Novel Functions of LBP-5 in Caenorhabditis elegans Fat Metabolism

SUPPLEMENTAL DATA (Xu et al., JBC/2011/227165.R2) Clean Version Novel Functions of LBP-5 in Caenorhabditis elegans Fat Metabolism Mo Xu, Hyoe-Jin Joo and Young-Ki Paik * Supplemental Table S1. A List of Oligonucleotides That Have Been Used for RNAi Primers Gene name Primers lbp-1 Sense: 5 -TACTCTAGAATGTGCGCTAAAATCGCT-3 Antisense: 5 -TACAAGCTTTTATGGGAGTCGTTTGTA-3 lbp-2 Sense: 5 -TACTCTAGAATGTCTTCGAAATTCCTC-3 Antisense: 5 -TACCTCGAGTTATGGGAGTCTCTTGTA-3 lbp-3 Sense: 5 -TACTCTAGAATGAATCTGTACTTAACT-3 Antisense: 5 -TACAAGCTTCTACTTCTTTCCGGTCGA-3 lbp-4 Sense: 5 -TACTCTAGAATGTCAGTGCCAGACAAG-3 Antisense: 5 -TACAAGCTTTCACTTCTGCTCGACTCT-3 lbp-5 Sense: 5 -TACTCTAGAATGTCTGCTGAACAATTT-3 Antisense: 5 -TACCTCGAGCTATTCACGAATATAGGC-3 lbp-6 Sense: 5 -TACTCTAGAGTTGGACGCTGGAAGCTC-3 Antisense: 5 -TACCTCGAGCGGATCCAGATTCAAGAG-3 lbp-7 Sense: 5 -TACTCTAGAATGGCATCTATGAATGAC-3 Antisense: 5 -TACAAGCTTTTATTCTCTCTCCCACTC-3 lbp-8 Sense: 5 -TACTCTAGAGAGTTTATTGGACGATGG-3 Antisense: 5 -TACAAGCTTTTTTGAAAGCGAGCTTGTTG-3 lbp-9 Sense: 5 -TACTCTAGAATGCCAATTCAAACCGATC-3 Antisense: 5 -TACCTCGAGTTAGGCAGCCTTCTCGTAG-3 Supplemental Table S2. Differential Efficiency of Ligand Displacement from LBP-5 by DAUDA Fatty Acid Ligand displacement a Oleic Acid (Δ9, 18:1) 68.9±5.1 Lauric Acid (C12:0) 31.6±3.4 Pentadecanoic Acid (C15:0) 32.3±5.0 Palmitic Acid (C16:0) 33.1±2.7 Stearic Acid (C18:0) 30.8±6.1 Arachidic Acid (C20:0) 26.1±4.8 Arachidonic Acid (C20:4, ω-6) 62.1±7.2 Alpha-Linolenic Acid (C18:3, ω-3) 4.6±0.7 a Values represent the percent drop in peak fluorescence emission (470 nm) of each ligand (oleic acid, lauric acid, pentadecanoic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid and alphalinolenic acid) in LBP-5-DAUDA complex when each fatty acid was added. Concentration of both DAUDA and LBP-5 was 1 μm; that of ligand (fatty acid) was 1 μm. The intensity of fluorescence emission was recorded at the peak emission wavelength (See also supplemental Fig. S5). 1

Figure S1 Figure S1. Multiple Sequence Alignment of lbp Genes. Small and hydrophobic residues (AVFPMILW) are shown in red; acidic residues (DE) are shown in blue; basic residues (RK) are shown in magenta; Hydroxyl + Amine + Basic residues (STYHCNGQ) are shown in green; and all other residues are shown in gray. Asterisks indicate residues that are identical in all sequences in the alignment. Colons indicate conserved substitutions. Periods indicate semi-conserved substitutions. Square frames indicate conserved fatty acid binding domain. 2

