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Originally published In Press as doi:10.1074/jbc.M408686200 on August 19, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45540-45545, October 29, 2004
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Cloning and Characterization of the Acid Lipase from Castor Beans*

Peter J. Eastmond{ddagger}

From the Department of Biology, University of York, York YO10 5DD, United Kingdom

Received for publication, July 30, 2004 , and in revised form, August 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A ~60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an {alpha}/{beta}-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima ~4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many eukaryotic organisms store (and transport) chemical energy in the form of triacylglycerol (TAG),1 which is contained in small (0.2–2 µm) oil bodies surrounded by a phospholipid monolayer (1, 2). In plants oil bodies have been found in various tissues (1, 2). They are particularly abundant in many seeds whereupon germination the TAG is broken down, and the carbon skeletons are used to fuel post-germinative growth (1, 2). The initial step in this metabolic process is catalyzed by lipase (EC 3.1.1.3 [EC] ), which hydrolyzes TAG at the oil/water interface to yield free fatty acids and glycerol (3). The free fatty acids are then transferred to the glyoxysome and activated to acyl-CoAs for subsequent catabolism by {beta}-oxidation. Much of the acetyl-CoA produced is ultimately converted to sugars by the glyoxylate cycle and gluconeogenesis. These latter pathways have been relatively well studied in plants, and many of the genes concerned have been cloned and characterized (4). In contrast, remarkably little is known about the process of lipolysis despite its fundamental importance.

Lipase activities have been studied at the biochemical level in a variety of seeds (3). In most, the activities are only detectable upon germination and increase concomitantly with the disappearance of TAG (3). These lipase activities are usually membrane-associated and can be found in the oil body, glyoxysome, or microsomal fractions of seed extracts, depending upon the species (3). Because lipases are interfacial enzymes, those that reside on the surface of the oil body might logically be expected to play a role in TAG breakdown. However, studies using electron microscopy have indicated that oil bodies are in close proximity with other organelles (particularly glyoxysomes), and it has long been hypothesized that an association between them may be required to facilitate fatty acid release and transfer (5, 6). Lipases have been purified to apparent homogeneity from the seeds of several plants such as maize (Zea maize) (7), castor (Ricinus communis) (8, 9), and ironweed (Vernonia galamensis) (10). However, most surprisingly none of the genes that encode these enzymes have been cloned and characterized. Consequently, many questions remain concerning the mechanism and regulation of lipolysis.

Probably the best studied lipase from plants is the castor bean acid lipase, which was first discovered over a century ago (11). The enzyme is unusual in that it is extremely active in mature seeds (prior to germination) and has a low pH optima of ~4.2. Nevertheless, it is associated with the oil body membrane (12), and its catalytic properties are quite well defined (13, 14) and broadly similar to lipases from mammals and fungi. Fuchs et al. (9) were able to partially solubilize the acid lipase from oil body membranes and purified a ~58-kDa protein using chromatographic methods. Subsequently, Altaf et al. (15) raised an antibody against a major oil body membrane protein of similar molecular weight and showed that it could immunoprecipitate acid lipase activity. The polypeptide composition of oil body membranes is relatively simple (1, 2), and here a proteomic approach has been used to identify, clone, and characterize the gene. The castor acid lipase exhibits homology with several fungal lipases, and orthologs are present in other plant species, defining a new family of putative oil body-associated lipases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant and Chemical Sources—Castor beans (R. communis var. gibsonii) were supplied by Chiltern Seeds (Ulverston, Cumbria, UK). The beans were soaked in running water for 1 day and germinated in the dark on moist vermiculite at 30 °C. All reagents were obtained from Sigma, except for glycerol tri[1-14C]oleate that was from Amersham Biosciences. Antibodies raised against the ~60-kDa band (RcOBL1) from castor bean oil body membranes (15) were provided by Dr. Mustak A. Kaderbhai, at the Institute of Biological Sciences, University of Wales, Aberystwyth, UK.

