reasonably constant ratio before and after the conversion. Detailed isotopomer analysis named “combinatomer analysis” was applied to the experiments with pulse labeling with 13C and chase in Anabaena sp. PCC 7118. The experimental results indicated that the labeled and unlabeled combinatomers in the precursor GlcDG were conserved in the product MGDG with limited production of molecules with exchanged hexose. This provides solid evidence of the epimerization of the glucose moiety of GlcDG. At the same time, direct desaturation of acyl groups at the sn-1 position from 18:1 to 18:2 was also confirmed.

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MATERIALS AND METHODS Cyanobacterial Strains. Anabaena variabilis strain IAMM3 was originally obtained from the Institute of Applied Microbiology of the University of Tokyo (the IAM culture collection has since been transferred to NIES, National Institute for Environmental Studies) and grown in KratzMyers’ Medium C (KM medium18) or BG-11 medium.19 5690

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Figure 2. Mass spectra of 18:1/16:0-GlcDG and 18:1/16:0-MGDG after photosynthetic labeling with [13C]bicarbonate and chase in A. variabilis M3. TOF-MS spectra of [M + Na]+ ions are shown. The monoisotopic mass is 779 for both molecular species. (A, C, and E) GlcDG and (B, D, and F) MGDG (A and B) after labeling for 30 min, (C and D) after labeling for 2 h, and (E and F) after chase for 2 h following the 2 h labeling. After the chase for 5 h, a low-abundance population almost disappeared in GlcDG and MGDG, and the labeled population of MGDG decreased to 5%.

medium at 38 °C. Labeling with 13C was performed for 2 h, and then the cells were washed as described above and chased for 5 h without cerulenin. Analysis of 13C-Labeled Lipids. Lipid analysis was performed as described in previous articles.12,20 Total lipids were extracted by the method of Bligh and Dyer.21 GlcDG and MGDG were fractionated by thin layer chromatography (TLC) using solvent 1 (chloroform/n-hexane/2-propanol/tetrahydrofuran/water, 25:50:40:0.5:1, by volume).12 Note that TLC is the only method that allows complete separation of these two classes of lipids (for TLC separation, see ref 12). Molecular species were separated by argentation TLC using solvent 2 (acetone/benzene/water, 90:30:8, by volume).12 Various molecular species of each class of lipid with unsaturation index (Un) values of 0, 1, and 2 were recovered from the plate and subjected to MALDI-TOF MS analysis or methanolysis.

(Cambridge Isotope Laboratories, Inc., 99% 13C), freshly dissolved in 10 mL of BG-11 medium, was added, and the cells were incubated for 45 min. Aliquots (10 mL) were withdrawn at 15 and 30 min, for lipid analysis. Then, the culture was divided into two parts. For each part, the cells were collected by centrifugation at 1500g for 4 min at 4 °C and resuspended in 3 mL of medium. The cells from one part were used for lipid extraction. The cells in another part were suspended in 200 mL of prewarmed BG-11 medium. Cerulenin (an inhibitor of fatty acid synthesis, obtained from Wako Pure Chemicals, Osaka, Japan) dissolved in ethanol (final concentration of 5 μg mL−1) and 1 mL of 0.9 M NaHCO3 were added, and the culture was transferred to a flat culture bottle for further incubation. After 4 h, the cells were collected by centrifugation for lipid extraction. Experiments in A. variabilis M3 were performed in a manner similar to that described above, but the cells were grown in KM 5691