Figure S2 A B LBP-2 LBP-3 LBP-4 LBP-9 LBP-5 LBP-6 LBP-7 LBP-8 LBP-1 77 57 55 46 42 43 42 41 LBP-2 55 51 44 39 43 43 43 LBP-3 71 48 43 42 44 44 LBP-4 42 44 42 41 43 LBP-9 50 54 50 46 LBP-5 69 62 61 LBP-6 68 62 LBP-7 70 Figure S2. Relationships of LBP Family Proteins. (A) Phylogenetic relationships of the LBP protein sequences of C. elegans. Relative distances are indicated. The phylogenetic tree was constructed as detailed in the Experimental Procedures and was visualized using Treeview. (B) Sequence identity of LBP family members. 3

Figure S3 A B 4

C Figure S3. (A) Sudan black staining of lbp(rnai) animals (adult). Scale bars, 50 μm. (B) Structural organization of the C. elegans lbp-5 and nhr-49 genes. Mutation sites are underlined for lbp-5(tm1618) and nhr-49(nr2041). (C) Rescue of the lbp-5(tm1618) mutant exhibiting high-fat phenotype. Lipid staining with either Oil Red O staining or Sudan Black staining (left panel) and quantitative analysis (right panel) indicate that high fat lbp-5 mutant is suppressed in the rescued worms. Shown here is wisualization of fat stores in fixed N2, lbp-5(tm1618), and lbp- 5(tm1618);Ex[lbp-5::gfp] worms using fixative dyes Oil Red O and Sudan Black. Scale bars, 50 μm. Triglyceride contents for wild type N2, lbp-5(tm1618), and lbp-5(tm1618);ex[lbp-5::gfp] worms. At least two independent experiments were performed. Error bars indicate the standard deviation. An asterisk (*) indicates a significant difference from the control sample (P < 0.05 as calculated by t- test). 5

Figure S4 A B C D Figure S4. Production of Recombinant GST-LBP-5. (A) 1D gel image of purified recombinant LBP-5 fusion protein. Separation and confirmation of the 6

size of purified recombinant LBP-5-GST fusion protein (42.5 kda including GST) by SDS-gel electrophoresis. (B) Map of LBP-5-GST fusion plasmid. (C) MALDI-TOF-MS/MS analysis of recombinant LBP-5. Purified LBP-5 protein was digested with trypsin (1:50, w/w) followed by analysis by MALDI-TOF-MS/MS (Applied Biosystems 4800) and then validated by peptide mass fingerprinting of the selected peaks of two representative peptides (top: EVGVAVLLR, bottom: EVGVAVLLR NTTLEFTLGVEFDETTPDGR) by NCBInr 20070407 Database (4815286 sequences: 1665828716 residues). For LBP-5, a total of 7 peaks were detected with 64% coverage. (D) MALDI-MS peptide mass spectrum of purified LBP-5 protein following tryptic digestion and trypsin autolytic fragments. indicates matched peptides. Matched peptides in sequence: MSAEQFVGRW KLVESENFED YLKEVGVGLL LRKAACAAKP TLEIKVNGNK WHVNQLSTFK NTTLEFTLGV EFDETTPDGR QFKSTITIED GKVVHVQKRI KDSDHDSVIT RWFEGEKLIT TLQSGSVISR RAYIRE. Experimental data were then compared to the peptide mass database Peptide Mass Fingerprint (PMF). After accurate determination of the peptide masses, databases were searched to identify the LBP-5 protein. 7

Figure S5 A B C D E F G 8

Figure S5. LBP-5 Binds Fatty Acids with Different Affinities. Binding of DAUDA to recombinant LBP-5 and competition with a fatty acid (lauric acid (A), pentadecanoic acid (B), palmitic acid (C), oleic acid (D), arachidic acid (E), arachidonic acid (F) and alpha-linolenic acid (G)). Fluorescence emission spectra (excitation at 345 nm) of 1 µm DAUDA alone or with 1 µm LBP-5 monomer. Also shown is the reversal of changes in DAUDA emission by competition with fatty acid (0.5, 1, 2, 4, and 8 µm) added to the LBP-5-DAUDA complex. 9