Preparation of Oil Body Membranes—Oil bodies were isolated from the endosperm of soaked castor beans by floatation centrifugation using the method of Hills et al. (16). For proteomic analysis, peripheral proteins were removed from the oil bodies by washing them sequentially with 2 M NaCl2 and 9 M urea according to Millichip et al. (17). The oil body membranes were delipidated by extraction with diethyl ether (16) and solubilized in SDS-loading buffer by heating at 70 °C for 10 min. The polypeptides were separated on a 10–20% (v/v) SDS-PAGE gradient gel from Bio-Rad as described by Laemmli (18), and the gel was stained using 0.1% (w/v) Coomassie Brilliant Blue R-250 in methanol:acetic acid:water (4:1:5, v/v/v).

For proteolytic treatments purified, urea washed, oil bodies were incubated in 20 mM Tris/HCl (pH 8) containing 10 µgml–1 of trypsin for 2 h at 37 °C. The oil body fraction was separated by centrifugation and washed, and the peptides were analyzed by SDS-PAGE using a 10–20% (v/v) gradient gel.

Tryptic Digestion and Peptide Analysis—In-gel tryptic digestion was performed (19), and the peptides were applied to the matrix-assisted laser desorption/ionization (MALDI) target plate. Positive ion MALDI mass spectra were obtained using an Applied Biosystems 4700 Proteomics Analyzer (CTS version, Applied Biosystems) in reflectron mode with an accelerating voltage of 20 kV. MS spectra were acquired with a total of 1000 laser pulses over a mass range of m/z 800–4000. Final mass spectra were the summation of 20 sub-spectra, each acquired with 50 laser pulses and internally calibrated using the tryptic peptides at m/z 842.509 and 2211.104. Monoisotopic masses were obtained from centroids of raw, unsmoothed data.

For collision-induced dissociation (CID)-MS/MS, a source 1 accelerating voltage of 8 kV, collision energy of 1 kV, and a source 2 accelerating voltage of 15 kV were used. Air was used as the collision gas at the "medium" pressure setting of the instrument with a recharge threshold of 9.9 x 10–7 torr, which produced a source 2 pressure of about 1 x 10–6 torr. The precursor mass window was set to ±10 "Da," and the metastable suppressor was enabled. The default calibration was used for MS/MS spectra.

Mass spectral data obtained in batch mode were submitted to data base searching by using a locally running copy of the Mascot program (20) (Matrix Science Ltd., version 1.7). Batch-acquired MS and MS/MS spectral data were submitted to a combined peptide mass fingerprint and MS/MS ion search through the Applied Biosystems GPS Explorer software interface (version 1.0) to Mascot. Search criteria included the following: maximum missed cleavages, 1; variable modifications, oxidation (M); peptide tolerance, 25 ppm; MS/MS tolerance, 0.2 Da. Peptide sequence tags were generated from CID-MS/MS spectra by manual interpretation or by using a de novo sequencing program supplied by Applied Biosystems.

Subcellular Fractionation, Western Blotting, and Immunocytochemistry—The endosperm from soaked beans was homogenized and fractionated according to the method of Hills and Beevers (21). Western blot analysis (22) was carried out on the fractions using anti-RcOBL1 anti-serum (15). Endosperm tissue was also prepared for electron microscopy, and immunocytochemistry was carried out according to the methods of Schmid et al. (23). Rabbit polyclonal anti-RcOBL1 (15) and goat anti-rabbit IgG conjugated to 10 nm gold were used at 1:1000 and 1:20, respectively.

RNA Extraction, cDNA Synthesis, and PCR—Total RNA from various tissues was isolated using the RNeasy kit from Qiagen Ltd. (Crawley, West Sussex, UK). The synthesis of single-stranded cDNA was carried out using SuperScriptTM II RNase H– reverse transcriptase from Invitrogen (Paisley, UK). Degenerate primers (5'-ttgatagtirtyagyttyaga and 5'-ctgtccraatgtrtaiarctt) corresponding to peptide sequence tags (DANLIVISFR and LLNVYTFGQPR) were designed and used to amplify a fragment of the RcOBL1 cDNA by using PCR. The following gene specific primers were used to obtain full-length cDNA sequences by 3'- and 5'-RACE, using the SMARTTM RACE cDNA amplification kit from Clontech: RcOBL1 (5'-gaccacttggtatgggcatatgatgg and 5'-catgtcattgcagtaaaccaccctga), RcOLE1 (5'-ttggcgacttctttgaacttggaagctact and 5'-aaatgccatcatagcaaggccaataacaac), RcOLE2 (5'-cgatggccttttgaaccaagacaaatctat and 5'-gtattgttacaggaaagcagccaccaggag), RcACT (5'-cgttctctccttgtatgccagtggtc and 5'-gagctgctcttggcagtctcaagttc). The same primers were also used to detect gene expression via RT-PCR.