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In Vivo Labeling and Chase Experiments Using 13C in A. variabilis M3. We then tried to use a stable isotope 13C to trace the conversion of GlcDG to MGDG in vivo. Because TLC is the sole method for clearly separating GlcDG and MGDG,12 these two classes of lipids were first separated and isolated by TLC, and then, GlcDG and MGDG were each fractionated into molecular species differing in unsaturation by argentation TLC. The fractionated molecular species were identified as Un = 0, Un = 1, Un = 2, and Un = 3 species. Because of the simplicity of the molecular species composition of glycolipids in this cyanobacterium, namely, C18 acids being found at the sn-1 position and C16 acids being found at the sn-2 position,23 Un = 0, Un = 1, Un = 2, and Un = 3 species consisted mainly of 18:0/16:0 (with a small amount of 16:0/16:0), 18:1/16:0 (with a small amount of 16:1/16:0), 18:2/16:0 (18:1/16:1 is a separate band below 18:2/16:0), and 18:3/16:0, respectively. These fractionated molecular species were used for mass spectrometry. Only the mass region in the C18/C16 species was considered, except in some cases in which C16/C16 became comparable to C18/C16 under particular experimental conditions. Figure 2 shows the results in A. variabilis M3. Because the amount of the Un = 0 fraction was very small under the experimental conditions, the primary product, 18:0/16:0GlcDG, was thought to be desaturated to 18:1/16:0-GlcDG rapidly,23 which was virtually the initial product. After labeling had been performed for 30 min, 18:1/16:0-GlcDG contained a population of highly labeled isotopomers, and this fraction was 39% of the total 18:1/16:0-GlcDG. The isotopic abundance (p) of the labeled population was 0.90 (see Figure S1 for the relationship between isotopic abundance and isotopomer distribution). This means that the carbon atoms that were used to produce 18:1/16:0-GlcDG during this time period contained 90% 13C and 10% 12C. The sodium bicarbonate used for this labeling contained 99% 13C. As the Calvin−Benson cycle is known to be rapidly equilibrated with the input carbon pool, this result indicates that unlabeled carbon was mixed during the synthesis of this lipid. If each of the three carbons in the product of the Calvin−Benson cycle (phosphoglycerate) contains 90% 13C, then we will expect that all the carbons in the resultant lipid contain equally 90% 13C. In other words, the product will consist of various molecules having different numbers of 13C atoms (called isotopomers) according to a binomial distribution, peaking at 38 (=43 × 0.90) 13C atoms. The real distribution was broader than such a theoretical distribution assuming a single p value, and we need a method of obtaining a good estimate. The method of calculation of p, as well as computational methods for the analysis of isotopomers, is described in Computational methods in the Supporting Information. At this time (30 min), no significant labeling was detected in 18:1/16:0-MGDG. When labeling was continued for 2 h, the unlabeled isotopomer was no longer detected in 18:1/16:0GlcDG, whereas the p value of the labeled population remained at 0.91. The peak at m/z 795 was ascribed to an unrelated, unlabeled compound, which was detected to a significant level because the isotopomer peaks of 18:1/16:0-GlcDG scattering over a wide range were relatively weak with respect to the contaminating peaks. At this time (2 h), however, MGDG contained a significant level (24%) of labeled population with a p of 0.90. This is a good indication that the 18:1/16:0-MGDG originated from 18:1/16:0-GlcDG by keeping the entire carbon skeleton. In other words, the virtually unchanged p value of

MALDI-TOF-MS analysis was performed as described previously.12 Each text file of the raw intensities (at a resolution of ∼0.07 mass unit) was processed by a perl script to estimate correct valleys and peaks and the level of baseline. Signals of (M + Na)+ ions were used for computational analysis to obtain the isotopomer distribution. Methanolysis was performed in 2.5% HCl in anhydrous methanol at 85 °C for 2.5 h, and the resultant fatty acid methyl esters were extracted with n-hexane and analyzed by GC−MS as described previously.20 Glycerol and methyl glycosides were recovered from the methanol phase and analyzed by GC−MS as trimethylsilyl derivatives as described previously.12 MS/MS Analysis. The 18:1/16:0 MGDG that was obtained from 13C-labeled A. variabilis M3 cells, as well as the 18:2/16:0 MGDG that was obtained from 13C-labeled Anabaena sp. PCC 7118 cells, was further analyzed by LC−MS/MS as previously reported.22 The mass resolution was ∼0.004 mass unit. The monoenoic (Un = 1) or dienoic (Un = 2) fractions of MGDG isolated by argentation TLC were used. For each (M + NH4)+ isotopomer peak of the initial LC−MS experiment (positive mode, from m/z 774.61 to 818.75 for 18:1/16:0 and from m/z 772.61 to 816.75 for 18:2/16:0), MS/MS spectra were obtained for the diacylglyceryl fragment (m/z 577 or 575) and the monoacylglyceryl fragments (m/z 339 for oleoyl, m/z 337 for linoleoyl, and m/z 313 for palmitoyl). The complete intensity data were converted to text files and processed to construct three-dimensional diagrams as shown in Figure S2 and Figure 6. Computer Analysis of Combinatomers. All the mass spectral data were processed digitally using perl scripts and the “C13dist” program developed in the C language with the GNU Scientific Library. Computation was performed in the UNIX environment of MacOS X running on Apple personal computers. The GNU Plot software (versions 4 and 5) was used for drawing spectra and histograms. The overview of the calculation is described in Computational methods in the Supporting Information. The software “C13dist” is available from the Web site of one of the authors (http://nsato4.c.utokyo.ac.jp/old/C13dist.html).