Figure S6 A B C D Figure S6. Relationship between lbp-5 and nhr-49 Gene Expression. (A) Gene expression levels of NHR-49 targets involved in mitochondrial β-oxidation in N2, lbp- 5(tm1618), nhr-49(nr2041), and lbp-5(tm1618); nhr-49(nr2041) animals by qrt-pcr. (B) Gene expression levels of NHR-49 targets involved in peroxisomal β-oxidation in N2, lbp-5(tm1618), nhr- 49(nr2041), and lbp-5(tm1618); nhr-49(nr2041) animals. (C) Gene expression levels of NHR-49 targets involved in fatty acid desaturation/elongation in N2, lbp-5(tm1618), nhr-49(nr2041), and lbp- 5(tm1618); nhr-49(nr2041) animals. (D) Gene expression levels of NHR-49 targets involved in gluconeogenesis in N2, lbp-5(tm1618), nhr-49(nr2041), and lbp-5(tm1618); nhr-49(nr2041) animals. Actin served as an internal control. At least three independent experiments were performed. Error bars indicate the standard deviation. 10

Figure S7 Figure S7. Relative Gene Expression of Non-nhr-49 Target Genes. Gene expression levels of non-nhr-49 targets involved in mitochondrial β-oxidation (ech-1 and T08B2.7), peroxisomal β-oxidation (acs-1 and F53A2.7) and gluconeogenesis (sdha-1) in N2, lbp- 5(tm1618), nhr-49(nr2041) and lbp-5(tm1618); nhr-49(nr2041) animals by qrt-pcr. Actin served as an internal control. At least three independent experiments were performed. Error bars indicate S.D. 11

Figure S8 Figure S8. Relative Fatty Acids Contents Measured by GC-MS. Total fatty acids were extracted from wild type N2 worms, lbp-5(tm1618), nhr-49(nr2041) and lbp- 5(tm1618); nhr-49(nr2041) mutant worms which were fed with fatty acid (oleic acid, palmitic acid, stearic acid, arachidonic acid and alpha-linolenic acid), then were analyzed by GC-MS. Worm of control group were fed with NP40. Relative content of each fatty acid in N2, lbp-5(tm1618), nhr- 49(nr2041) and lbp-5(tm1618); nhr-49(nr2041) mutant worms indicates any fatty acid was actually taken up by the worms. In detail, each determined contents of fatty acid (oleic acid, palmitic acid, stearic acid, arachidonic acid and alpha-linolenic acid) in each control group (N2, lbp-5(tm1618), nhr- 49(nr2041) and lbp-5(tm1618); nhr-49(nr2041) worms fed with NP40) was as a baseline, while the content of each specific fatty acid of each group worms fed with this specific fatty acid was just compared with its baseline. Pentadecanoic acid (C15:0) served as an internal standard. Three replicates of fatty acid methyl esters were prepared and three independent experiments were performed. Error bars indicate S.D. 12

Figure S9 A B C D 13

E F G H I J 14

K L M N O P 15

Q R S Figure S9. nhr-49 Target Gene Expression in Different Conditions. Gene expression levels of nhr-49 targets involved in mitochondrial β-oxidation, peroxisomal β- oxidation, fatty acid desaturation/elongation and gluconeogenesis in N2, lbp-5(tm1618), nhr- 49(nr2041) and lbp-5(tm1618); nhr-49(nr2041) worms fed with oleic acid, palmitic acid, arachidonic acid and alpha-linolenic acid by qrt-pcr. Actin served as an internal control. At least three independent experiments were performed. Error bars indicate S.D. 16

Figure S10 Figure S10. Relative expression levels of lbp-5 at different stages of N2 and dauer animals. Actin served as an internal control. Graph shows the mean values (± S.D.) from three independent experiments. 17