Expression of RcOBL1 and Determination of Lipase Activity—A truncated version of the RcOBL1 cDNA that lacks the hydrophobic N terminus was amplified using primers 5'-gaattcgtgtcgcaccaggcagacgaagtgatttca and 5'-tctagactagtaaccttgtgccatcattttcagag and cloned into the pCR 2.1-TOPO vector from Invitrogen (Paisley, UK). The insert was then excised by using EcoRI and XbaI and cloned into the pMAL-c2E vector from New England Biolabs (Hitchin, Hertfordshire, UK). The cMBP fusion protein was expressed in BL21-Codon Plus-RIL cells from Stratagene (La Jolla, CA). The cells were cultured at 30 °C and induced using 0.4 mM isopropyl-{beta}-D-thiogalactopyranoside (IPTG). The culture was centrifuged at 700 x g for 10 min, and the pellet was resuspended in 200 mM sodium acetate (pH 4.2) plus 2 mM dithiothreitol. The cells were lysed by sonication and the cell debris removed by centrifugation at 21,000 x g for 10 min. Western blot analysis (22) was carried out on the supernatant by using anti-RcOBL1 antiserum (15). Protein content was determined as described by Bradford (24) using bovine serum albumin as a standard.

Assays were performed on the supernatant by using an emulsified substrate essentially according to the method of Fuchs et al. (9). Reactions were carried out at 30 °C in a 100-µl reaction mixture consisting of 200 mM sodium acetate (pH 4.5), 2 mM DTT, and substrate. The substrates were emulsified in 5% (w/v) gum arabic using sonication (9), and 10 µl was added to the assay mixture. Reactions using [14C]triolein as the substrate were stopped by the addition of 1 ml of chloroform: methanol:heptane (1.25:41:1, v/v/v) and 72 µl of 0.2 M NaOH, 150 mM

NaCl2 plus lipid carrier (50 µg of oleic acid, mono-, di-, and trioleoylglycerol). The reactions were vortexed and centrifuged for 5 min at 10,000 x g. 0.4 ml of the upper phase was removed and subjected to liquid scintillation counting. Alternatively to analyze the products, the assays were stopped with 1 ml of chloroform:methanol (2:1, v/v), and the total lipids were extracted according to Folch et al. (25), and the distribution of 14C in acylglycerols and free fatty acids was determined by thin layer chromatography (26).

For assays using various nonradiolabeled substrates, the reactions were conducted in essentially the same manner as described above, but they were stopped with 200 µl of isopropyl alcohol and dried, and the pellet was resuspended in water. Free fatty acid content was measured with the NEFA colorimetric kit (Wako Chemicals, Neuss, Germany) according to the manufacturer's instructions. The liberation of p-nitrophenol from p-nitrophenyl esters was monitored spectophotometrically at 405 nm (9). The release of CoA from oleoyl-CoA was also monitored colorimetrically at 412 nm via detection of the complex formed following the addition of 1 mM 5,5'-dithiobis(2-nitrobenzoate) (26).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Oil Body Membrane Proteins—Castor beans contain a well defined acid lipase activity, which is associated with the oil body membrane (12–15). To identify this enzyme, oil bodies were isolated from the endosperm of castor beans by floatation centrifugation (16) and stripped of peripheral proteins by washing sequentially with 2 M NaCl2 and then 9 M urea (17). The polypeptides that are associated with the oil body membrane were solubilized in SDS loading buffer and separated by SDS-PAGE (Fig. 1). Three major bands of ~60, ~16 and ~14 kDa were visible. The bands were excised and subjected to tryptic digestion, and the resulting peptides were analyzed by MALDI-MS and CID tandem MS. The combined data were used to query the NCBI nonredundant and EST data bases with Mascot software (20). The ~60-kDa band matched most closely to an Arabidopsis thaliana lipase-like protein (At3g14360). The ~16- and ~14-kDa bands matched two castor oleosin ESTs (GenBankTM accession numbers T14916 [GenBank] and T14903 [GenBank] , respectively). Full-length cDNAs were obtained for each oleosin by 3'- and 5'-RACE. The sequences were designated RcOLE1 and RcOLE2 and submitted to GenBankTM. The total ion scores for the ~16-kDa band and the ~14-kDa band, when matched to RcOLE1 and RcOLE2, were 134 and 249, respectively (25 = p < 0.05) (Fig. 1).