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RESULTS AND DISCUSSION Conversion of Exogenous [14C]GlcDG to MGDG. As a first step for analyzing the conversion of GlcDG to MGDG, we tried to add radiolabeled GlcDG exogenously to the cells of A. variabilis M3 (Figure 1). During the incubation, the radioactivity was incorporated into the cells (panel B), and a significant part of the incorporated radioactivity was recovered in MGDG (panel C). This suggests that the incorporated GlcDG was converted to MGDG. Then, the GlcDG and MGDG were recovered from the TLC plate and subjected to methanolysis. The proportion of the radioactivity in the fatty acid methyl esters and that in the polar (glycerol and methyl glycoside) fraction were measured (panel D). Although the labels in both parts moved from GlcDG to MGDG, the fact that the fatty acid part was significantly more labeled in MGDG than in GlcDG might be interpreted in various ways: during the conversion in vivo, the label in the sugar part could be lost by exchange with unlabeled sugar, or the fatty acids in the substrate GlcDG were hydrolyzed and transferred to unlabeled MGDG by acyl exchange. Because the experiment of direct conversion of GlcDG to MGDG was not successful in vitro, as in the isolated membrane fraction, we had to take another approach using in vivo labeling and chase. 5692

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Figure 3. Scheme for the combinatomer analysis. A combinatomer is defined as GlcDG or MGDG molecules consisting of a combination of their four parts (C18, C16, glycerol, and hexose) that are either fully labeled or unlabeled. The labeling state of the parts is shown by “L” or “U”, for labeled or unlabeled, respectively. Therefore, the combinatomer of GlcDG or MGDG is shown by a combination of four symbols, such as UUUL, indicating the molecule has only labeled hexose. Let us assume that the initial GlcDG is a mixture of fully labeled (LLLL) and unlabeled (UUUU) molecules. If this pool of GlcDG is directly epimerized (case 1), then the resultant MGDG will be fully labeled (LLLL) and unlabeled (UUUU) molecules. The UUUU type of MGDG is also present from the beginning of the experiment. If glucose is released from GlcDG as the UDP derivative, for example, then epimerized, and finally recombined with the DAG, then four types of combinatomers will be produced: UUUU, UUUL, LLLU, and LLLL (case 2). Under the experimental conditions, the proportion of UUUL and LLLU will be high with respect to UUUU and LLLL. The figure also shows two other cases. Case 3 indicates incorporation of a new hexose into MGDG. Case 4 indicates acyl exchange at the sn-1 position. Acyl exchange of sn-2 is equally possible.

43 carbon atoms). The isotopomer of GlcDG containing, for example, 26 13C atoms (this means p = 26/43 = 0.60, if all carbons are uniformly labeled) might be a “uniformly labeled” molecule, namely, the molecule consisting of equally labeled component parts. In this situation, C18 acid contains 11 13C atoms (=18 × 0.60), C16 acid contains 10 13C atoms (=16 × 0.60), glucose contains 3 13C atoms (=6 × 0.60, rounded down to make the sum 26), and glycerol contains 2 13C atoms (=3 × 0.60), but we can imagine a situation of unequal labeling, in which only some of the component parts are labeled. For example, C18 acid contains 17 13C atoms, glucose contains 6 13 C atoms, glycerol contains 3 13C atoms, and C16 acid contains no 13C. To distinguish these and other situations, we introduce a nomenclature for the different types of molecules formed by the combination of labeled (L) or unlabeled (U) parts (sn-1 acyl, glycerol, sn-2 acyl, and hexose) that we call “combinatomers” and designate them with a set of four symbols (in the same order), such as UUUL for the molecule labeled in only the hexose part (Figure 3). If the original GlcDG is a mixture of LLLL (uniformly labeled) and UUUU (unlabeled or

0.90 indicates that, during the conversion, all the carbon atoms constituting the basic structure were kept unchanged. During the chase, low-abundance isotopomers with p values of 0.10− 0.22 appeared in 18:1/16:0-GlcDG. Smaller but similar peaks appeared also in 18:1/16:0-MGDG. These could arise from transitory dilution of 13C after the shift to ordinary, unlabeled CO2. The fact that there was no labeling of MGDG after the 30 min labeling, but 18:1/16:0-MGDG that gained 24% of the label at the 2 h time point might suggest that the lipid biosynthesis in general and conversion of GlcDG to MGDG was enhanced during this time. One probable reason is that labeling was performed in 10 mM bicarbonate under 100 μE m−2 s−1 light, whereas the standard culture condition was 1% CO2 under 40 μE m−2 s−1 light. This favorable condition could accelerate growth during the labeling time and enhanced the conversion of GlcDG to MGDG. Initial Analysis of Combinatomers. To proceed further, we have to distinguish two types of “partially labeled” isotopomers of GlcDG or MGDG (these glycolipids contain 5693

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Figure 4. TOF-MS spectra and isotopomer distribution in the experiment with Anabaena sp. PCC 7118. Panels on the left (A−I) show the results after labeling for 45 min, whereas the panels on the right (J−P) show the results after the chase for 4 h following labeling for 45 min. Panels A−E and J−L show TOF-MS spectra: (A) Un = 0 fraction of GlcDG, (B) Un = 1 fraction of GlcDG, (C and J) Un = 2 fraction of GlcDG, (D and K) Un = 1 fraction of MGDG, and (E and L) Un = 2 fraction of MGDG. The remaining graphs show isotopomer distributions: (F) 18:0/16:0-GlcDG before chase, (G) 18:1/16:0-GlcDG before chase, (M) 18:2/16:0-GlcDG after chase, and (N) 18:2/16:0-MGDG after chase. Panels H, I, O, and P show results for experiment 2 corresponding to panels F, G, M, and N for experiment 1, respectively.