Supplementary Experimental Procedures Identification of Fatty Acid Binding Domain in C. elegans LBP Family Members-C. elegans lipid binding protein (LBP) family members (lbp-1 through -9) were previously reported (1). In this study, the sequences of nine lbp were obtained from WormBase (http://www.wormbase.org/) and the conserved fatty acid binding domain of all these nine LBP proteins as well as their protein sequences were aligned by Vector NTI software package. Information on the conserved sequences among LBPs was obtained using ScanProsite (http://www.expasy.org/tools/scanprosite/). RNAi by Feeding-The primers used to construct lbp RNAi vectors are shown in supplemental Table S1. These clones were digested with the appropriate restriction enzymes and inserted into the ppd129.36 (L4440) vector containing two convergent T7 polymerase promoters in opposite orientations and separated by a multiple cloning site. Plasmid DNA was transformed into E. coli HT115 (DE3) cells. HT115, which carries the ppd129.36 plasmid with no insert, was used as a control. Cells carrying plasmid DNA were directly applied onto agar plates composed of standard NGM/agar medium supplemented with 50 µg/ml ampicillin and 0.5 mm isopropyl beta-d-1- thiogalactopyranoside (IPTG), and then cultured overnight at room temperature. Beginning the next day, N2 worms were grown on the plates containing transformed E. coli HT115 (DE3) cells producing lbp dsrna. Expression of the corresponding endogenous C. elegans gene was knocked down by RNAi (2). Nile Red Lipid Staining-As a preliminary examination procedure, Nile red was used to stain and assess the amount of lipid present in worms (3). Nile red powder (Molecular Probes, Eugene, OR) was dissolved in acetone as a stock solution of 500 µg/ml, and diluted to 1 µg/ml in M9 buffer before 200 µl was added to E. coli (OP50)-seeded 50-mm NGM plates, resulting in a final concentration of 0.05 µg/ml. Synchronized cohorts of C. elegans were transferred to these plates, left overnight, and the fluorescence intensity was measured on the following day using a rhodamine filter. Nile red was visualized using a Zeiss AX10 microscope (Jena, Germany). All Nile red images were acquired using identical settings and exposure times. Source of lbp-5(tm1618) and nhr-49(nr2041)-the lbp-5(tm1618) mutant was created with 940-bp deletion and 20-bp insertion at the 3 region, which was prepared by NBRP, Japan. The nhr- 49(nr2041) mutant has an 893-bp deletion and was a gift from Carl Johnson at Nemapharm Pharmaceuticals. Oil Red O Staining-Worms were washed three times with 1 x PBS and then suspended in 200 µl of PBS to which an equal volume of 2X MRWB buffer (160 mm KCl, 40 mm NaCl, 14 mm Na 2 EGTA, PIPES ph 7.4, 1 mm spermidine, 0.4 mm spermine, 30 mm, 2% paraformaldehyde, 0.2% 18

beta-mercaptoethanol) was added. The worms were taken through 1 freeze-thaw cycle between liquid nitrogen and warm running tap water, followed by spinning at 14000g and washing once in PBS to remove paraformaldehyde. After fixation, worms were resuspended and dehydrated in 60% isopropanol. Approximately 450 ml of 60% Oil Red O stain (Cat. No. O9755, Sigma-Aldrich, St. Louis, MO, USA) was added to each sample, and samples were incubated overnight at room temperature (4). Animals were mounted and imaged with a Zeiss AX10 microscope (Jena, Germany). Construction of LBP-5 Expression Plasmid and Recombinant LBP-5-Primers were designed to PCR amplify the LBP-5 coding region from the reverse-transcribed C. elegans RNA. The PCR fragment was sub-cloned into the pgex4t3 expression plasmid, and transformed first into DH5α cells and then into BL21 (DE3) E. coli for recombinant protein expression. Transformed E. coli BL21 (DE3) were cultured overnight at 37 C in Luria Bertani medium containing 50 µg/ml ampicillin. The stock culture was diluted 1:100 in the same medium and the bacteria were grown to A 600 =0.5 before induction with 1 mm isopropyl beta-d-1-thiogalactopyranoside (IPTG). After 4 hr at 37 C, the cells were collected by centrifugation at 4,000 g and resuspended in pre-cold PBS plus lysozyme at a final concentration of 0.2 mg/ml, then sonicated on ice for five 1-min bursts. Following centrifugation at 15,000 g, the supernatant was applied to glutathione Sepharose 4B (GE Healthcare) packed in a 2-ml column. The washing procedure and elution of the fusion protein were carried out according to the manufacturer s instructions (www.embl.de/externalinfo/pepperko/assets/applets/pdf-gstpurification.pdf). The concentration of the protein solution was measured using a Bradford assay. Aliquots of protein solution were subjected to SDS-PAGE analysis on 8% gels followed by staining with Coomassie Brilliant Blue R- 250 (Amersham Pharmacia). The recombinant LBP-5 was confirmed by MS/MS analysis and further characterized by various chemical methods. Identification of Recombinant LBP-5 Protein by MALDI-TOF-Protein bands that were not present in control samples on Coomassie blue-stained SDS-PAGE gels were excised, destained, and digested by 10 μg/ml trypsin (Promega, Southampton, UK) in 50 mm ammonium bicarbonate. Mixtures were processed for mass spectrometry analysis as previously described (5). Recovered peptides were prepared for MALDI-TOF MS by mixing with CHCA, 1% formic acid in 50% ACN, and droplets were allowed to dry on the MALDI sample plate. The peptide mass fingerprinting (PMF) was analyzed with the 4800 MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). The mass spectra were obtained in reflect/delayed extraction mode with an accelerating voltage of 20 kv and sum data from 1,000 laser pulses. Proteins were identified from the PMF using MASCOT (http://mascot.proteomix.org/search_form_select.html) in search of protein database and NCBInr database with the Matrix Science search engine. Circular Dichroism Analysis-CD spectra were collected using a Jasco J810 19