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  		FIG. 1.
Analysis of oil body membrane polypeptides from castor bean endosperm. Polypeptides were separated on a 10–20% (v/v) SDS-PAGE gradient gel. Molecular weight (Mr) markers, lane 1; crude extract, lane 2; oil bodies purified by floatation centrifugation, lane 3; NaCl2-washed oil bodies, lane 4; and urea washed oil bodies, lane 5. The ~60, ~16, and ~14-kDa bands were subjected to tryptic digestion, and the peptides were analyzed by MALDI and CID-MS/MS. The corresponding cDNAs were cloned, and the Mascot (20) total ion scores from combined peptide mass fingerprint and MS/MS ion searches are shown in parentheses. Scores greater than 25 are statistically significant (p < 0.05).

 
Cloning of Oil Body Lipase 1 (OBL1)—In order to clone a cDNA encoding the ~60-kDa lipase-like protein, peptide sequences tags were interpreted from the CID-MS/MS spectra. Degenerate primers were designed, based on two peptide sequences (DANLIVISFR and LLNVYTFGQPR), and a cDNA fragment was amplified by RT-PCR. Gene-specific primers were then used to obtain a full-length cDNA sequence by 3'- and 5'-RACE. This sequence was designated Oil Body Lipase 1 (OBL1) and submitted to the GenBankTM data base. The cDNA is 1870 bp and contains a 1578-bp putative open reading frame. The deduced RcOBL1 protein is 525 amino acids (Fig. 2), has a calculated molecular mass of 59.6 kDa and a predicted pI of 6.68. The total ion score for the ~60-kDa band, when matched to RcOBL1, was 326 (25 = p < 0.05), and both peptide sequences tags were present (Fig. 1).



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  		FIG. 2.
The amino acid sequences of RcOBL1, RcOLE1 and RcOLE2. In RcOBL1 the conserved lipase motif (PS00120: (LIV)X(LIVFY)(LIVMST)G(HYWV)SXG(GSTAC)) surrounding the catalytic serine residue is shown in italics, and the putative catalytic triad (SDH) is shown in boldface. In RcOLE1 and RcOLE2, the regions predicted to be hydrophobic (28) are underlined, and the three conserved proline residues that define the "proline knot" motif (36) are also shown in boldface.

 
A similarity search revealed that the C-terminal region of RcOBL1 (amino acid residues ~220 to ~410) shares a low level of identity (31–28%) with the well characterized lipases from the filamentous fungi Rhizomucor miehei, Rhizopus oryzae, and Thermomyces lanuginosa. Lipases from different organisms can vary considerably in their primary amino acid sequences. However, all lipases are "serine esterases" and exhibit a common structural feature called an {alpha}/{beta} fold that consists of a central {beta}-sheet composed of parallel strands that are linked via {alpha}-helices (27). The nucleophilic serine forms part of a catalytic triad (S(DE)H) and is located in a sharp turn (nucleophilic elbow). A conserved signature surrounds the serine residue (PROSITE accession number PS00120: (LIV)X(LIVFY)(LIVMST)G(HYWV)SXG(GSTAC)). The RcOBL1 amino acid sequence (Fig. 2) contains both a putative catalytic triad (Ser-340, Asp-404, and His-497) and all but the first residue of the conserved signature (FVVTGHSLGG).

Association with the Oil Body—Acid lipase activity has been shown previously (15) to be associated exclusively with the oil body fraction from castor bean endosperm homogenates. Antibodies raised against the ~60-kDa band from purified castor bean oil body membranes (15) were used to investigate the subcellular localization of RcOBL1 in endosperm tissue from soaked beans. Fractionation of a homogenate was performed by centrifugation (21). The proteins from the oil pad, the 10,000 x g pellet, the 100,000 x g pellet, and the 100,000 x g supernatant were then separated by SDS-PAGE, and the presence of RcOBL1 was determined by Western blotting. RcOBL1 was only detected in the oil pad (Fig. 3A). To investigate the localization of RcOBL1 in intact tissue, immunogold labeling was performed on EM sections. Gold particles were found to be associated with the surface of oil bodies (Fig. 3B).