value of a population, rather than a single isotopomer representing the labeled or unlabeled molecule. LC−MS/MS analysis of 18:1/16:0-MGDG after the 2 h labeling (Figure S2) showed that the p = 0.90 peak consisted of a uniformly labeled population (LLLL), whereas the lowintensity peaks from (M + 17 + NH4)+ to (M + 25 + NH4)+ consisted of isotopomers having labeled component parts and unlabeled component parts, such as labeled 16:0 and glycerol (and presumably galactose) and unlabeled 18:1 (ULLL). The presence of a small amount of labeled and/or unlabeled hybrid molecules at the 2 h time point suggests that some exchange of constituent parts (presumably acyl groups) occurred during the synthesis of 18:1/16:0-MGDG. The fact that the amount of ULLL (2.7 ± 0.4%, average ± standard deviation of the estimations from the three partial spectra) was significantly larger than the amount of LLUL (1.0 ± 0.4%) whereas the

natural abundance), this will give MGDG consisting of a mixture of LLLL and UUUU by direct epimerization (case 1 in Figure 3). If glucose is released and epimerized as a UDP or other derivatives (case 2), the product will contain LLLU and UUUL as well as LLLL and UUUU. If galactose is provided from an unknown internal pool (case 3), the product will include LLLU. If the acyl group at the sn-1 position is replaced by another acyl group (case 4), ULLL will be produced. The notion of a combinatomer might be difficult to grasp, but it is critically important, because the production of partially labeled combinatomers or lack thereof will distinguish the different mechanisms of conversion of GlcDG to MGDG. Note that, in this notation, L and U each denote a population of molecules consisting of various isotopomers according to the distribution specified by isotopic abundance p. We must consider the p 5694

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Figure 5. Isotopomer distributions of representative labeled molecular species of GlcDG and MGDG after photosynthetic labeling for 45 min and chase for 4 h in the presence of cerulenin in Anabaena sp. PCC 7118. Isotopomer distributions estimated from TOF-MS spectra of GlcDG and MGDG are presented in panels A, F, K, and P. The graphs are enlarged to show the distribution of the labeled population. Isotopomer distributions of C18 acids at sn-1 (B, G, L, and Q), 16:0 at sn-2 (C, H, M, and R), glycerol (D, I, N, and S), and hexoses (E, J, O, and T) are shown. (A−E) 18:0/ 16:0-GlcDG and the constituents of the Un = 0 fraction of GlcDG after labeling for 45 min. (F−J) 18:1/16:0-GlcDG and the constituents of the Un = 1 fraction of GlcDG after labeling for 45 min. (K−O) 18:2/16:0-GlcDG and the constituents of the Un = 2 fraction of GlcDG after chase for 4 h following labeling for 45 min. (P−T) 18:2/16:0-MGDG and the constituents of the Un = 2 fraction of MGDG after chase for 4 h following labeling for 45 min. Results of experiment 1 of the two independent series of experiments are shown.

In Vivo Labeling and Chase Experiments Using 13C in Anabaena sp. PCC 7118. Anabaena sp. PCC 7118, which was known to be identical to strain M3 ∼50 years ago, was found to contain a higher level of GlcDG.12 Strain PCC 7118 is, therefore, a suitable organism for studying the conversion of GlcDG to MGDG. The cells were labeled with 13C for 45 min and then chased in the presence of cerulenin, which is necessary to prevent incorporation of the diluted carbon isotope during the chase period as seen in the previous experiment (Figure 2E,F). Two series of experiments were performed independently. The top part of Figure 4 (A−E and J−L) shows the raw

amount of LUUU (0.7 ± 0.2%) was larger than the amount of UULU (0.2 ± 0.2%) suggests that the exchange at the sn-1 position (C18 fatty acids) was more frequent than the exchange at the sn-2 position. Labeling and chase experiments were repeated in strain M3 with or without cerulenin (inhibitor of de novo fatty acid synthesis to stop labeling at low p values during the chase) at 38 or 30 °C (for a changing desaturation rate), but we were not able to find a good condition for labeling and chase for further detailed analysis of isotopomers, mainly because the level of GlcDG was too low in strain M3 as in most cyanobacteria. 5695

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Figure 6. MS/MS spectra of 18:2/16:0-MGDG after chase for 4 h following labeling for 45 min in Anabaena sp. PCC 7118. Two-dimensional spectra of the representative regions are shown in panels A−C. Panel D represents a LC−MS spectrum of 18:2/16:0-MGDG (as NH4 adducts or [M + 17]+ ions). Each of the peaks in the LC−MS spectrum was ionized individually: (A) diacylglyceryl ion, (B) linoleoylglyceryl ion, and (C) palmitoylglyceryl ion. The shaded triangular areas represent regions in which the ions in question should not be observed theoretically; in other words, the peaks found in these areas are unrelated labeled ions.