spectropolarimeter with a thermostatically controlled cell holder. A fused quartz cell with a path length of 0.1 cm was used for CD measurements, using 0.67 mg/ml of recombinant LBP-5. All measurements were made in 10 mm potassium phosphate buffer at ph 7.4 and ambient temperature. The spectra measured in the far UV-region 190-250 nm were averages of four scans and were normalized by subtracting the baseline of the buffer (6,7). Fluorescent Probes and Competitors-The fluorescent fatty acid analogue 11-((5- dimethylaminonaphthalene-1-sulfonyl) amino) undecanoic acid (DAUDA) was obtained from Molecular Probes. Oleic acid (Δ9, 18:1), lauric acid (12:0), pentadecanoic acid (15:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (C20:0), arachidonic acid (C20:4, ω-6) and alpha-linolenic acid (C18:3, ω-3) were obtained from Sigma. The fluorescent compound DAUDA was prepared as a 10 mm stock solution in ethanol, stored in the dark at 20 C, and freshly diluted in PBS for use in the fluorescence experiments. Competitors of fluorescent fatty acid binding were prepared as stock solutions in ethanol at approximately 10 mm and diluted in PBS for use. Spectrofluorimetry Analysis-Fluorescence intensities were measured at 20 C in a Jasco FP- 6500 Spectrofluorimeter using 200 μl samples in a quartz cuvette. Raman and background scattering by the solvent was corrected for using appropriate blank solutions, if necessary. Binding Affinity Assay-The ligand binding capacity of the recombinant LBP-5 was investigated with DAUDA. This fluorescent lipid shows a blue shift of emission wavelength maximum from 535 nm to 470 nm when it binds to protein. Fixed amounts (1 μm) of recombinant LBP-5 were incubated with 0-10 μm DAUDA in PBS to a total volume of 200 μl. Fluorescence intensity was measured (Ex345nm, Em420-600nm) after equilibration at 25 C for 20 min. The fluorescence enhancement that occurred with the probe binding to LBP-5 was compared to the amount of fluorescence of the same probe in buffer alone. The data were subjected to Scatchard analysis. Apparent dissociation constants [K d ] and Bmax were calculated from the Scatchard Curves. If the fluorescent probe bound to the fatty acid-binding site of LBP-5 caused displacement of the probe, this was detectable as a concomitant decrease in the fluorescence intensity with increasing fatty acid concentration. We assumed that the maximal fluorescence achieved in the presence of excess DAUDA represented 100% binding and that the amount of DAUDA bound was proportional to the relative intensity (1,8-10). Fatty Acid Analysis by GC-MS-For quantitative analysis of the fatty acid composition, worms were washed with S-basal medium for three times and the buffer should be completely removed. Fatty acid methyl esters were prepared as described (11,12). As an internal standard, 50 µg of pentadecanoic acid (C15:0) was added to each worm sample. About 1 ml of 2.5% methanolic H 2 SO 4 was then added, and the worm samples were boiled at 90 C for 1 hr. After cooling the samples, 1 ml of hexane and 1.5 ml H 2 O were added into the sample and mixed thoroughly. Methyl 20