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  		FIG. 3.
Association of RcOBL1 with the oil body. A, subcellular fractionation of homogenized endosperm tissue. Protein was separated by SDS-PAGE, Western-blotted, and probed with antibodies against RcOBL1. OP is oil pad; P1 is 10,000 x g pellet; P2 is 100,000 x g pellet; and S2 is 100,000 x g supernatant. B, EM of castor seed endosperm. Sections were labeled with 10 nm gold particles using RcOBL1 antibodies. Bar = 200 nm. OB is oil body, and OBM is oil body membrane. C, a hydropathicity plot of RcOBL1 using the Hopp and Woods scale (28), with a window size of 17 amino acids. D, protease treatment of purified urea-washed oil bodies using trypsin. After repeated washes the peptides retained by the oil bodies were solubilized, separated by SDS-PAGE, and analyzed using MALDI and CID-MS/MS. The ~6-kDa band contains peptide sequence tags from the region marked in C.

 
Oleosins (such as RcOLE1 and RcOLE2; Fig. 2) are anchored to the oil body by a single central hydrophobic domain consisting of ~70 amino acids, which is flanked by more hydrophilic regions (1, 2). Hydropathicity predictions using a variety of standard algorithms suggest that RcOBL1 is relatively amphipathic but does not contain a hydrophobic region as prominent as that found in oleosins. Of the algorithms that were used, the Hopp and Woods scale (28) (with a window size of 17 amino acids) predicted the longest hydrophobic stretch of amino acids in RcOBL1, situated near the N terminus (Fig. 3C).

Although the N terminus of RcOBL1 does not show significant sequence homology to oleosins, the prediction of a relatively hydrophobic region suggests that it might play a role in anchorage. To address this, purified urea washed oil bodies were subjected to proteolytic treatment using trypsin (29). The oil bodies were recovered by centrifugation and washed, and the remaining peptides were separated by SDS-PAGE (Fig. 3D). A major band of ~8-kDa and a minor ~6-kDa band were visible following proteolysis. The bands were further digested and analyzed using MALDI and CID-MS/MS. The ~8-kDa band contained peptide sequences tags from the hydrophobic regions of RcOLE1 and RcOLE2, and the ~6-kDa band contained tags from the predicted N-terminal hydrophobic region of RcOBL1 (Fig. 3D).

Determination of Enzyme Activity—To characterize further RcOBL1, the protein was expressed heterologously in Escherichia coli. Acid lipase activity from purified castor oil body membranes is poorly solubilized in detergents, and the protein has a strong tendency to aggregate (9, 15). Therefore, to maximize the likelihood that the recombinant protein would be soluble, a truncated version (lacking the first 135 amino acids) was produced, fused to maltose-binding protein (cMBP). The ~86-kDa fusion protein was detected by Western blotting, in soluble extracts from E. coli harboring the expression vector, following induction by IPTG (Fig. 4A).



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  		FIG. 4.
Expression of a cMBP-RcOBL1 fusion protein in E. coli and determination of acid lipase activity. A, Western blot of soluble extracts from induced () (+0.4 mM IPTG) and uninduced ({circ}) cells probed using RcOBL1 antibodies. B, a time course of [14C]triolein hydrolysis at pH 4.5. C, the effect of the amount of emulsified substrate on the rate of hydrolysis. D, the effect of pH on the rate of hydrolysis. For assays the values are the mean ± S.E. of measurements from four separate incubations. The standard amount of substrate used was 9 mg ml–1 (~10 mM), except in C where the amount of substrate was varied. The rate of hydrolysis is consistently ~5 nmol mg total protein–1 min–1 at pH 4.5 using 10 mM triolein.

 
To investigate whether this protein had lipase activity, the capacity of extracts to hydrolyze [14C]triolein emulsified with gum arabic was measured (9). No activity was detected in extracts from cells that were expressing MBP alone (data not shown). However, in those that contained the cMBP-RcOBL1 vector, induction by IPTG resulted in a >10-fold increase in acid lipase activity (Fig. 4B). The rate of hydrolysis was proportional to the amount of protein (data not shown) and also to the amount of emulsified substrate, up to ~18 mg ml–1 (equivalent to ~20 mM) (Fig. 4C). When the pH of the assay medium was varied, the optimum was found to be between 4 and 4.5 (Fig. 4D). Essentially no activity was detected above pH 6.