data of mass spectra from experiment 1. Processed data showing the isotopomer distribution of selected molecular species in the two experiments are shown below (F−I and M− P). After the labeling, the Un = 0 fraction of GlcDG (mostly 18:0/16:0) consisted of uniformly labeled isotopomers of 18:0/ 16:0 species (∼81%) as indicated by the large cluster of peaks at m/z 815−822 (panel A). The Un = 1 fraction of GlcDG consisted mainly of 18:1/16:0 species labeled to ∼34% (panel B). In contrast, the Un = 2 fraction of GlcDG (18:2/16:0), the Un = 1 fraction of MGDG (18:1/16:0 and 16:1/16:0), and the Un = 2 fraction of MGDG (18:2/16:0) were not labeled significantly (panels C−E, respectively). The level of the Un = 0 fraction of MGDG was very low throughout the experiment and was not analyzed. After the chase, the levels of the Un = 0 and Un = 1 molecular species decreased drastically because of desaturation of the C18 fatty acids (Figure S3). Un = 2 species of GlcDG and MGDG contained small proportions of labeled isotopomers (Figure 4M,N). Analysis of the isotopomer distribution showed that the isotopic abundance (p) of the labeled population of isotopomers was approximately 0.78−0.80 in experiment 1 and approximately 0.71 in experiment 2. The constant p values within each experiment suggest that the labeled isotopomers were converted from Un = 0 species of GlcDG to Un = 2 species of MGDG without a significant change in the isotopomer composition, although more detailed analysis would be necessary as described below.

Figure 5 shows the isotopomer distribution of intact glycolipids (panels A, F, K, and P) and their component parts, namely, C18 fatty acids at the sn-1 position (panels B, G, L, and Q), 16:0 at the sn-2 position (panels C, H, M, and R), glycerol (panels D, I, N, and S), and hexose (glucose in panels E, J, and O or galactose in panel T) in experiment 1. All component parts consisted of the labeled population of isotopomers with p values of 0.78−0.80. As a first approximation, we can assume that the peaks of isotopomers in the four parts correspond to the peak of highly labeled isotopomers of GlcDG or MGDG. Via comparison of panels A−E, for example, the stearic acid containing 14 13C atoms (panel B), the palmitic acid containing 12 13C atoms (panel C), the glycerol containing 3 13C atoms (panel D), and the glucose containing 6 13C atoms (panel E) make up GlcDG containing 35 13C atoms. The real peak of GlcDG was indeed an isotopomer containing 35 13C atoms (panel A). This good matching of the peaks suggests that the highly labeled GlcDG consists of a combination of labeled parts. However, the situation might not be simple for the isotopomers containing ∼25 13C atoms (panels F, K, and P), which are crucial for discriminating different mechanisms (Figure 3). These isotopomers could consist of combinations of equally partially labeled component parts, each having ∼58% (=25/43) 13C, or of combinations of labeled parts and unlabeled parts. To solve this problem, we performed LC−MS/MS analysis as described in the next section. 5696

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Biochemistry

Figure 7. Combinatomer analysis. (A) Isotopomer distributions of components (C18, glycerol, C16, and hexose) are decomposed into distributions of labeled (magenta) and unlabeled (green) populations. U and L refer to unlabeled and labeled, respectively. In the case of glycerol, the computed distributions with p = 0.03 and p = 0.96, which matched best with the observed isotopomer distribution, were used as the distributions for U and L, respectively. Therefore, there is a small overlap of the two distributions, which is not evident in the figure. In other cases, the observed isotopomer distribution was simply separated into U and L, because the overlap of the two distributions was negligible. (B) Estimated 1D distributions of all possible combinatomers consisting of the labeled and unlabeled populations in panel A. The set of four characters represents the labeled status of the four components, C18, glycerol, C16, and hexose, in this order. (C) Estimated 2D distributions of all possible combinatomers (linoleoylglyceryl vs MGDG) consisting of the labeled and unlabeled populations in panel A. Similar sets of 2D distributions of combinatomers were also calculated for 5697

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Biochemistry Figure 7. continued

diacylglyceryl vs MGDG and palmitolglyceryl vs MGDG (not shown). (D) Fitting of the linoleoylglyceryl part of the MS/MS spectrum with a linear combination of the 16 combinatomers. MSMS, 2D map obtained from the observed spectra (Figure 6B) with corrections for the contributions of multiple peaks (mode 3 in C13dist); Synthesized, result of fitting; Difference, residual of fitting, namely, MSMS − Synthesized (enlarged with respect to other panels).