esters in the hexane layer were analyzed using an Agilent HP6890 gas chromatograph (GC)-HP5973N mass selective detector (MSD) system (Agilent, Palo Alto, CA, USA) equipped with an HP-5 column (30 m 0.25 mm, 0.25 µm). Three replicates of fatty acid methyl esters were prepared and analyzed for each worm sample. Life Span Assay-Life spans were measured in synchronized populations from the onset of the L4 stage. For each experiment, five animals per strain were placed on each 50-mm seeded NGM plate and checked daily until expiration. During the egg-laying period, animals were transferred daily to fresh plates. At all other times, they were transferred approximately once per week to prevent starvation. Worms were classified as dead when no movement was detected following a gentle prod to the anterior end with a worm pick. Worms that died because they crawled off the plates, exploded (i.e., had a gonad extruding through their vulva), or bagged (i.e., experienced internal hatching) were excluded from this study, because they did not die of old age. Brood Size Assay-The brood sizes of mutant and wild-type strains were determined by placing synchronized late-l4 animals on seeded NGM plates at 20 C and recording when egg laying began. From this point onward, animals were transferred daily to fresh plates, and the number of eggs laid per day was scored. Plates containing the eggs were checked a few days later to determine the proportion that had hatched and developed (13). Supplemental References 1. Plenefisch, J., Xiao, H., Mei, B., Geng, J., Komuniecki, P. R., and Komuniecki, R. (2000) Mol Biochem Parasitol 105(2), 223-236 2. Kim, S., and Paik, Y. K. (2008) Biochem Biophys Res Commun 368(3), 588-592 3. Ashrafi, K., Chang, F. Y., Watts, J. L., Fraser, A. G., Kamath, R. S., Ahringer, J., and Ruvkun, G. (2003) Nature 421(6920), 268-272 4. Soukas, A. A., Kane, E. A., Carr, C. E., Melo, J. A., and Ruvkun, G. (2009) Genes Dev 23(4), 496-511 5. Cho, S. Y., Park, K. S., Shim, J. E., Kwon, M. S., Joo, K. H., Lee, W. S., Chang, J., Kim, H., Chung, H. C., Kim, H. O., and Paik, Y. K. (2002) Proteomics 2(9), 1104-1113 6. Provencher, S. W., and Glockner, J. (1981) Biochemistry 20(1), 33-37 7. Sreerama, N., and Woody, R. W. (1993) Anal Biochem 209(1), 32-44 8. Motola, D. L., Cummins, C. L., Rottiers, V., Sharma, K. K., Li, T., Li, Y., Suino-Powell, K., Xu, H. E., Auchus, R. J., Antebi, A., and Mangelsdorf, D. J. (2006) Cell 124(6), 1209-1223 9. Prior, A., Jones, J. T., Blok, V. C., Beauchamp, J., McDermott, L., Cooper, A., and Kennedy, M. W. (2001) Biochem J 356(Pt 2), 387-394 10. Kennedy, M. W., Britton, C., Price, N. C., Kelly, S. M., and Cooper, A. (1995) J Biol Chem 270(33), 19277-19281 11. Watts, J. L., and Browse, J. (2002) Proc Natl Acad Sci U S A 99(9), 5854-5859 12. Joo, H. J., Yim, Y. H., Jeong, P. Y., Jin, Y. X., Lee, J. E., Kim, H., Jeong, S. K., Chitwood, D. J., and Paik, Y. K. (2009) Biochem J 422(1), 61-71 13. Janssen, D., and Barrett, J. (1995) Biochem J 311(Pt 1), 49-57 21