The activity of cMBP-RcOBL1 was also tested against a variety of other substrates (Table I). cMBP-RcOBL1 exhibited the greatest activity on TAGs containing short to medium chain saturated fatty acids, but it was also active on those with long chain saturated and unsaturated fatty acids (Table I). Castor oil, which contains the unusual hydroxylated fatty acid ricinoleic acid, is also hydrolyzed (Table I). Analysis of the products of [14C]triolein hydrolysis after a 10-min incubation revealed that very little 1,3-dioleoylglycerol is produced versus mono-oleoylglycerol and 1,2(2,3)-dioleoyl-sn-glycerol (data not shown). In addition to TAGs, the enzyme is also capable of hydrolyzing artificial p-nitrophenyl fatty acid esters, but it has essentially no activity on oleoyl-CoA or a representative phospholipid (1,2-dioleoyl-3-phosphatidylcholine) (Table I).


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  		TABLE I
Substrate specificity of lipase activity from extracts of E. coli expressing cMBP-RcOBL1 fusion protein

The values are the mean ± S.E. of measurements from four separate incubations. All rates were determined as the difference between the activity from induced and uninduced cultures. Reactions contained 200 mM sodium acetate (pH 4.5), 2 mM dithiothreitol, cell extract and were initiated by the addition of substrate. The final concentration used was equivalent to 10 mM for all substrates with the exception of p-nitrophenyl esters and oleoyl-CoA, which were 100 µM. ND indicates not detected.

 
The effect of a variety of known inhibitors and cofactors of lipases was investigated by using p-nitrophenyl butyrate as a convenient substrate (Table II). The activity was sensitive to preincubation with a 100-fold molar excess of the classical serine esterase inhibitor diethyl-p-nitrophenyl phosphate. The thiol-directed reagent p-chloromercuribenzoic acid and HgCl2 also blocked hydrolysis. The addition of NaCl2 did not stimulate activity. Of the divalent cations tested, Ca2+ enhanced activity slightly. The inclusion of EDTA leads to a small decrease in activity.


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  		TABLE II
The effect of inhibitors and cofactors on lipases activity from extracts of E. coli expressing cMBP-RcOBL1 fusion protein

Assays were performed using 100 µM p-nitrophenyl butyrate as the substrate in a basic reaction mixture consisting of 200 mM sodium acetate (pH 4.5) plus cell extract. Rates were determined as the difference between the activity from induced and uninduced cultures. Effectors were preincubated with cell extract for 30 min before the addition of the substrate. The values are the mean ± S.E. of measurements from four separate incubations. Activity with the addition of no effectors was 22.5 ± 1.9 nmol mg protein-1 min-1. ND indicates not detected.

 
Gene Expression and Protein Levels—RT-PCR was used to show that RcOBL1 transcripts are present in the endosperm of mature seed but that the level declines prior to germination (Fig. 5A), which occurs ~2 days after soaking. The expression of RcOBL1 was not detected in the leaves, stems, or roots of mature plants (Fig. 5A). Western blots of total protein also show that RcOBL1 is only detected in the endosperm, but the protein persists in this tissue for several days following germination, before it is eventually lost (Fig. 5B).



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  		FIG. 5.
Developmental and tissue-specific distribution of RcOBL1. A, RT-PCR analysis of the expression of RcOBL1. Expression levels are relative to RcACT, which was used as a constitutive control. B, Western blot analysis of RcOBL1. Lanes were loaded with 10 µg of total protein. D is dry seed endosperm; S is soaked seed endosperm; DAS is days after soaking; R is root; S is stem; and L is leaf. The data shown are representative of three replicate experiments performed on separate RNA or protein extracts.

 
Identification of RcOBL1 Orthologs—An examination of the Arabidopsis genome sequence revealed that there are more than 20 lipase-like proteins with significant homology to RcOBL1. In the majority of cases the sequence similarity is limited to the lipase catalytic domain near the C terminus (particularly surrounding the nucleophilic elbow region). However, five lipase-like proteins displayed significant similarity over the whole length of RcOBL1, including the relatively hydrophobic N-terminal region (Fig. 6). A search of all available higher plant sequences suggests that there are RcOBL1 orthologs present in a taxonomically diverse set of species including soybean (Glycine max), tomato (Lycopersicon esculentum), potato (Solanum tuberosum), lettuce (Lactuca sativa), peach (Prunus persicarice), rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), and loblolly pine (Pinus taeda).