chase in all one-dimensional (1D) analyses. For two-dimensional (2D) analyses (see below), the distributions after the chase were also used. We first performed combinatomer analysis in one dimension using the isotopomer distribution of various molecular species of GlcDG or MGDG (as shown in Figure 5A,F,K,P). The estimated theoretical isotopomer distributions of various combinatomers are shown in Figure 7B. The fitting results are shown in Figure 8A−H. The 18:0/16:0-GlcDG after labeling consisted mostly of LLLL (panel A), and the 18:1/ 16:0-GlcDG consisted of UUUU and LLLL (panel B). A small amount of LULL was detected maybe because of inaccuracy in the fitting, because it is improbable that only glycerol was exchanged. The more unsaturated GlcDG and MGDG were mostly unlabeled at this time (panels C−E). After the chase, 34:2-GlcDG contained a small proportion of LLLL (panel F). 34:1- and 34:2-MGDG apparently contain various combinatomers, but this could be due to contaminating peaks in the original mass spectra (panels G and H). 34:2-MGDG was analyzed by LC−MS/MS as described above, and the results were subjected to 2D combinatomer analysis. Figure 7C shows the theoretical 2D isotopomer distributions of combinatomers constructed from isotopomer distributions of the four parts (Figure 7A). Figure 7D shows the result of fitting. The calculation was performed for each of the three kinds of MS/MS spectra of the two experiments (Table S1). The results of the fittings using the three different parts of MS/MS were essentially similar, though not completely identical. The final result (Figure 8I) indicated that LLLL was the major labeled combinatomer. The very small amounts of UUUL and LLLU suggest that the exchange of hexose (case 2 or 3 in Figure 3) did not happen during the conversion of GlcDG to MGDG. The index (UUUL + LLLU)/LLLL was ∼0.1 (Table S1), strongly in favor of direct epimerization (case 1). The small amount of LULU and LULL could represent errors due to imprecise fitting as described above. Direct Epimerization (and direct desaturation). Although various mechanisms can account for the conversion of GlcDG to MGDG, there are two biochemically plausible ones: exchange of sugars with epimerization as UDP or other derivatives (case 2 in Figure 3) and direct epimerization of the sugar moiety (case 1). All the results are consistent with the original hypothesis that the conversion of 18:0(or 18:1)/16:0GlcDG to 18:2/16:0-MGDG occurs by direct epimerization of the glucose moiety at C-4 and, at the same time, by the direct desaturation at the sn-1 position. As explained in the introductory section, the reaction of UDP-glucose epimerase is the only 4-epimerization of glucose that is known to date. In this respect, the epimerization of GlcDG to MGDG as reported here provides a novel example of 4-epimerization within a glycolipid. Although the index (UUUL + LLLU)/LLLL was as low as 0.1, we will have to be cautious about a possible pitfall, such as the nonmixing of intermediates: namely, even in the mechanism of case 2, this index could be low if the reaction intermediate is not released from the enzyme complex and is not mixed with each other. This problem will have to be solved

Comparison of the data before and after the chase indicated that all parts of the 18:0/16:0- and 18:1/16:0-GlcDG present before the chase were equally shifted to the 18:2/16:0-GlcDG and 18:2/16:0-MGDG after the chase, accompanied by the desaturation of 18:0 to 18:1 and 18:2 at the sn-1 position and the conversion of glucose to galactose. The distribution of isotopomers in the fatty acids was not completely smooth, but this can be explained by uneven labeling in the precursor pools (see the last section). This effect, however, does not seriously affect the analysis of the results. Note also that the Un = 1 fraction of MGDG contained a significant proportion of 16:1/ 16:0 species after the chase with cerulenin (Figure 4D,K), probably because the 18:1/16:0 species was no longer synthesized from the 18:0/16:0 species but efficiently desaturated to the 18:2/16:0 species, whereas the desaturation of 16:1/16:0 species was slow. Similar results were obtained in experiment 2 with a slightly lower p value (Figure 4H,I,O,P). The distribution of isotopomers in the labeled population of 18:2/16:0-MGDG after the chase (Figure 5P) was not very smooth because the content of the labeled population was very low in this fraction. This fraction was further analyzed by LC− MS/MS (Figure 6) to determine the isotopomer distribution more precisely. Because of the high sensitivity of the instrument used, the isotopomer distribution as detected after the initial MS was smoother (Figure 6D). The peaks at m/z 813−815 were unrelated signals, because no MS/MS signals were detected in the acylglyceryl (Figure 6B,C) or diacylglyceryl fragments (Figure 6A). Combinatomer Analysis. As a rough estimate (Figure S3), ∼15 and ∼28 nmol of Un = 0, Un = 1, and Un = 2 fractions of GlcDG were converted to MGDG during the chase period in experiments 1 and 2, respectively, and in both experiments, the consumed GlcDG consisted of ∼17% LLLL and ∼83% UUUU. On the basis of the scheme in Figure 3, it is expected that these mixtures should be converted to UUUU and LLLL combinatomers of MGDG, keeping the same ratio in the direct epimerization (case 1), whereas in case 2 (epimerization of released hexose), the product should consist of 68% UUUU, 14% UUUL, 14% LLLU, and 3% LLLL. Because we cannot distinguish the originally existing UUUU and the newly converted UUUU in MGDG, we will have to use the (UUUL + LLLU)/LLLL ratio as an index of the contribution of mechanisms other than direct epimerization: the index will be 0.0, 9.3, and 4.7 in cases 1−3, respectively. The composition of combinatomers was estimated by fitting the isotopomer distribution of GlcDG or MGDG with a set of theoretical distributions of 16 combinatomers, which are constructed from labeled or unlabeled populations of the C18 acyl group (at sn-1), glycerol, the C16 acyl group (at sn-2), and hexose (glucose or galactose). See Materials and Methods and the Supporting Information for details. The isotopomer distribution of labeled and unlabeled parts (C18, glycerol, C16, and hexose) was estimated from the data in Figure 5 as shown in Figure 7A. Because the distributions were not quite different in different molecular species, we used the isotopomer distributions of the component parts of 34:1-GlcDG before the 5698

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Biochemistry

the acyl exchange reaction, also suggested in the experiment depicted in Figure 2. The mgdA gene encoding the glucosyltransferase involved in the formation of GlcDG is encoded in all the cyanobacterial genomes analyzed to date.4 The conversion of GlcDG to MGDG occurs in all cyanobacteria analyzed to date.12 The mgdE gene is involved in the conversion of GlcDG to MGDG in most cyanobacteria.4,13 These lines of evidence suggest that the conversion of GlcDG to MGDG is an essential step in the cyanobacterial synthesis of MGDG. The question of why glucose should be epimerized on the glycolipid rather than as a UDP derivative is now asked. Cyanobacteria can synthesize UDP-galactose, which is indeed used in the synthesis of DGDG by galactosylation of MGDG.4 Putative genes for UDP-glucose epimerase are also known, such as sll0576, slr0583, slr0809, and slr1617 in Synechocystis sp. PCC 6803. The direct epimerization of GlcDG is not, however, conserved in chloroplasts of algae and plants that are thought to be descendants of a cyanobacterial endosymbiont. We still do not know the origin of this unique system specific to cyanobacteria. Mechanism of Epimerization. Another question is asked with respect to the reaction mechanism of the epimerization. Because the single gene mgdE is sufficient for the conversion of GlcDG to MGDG in Escherichia coli cells harboring the mgdA gene, the MgdE epimerase must catalyze a single-step conversion of glucose to galactose on the glycolipid. The conserved C-terminal domain of MgdE contains the Rossman fold, in which many of the important motifs involved in the binding of NAD are present14 with some differences (Figure S4). The motifs involved in the binding of hexose (the “hexagonal box” and the “gatekeeper”) in the UDP-glucose epimerases were not conserved. Homology modeling of the Cterminal epimerase domain of MgdE in Anabaena sp. PCC 7120 (the mgdE gene sequence is identical in PCC 7118 and PCC 7120) indicated a similarity to the corticosteroid 11βdehydrogenase (Figure S5). The position and orientation of the NAD+ in the active center are different in UDP-glucose epimerase (GalE, panel A) and 11β-dehydrogenase (11-DH, panel B). GalE has a large UDP-binding domain above the epimerase domain, whereas 11-DH has a small substratebinding domain. The interaction of NAD+ and the substrate might not be identical in GalE and 11-DH. As the N-terminal domain of MgdE is not modeled currently, we are unable to estimate the binding site of GlcDG in the MgdE structure (panel C). An interesting feature of MgdE is the apparent irreversibility of the reaction. GalE has a mechanism of rotating the sugar group during the epimerization reaction involving successive oxidation and reduction, with similar affinity for both UDP-glucose and UDP-galactose.14 MgdE might be different in this respect. Structural studies of the MgdE enzyme will answer these questions about the reaction mechanism of GlcDG epimerization. General Usefulness of Isotopomer Analysis in Combination with Photosynthetic Labeling. Use of a stable isotope in metabolic analysis has become popular in recent years, but in many cases, a labeled compound containing 13 C at a specified position is used. Such exogenous carbon compounds, except for glucose for heterotrophic cells, could disturb the metabolism of the cells. We use photosynthesis as a means to label metabolites within the photoautotrophic cells under normal physiological conditions. A drawback of this method is the fact that various isotopomers are produced as a result of random incorporation of many isotopes within a