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  		FIG. 6.
Arabidopsis proteins that have homology to RcOBL1. A phylogenetic analysis was performed using ClustalW (43) and displayed with TreeView version 1.6.6. Sequences were obtained from the TIGR Arabidopsis data base. The existing annotations of At3g14360, At5g42930, and At1g56630 were modified based on alignments with RcOBL1. The percentage identity shared with RcOBL1 is shown in parentheses.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Many germinating oilseeds have been reported to contain lipase activity associated with the oil body membrane (3). It has been suggested that this activity is likely to initiate the breakdown of the TAG, releasing carbon skeletons to drive post-germinative growth (3). The genes that encode these enzymes have yet to be defined. Perhaps the best studied example of an oil body-associated lipase is the acid lipase from castor beans, which has been investigated by many researchers since its discovery over a century ago (9, 11–15). In this report the castor acid lipase has been cloned and characterized. Genes that encode acyl-hydrolases have been identified previously in plants (see for example Refs. 30 and 31). However, this is the first study to reveal the molecular identity of an oil body-associated lipase with relatively well studied TAG hydrolase activity (9, 11–15).

The castor acid lipase (RcOBL1) is homologous to lipases from several filamentous fungi and exhibits the characteristic features of an {alpha}/{beta}-hydrolase. At the level of the primary amino acid sequence, these features include a putative catalytic triad (SDH) with a conserved motif (GXSXG) surrounding the nucleophilic serine residue (27). Overexpression of the catalytic domain in E. coli confirms that the protein can hydrolyze an emulsion of triolein and therefore fulfills the definition of a lipase (32). Furthermore, the activity is sensitive to the classical serine reagent diethyl-p-nitrophenyl phosphate, providing experimental evidence that RcOBL1 is a serine esterase (26). The enzyme is also inhibited by p-chloromercuribenzoic acid and mercuric ions, which react with sulfhydryl groups.

The catalytic properties of the castor bean acid lipase from purified oil body membranes have been defined previously in some detail (9, 11–15). In these preparations RcOBL1 is likely to be the most abundant enzyme (Fig. 1). The most distinctive feature of the acid lipase is its sharp pH optima of ~4.5 and its inactivity above pH 6 (13). The acid lipase is known to be capable of hydrolyzing a variety of natural and artificial esters (9, 11–15). When using triolein emulsified in gum arabic as a substrate, the rate of hydrolysis increases linearly with amount, up to at least ~18 mg ml–1 (9). The acid lipase exhibits apparent typo-selectivity toward TAGs containing short chain and medium chain saturated fatty acids but is also active on a variety of long chain saturated and unsaturated substrates (13). This includes the "unusual" hydroxylated fatty acid ricinoleic acid that is the principal fatty acid in castor oil (13). The enzyme has been reported to have some regio-selectivity toward fatty acyl groups at the sn-1 and sn-3 positions (33). However, in prolonged incubations it is capable of the complete hydrolysis of TAG (13). It has been proposed that acyl migration, promoted by the relatively acidic conditions of the assay, could account for this apparent discrepancy (33). Finally, the acid lipase is not active on phospholipids (13). All the characteristics of the acid lipase activity from purified oil bodies that are listed above are also evident in recombinant cMBP-RcOBL1.

Previous studies (9, 11–15) have reported that acid lipase activity is associated with castor oil body membranes. Here immunological evidence for the localization of the protein has been provided both from subcellular fractionation experiments and from gold labeling of EM sections. Other proteins that have been identified from plant oil bodies include oleosin, caleosin, steroleosin, a putative glycosylphosphatidylinositol-anchored protein, and a putative aquaporin (34). However, the mechanism by which proteins are targeted and anchored to the oil body has only been studied in detail for oleosins.