Figure 8. Combinatomer compositions of various molecular species of GlcDG and MGDG. (A−H) Combinatomer compositions estimated by 1D analysis based on TOF-MS: blue for experiment 1 and red for experiment 2. The distributions of parts (C18, glycerol, C16, and hexose) for 34:1 (or 18:1/16:0)-GlcDG before chase were used for all calculations, because the distributions were not quite different in different molecular species at different time points. (I) Combinatomer composition of 34:2 (or 18:2/16:0)-MGDG after chase estimated by 2D analysis based on MS/MS. The average ± standard deviation of six different estimates in the two experiments is shown. The six estimates include calculations using diacylglyceryl, linoleoylglyceryl, and palmitoylglyceryl ions with distributions of parts for 34:1-GlcDG before chase and 34:2-MGDG after chase. See Table S1 for the original values. In spite of a small difference in the p value, the two series of experiments gave very similar estimates of combinatomers.

by in vitro experiments in the future. Assuming the direct epimerization, the results of conversion of exogenously added radioactive GlcDG (Figure 1) could be interpreted to show that a significant proportion of added GlcDG was degraded to give fatty acids that were incorporated into the existing MGDG by 5699

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Biochemistry molecule. To overcome this problem, we use computational analysis of the distribution of isotopomers. As described earlier, the population of molecules (with n carbon atoms) that are labeled with 13C at an isotopic abundance p contains various isotopomers according to a binomial distribution peaking at an isotopomer containing np 13C atoms. In the actual photosynthetic labeling, a deviation could result from uneven labeling of different precursor pools during the initial phase of labeling. For example, the even-numbered isotopomers are more abundant than the odd-numbered isotopomers in the fatty acid pools (see Figure 5B,C,G,H, after labeling for 45 min). This is easily explained by assuming that there are two precursor pools of acetyl-CoA, one with a high p value and the other with a low p value. A simple simulation is given in Figure S6, which supports this intuitive inference. As a corollary, a potential use of photosynthetic incorporation of 13C is a “double labeling” of different pools with different p values. The analytical method that we introduced can be adapted to multiple pools with distinct p values, which can be traced as different pools. General Utility of Combinatomer Analysis. Combinatomer analysis is a new application of a stable isotope. It is, in principle, capable of distinguishing between mechanisms of the biochemical reaction involving complex molecules consisting of several parts: direct conversion or exchange of parts. A simple version of this logic was used in the famous experiment on the semiconservative replication.24 Combinatomer analysis is a more general way of analyzing biochemical reactions not only in vitro but also in vivo as described here if appropriate experimental settings are available.

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AUTHOR INFORMATION

Corresponding Author

*Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan. E-mail: [email protected]. Phone: 81-3-5454-6631. Funding

This work was supported in part by the Japan Advanced Plant Science Network, by a Grant-in-Aid for Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency, and by a Grant-in-Aid for Scientific Research (24570043) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. K. Awai of the University of Shizuoka (Shizuoka, Japan) for invaluable discussion.

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ABBREVIATIONS DAG, diacylglycerol; GC−MS, gas chromatography−mass spectrometry; GlcDG, monoglucosyl diacylglycerol; LC−MS/ MS, liquid chromatography−mass spectrometry/mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MGDG, monogalactosyl diacylglycerol; TLC, thin layer chromatography; UDP, uridine diphosphate; Un, unsaturation (to denote molecular species differing in unsaturation).

CONCLUSION Epimerization of GlcDG to MGDG is a pivotal step in the biosynthesis of cyanobacterial MGDG. This pathway was proposed more than 30 years ago by one of the authors,11 but the evidence remained elusive for a long time. In parallel with the identification of the responsible gene mgdE,13 we were trying to establish the nature of the conversion of GlcDG to MGDG in terms of (bio)chemistry. The use of a stable isotope in conjunction with computational tools that we developed recently, as well as the use of high-sensitivity mass spectrometers, allowed us to finally identify the direct epimerization of GlcDG to MGDG in vivo. All available evidence indicates now that it is quite probable that the MgdE protein is a GlcDG epimerase. Further confirmation could be achieved by an in vitro enzyme assay that will have to be developed from scratch. In addition, the methodologies that we developed here may be useful in metabolic studies using stable isotopes in various pathways. The computational tools for the conversion of mass spectra to isotopomer distributions, as well as those for the combinatomer analysis, especially in two dimensions, will certainly facilitate analysis of various components of photosynthetically labeled organisms, which has yet to be exploited.

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for 2 h in A. variabilis M3 (Figure S2), amounts of molecular species of GlcDG and MGDG before and after chase in Anabaena sp. PCC 7118 (Figure S3), putative motifs of NAD-dependent epimerase in MgdE (Figure S4), homology modeling of MgdE (Figure S5), effects of mixing of labeled and unlabeled precursor pools on the isotopomer distributions of the fatty acid products (Figure S6), 2D combinatomer analysis of 18:2/16:0MGDG after chase for 4 h (Table S1), and references for the Supporting Information (PDF)

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00769. Isotopic abundance and isotopomer distribution (Figure S1), MS/MS spectra of 18:1/16:0-MGDG after labeling 5700

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