Oleosins are the major protein constituent of oil body membranes, and their primary function is believed to be preventing coalescence by coating the oil body surface (1, 2). Oleosins are initially targeted to the endoplasmic reticulum by the signal recognition particle-mediated pathway (35), and the central hydrophobic domain, consisting of ~70 amino acids, is required for both endoplasmic reticulum trafficking and subsequent anchorage to the oil body (36). A model has been proposed in which this domain forms a "hair pin" structure that inserts through the phospholipid monolayer into the TAG matrix and consists of two antiparallel {beta}-sheets connected by a proline-rich turn (proline knot) (1). However, some experimental evidence has led to the development of alternative models, and the precise structure of oleosins is currently unresolved (37, 38). The proline knot motif is unnecessary for oleosin endoplasmic reticulum membrane integration, but it is required for oil body targeting (36).

Hydropathicity analysis using standard algorithms indicates that RcOBL1 is a relatively amphipathic protein, like oleosins. Its amphipathic nature can also be demonstrated experimentally using the method of Jolivet et al. (34). RcOBL1 partitions entirely at the interface between the aqueous and organic phases when purified oil body membranes are extracted with 6:3 (v/v) chloroform:methanol (data not shown). It is possible that the N terminus of RcOBL1 might play a role in anchorage. It is predicted to contain a relatively long stretch of hydrophobic amino acids by some hydropathicity algorithms, such as the Hopp and Woods scale (28), and this same region is retained on purified oil bodies following proteolytic treatment. However, the hydrophobicity of the region is not as pronounced as that characterizing oleosins, and it also appears to contain no proline knot motif. Targeting, trafficking, and attachment of the acid lipase to the oil body may differ substantially from that of oleosins and will require further study.

The physiological function of the castor acid lipase is uncertain. No other seed has been reported to contain so much lipase activity (3). This activity clearly reflects the unusually high abundance of the protein (Fig. 1). The acid lipase is effectively inactive at physiological (neutral) pH and is most abundant prior to germination when no TAG breakdown is occurring (39). These data appear inconsistent with a role for the enzyme in TAG mobilization during post-germinative growth (39). It is possible that the acid lipase functions as an emulsifier and/or storage protein like oleosin, but this explanation cannot account for its catalytic activity. Alternatively, the acid lipase might play a role in defense against predation. The potato tuber storage protein patatin exhibits acyl hydrolase activity (40), and this activity has been shown to be insecticidal (41).

Because lipase catalyzes the initial step in TAG breakdown, it is a logical target for the developmental and metabolic regulation of this important process that governs early seedling growth (3, 4). Lipases from animals are known to play a central role in regulating carbon metabolism (42). In most seeds lipase activity exhibits a neutral or alkaline pH optima and is only detectable upon germination, increasing concomitantly with the onset of TAG breakdown (3). In fact, castor beans have been reported to contain a second oil body-associated lipase with precisely these characteristics (16, 39). The neutral lipase is ~40-fold less active than the acid lipase, and therefore probably far less abundant. However, its activity is sufficient to account for the in vivo rate of lipolysis (39). In light of the discovery that many plants possess families of RcOBL1 orthologs, it is possible that some encode the enzymes that are responsible for TAG breakdown following germination. Hence discovering the molecular identity of the castor acid lipase may prove a crucial first step to better understand the mechanism and regulation of lipolysis in germinated seeds.


   FOOTNOTES
 
* This work was supported by the Biotechnology and Biological Sciences Research Council through David Phillips Research Fellowship 87/JF/16985 (to P. J. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY360218 [GenBank] , AY360219 [GenBank] , AY360220 [GenBank] , and AY360221 [GenBank] . Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-01904-328751; Fax: 44-01904-328762; E-mail: pje4{at}york.ac.uk.

1 The abbreviations used are: TAG, triacylglycerol; MALDI, matrix-assisted laser desorption/ionization; CID, collision-induced dissociation; IPTG, isopropyl-{beta}-D-thiogalactopyranoside; MBP, maltose-binding protein; OBL, oil body lipase; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; RT, reverse transcription; MS, mass spectrometry. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jerry Thomas, Michael Hodgkinson, Dr. Peter O'Toole, and Meg Stark, from the Technology Facility at the University of York, Biology Department, for their assistance in performing the proteomic analysis and immunocytochemistry. Dr. Mustak A. Kaderbhai (the Institute of Biological Sciences, University of Wales, Aberystwyth, UK) kindly provided the antibodies.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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