15-lipoxygenases

ARTICLE IN PRESS Prostaglandins, Leukotrienes and Essential Fatty Acids 82 (2010) 121–129

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Cloning, purification and characterization of non-human primate 12/15-lipoxygenases$ M. Johannesson a, L. Backman a, H.-E. Claesson a,b, P.K.A. Forsell a,n a b

Orexo AB, P.O. Box 303, 751 05 Uppsala, Stockholm, Sweden Department of Medical Biophysics and Biochemistry, Karolinska Institutet, Stockholm, Sweden

a r t i c l e in fo

abstract

Article history: Received 19 August 2009 Received in revised form 7 October 2009 Accepted 29 November 2009

The enzyme 15-lipoxygenase-1 (15-LO-1) possesses mainly 15-LO activity and has so far only been described in human cells and rabbit reticulocytes. The animal ortholog, except rabbit reticulocytes, is an enzyme with predominantly a 12-lipoxygenase activity, commonly referred to as 12/15-LO. We describe herein the characterization of the 12/15-LOs in Macaca mulatta (rhesus monkey) and in Pongo pygmaeus (orang-utan). The rhesus and the orang-utan enzymes have mainly 12-lipoxygenase and 15-lipoxygenase activity, respectively, and they display 94% and 98% identity to the human 15-LO-1 protein. The rhesus enzyme was functionally different from the human enzyme with respect to substrate utilization in that anandamide was used differently and that the rhesus enzymes positional specificity could be affected by the substrate concentration. Furthermore, genomic data indicate that chimpanzees express an enzyme with mainly 15-lipoxygenase activity whereas marmosets express an enzyme with mainly 12-LO activity. Taken together, the switch during evolution from a 12-lipoxygenating enzyme in lower primates to a 15-lipoxygenating enzyme in higher primates and man might be of importance for the biological function of this enzyme. & 2009 Elsevier Ltd. All rights reserved.

Keywords: 15-Lipoxygenase 12-Lipoxygenase Phylogeny Arachidonic acid Anandamide Endocannabinoid Inflammation

1. Introduction Lipoxygenases (LOs) are enzymes catalyzing the positional as well as stereo-specific introduction of molecular oxygen into 1,4-pentadiene structures found in unsaturated fatty acids such as arachidonic acid or linoleic acid [1–3]. The positional specificity of the introduction of molecular oxygen in arachidonic acid is commonly used to classify lipoxygenases. Thus, 5-LO introduces oxygen at carbon 5 of arachidonic acid and 12-LO at carbon 12. Lipoxygenases are distributed throughout the animal, plant and fungi kingdom as well as in several prokaryotes [4–6]. The mammalian 15-LO (EC 1.13.11.33) was first purified and characterized 1975 [7]. Later on it was demonstrated that the enzyme showed a fairly restricted expression pattern with the airway epithelium [8], eosinophils [9], reticulocytes [7], macrophages [10], dendritic cells [11], human mast cells [12] and Hodgkin lymphoma [13] as being the most prominent sites of expression. It has also been shown that the expression can be regulated by inflammatory cytokines such as interleukin-4 (IL-4) and IL-13 [8,10,11,13]. The regulation of 15-LO-1 by

$ Grant support: This work was financially supported by Orexo AB, Karolinska Institutet and by the European Commission FP6 Grant LSHM-CT-2004-005033. n Corresponding author. Tel.: + 46 18 7808954; fax.: +46 18 7808999. E-mail addresses: [email protected], [email protected] (P.K.A. Forsell).

0952-3278/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2009.11.006

pro-inflammatory cytokines is in line with the role of 15-LO-1 in the formation of pro-inflammatory mediators such as the eoxins [14]. These potent mediators have been described to be formed in cells involved in airway inflammation such as eosinophils and mast cells [14]. Several studies have demonstrated increased expression and activity of 15-LO-1 in the bronchial mucosa of patients with asthma compared with control subjects [15,16]. Furthermore, mice deficient of 12/15-LO had an attenuated allergic airway inflammation compared to wild-type controls [17,18]. On the other hand, the enzyme can also under certain conditions be involved in the formation of lipoxins which possess anti-inflammatory actions [19]. A second 15-LO was discovered in 1997 and it is hence named 15-LO-2 whereas the first reported 15-lipoxygenase is referred to as 15-LO-1. The 15-LO-1 has so far only been found in humans and rabbit reticulocytes [3,20] and the rabbit 15-LO-1 protein demonstrate 81% identity to the human 15-LO-1. However, rabbit monocytes express an enzyme with mainly 12-LO activity [20,21]. The high degree of sequence conservation between these two rabbit enzymes (more than 99%) indicates that these genes might originate from gene duplication. The lipoxygenase activity of 12/ 15-LO in other species is thought to be an enzyme with primarily 12-activity, earlier named leukocyte type 12-lipoxygenase. The crystal structure of rabbit 15-LO-1 enzyme was solved and it was demonstrated that the enzyme is composed of an

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N-terminal b-barrel, similar to a domain in mammalian lipases [22]. The substrate binding site was suggested to be a hydrophobic pocket in which the fatty acid is docked with the methyl end extending down into the pocket. The carboxyl group of the fatty acid functions to tether and position the fatty acid by binding to Arg402. The hydrophobic pocket is defined at its base by the side chains of Phe353, Met419, Ile418 and Ile593. Mutagenesis studies have demonstrated that variation of the size of the hydrophobic pocket affects the positional specificity of the enzyme [23–25]. Thus, enlargement of the pocket of 15-LO-1 increased the amount of 12-lipoxygenase products. Only a few reports describe the formation of lipoxygenase products in cells or tissues from monkeys. Liminga et al. [26] demonstrated the formation of both 12-HETE and 15-HETE in monkey corneal epithelium and Kulkarni et al. [27] demonstrated the formation of 5-HETE and 12-HETE in both cynomolgus and rhesus monkey . Smith et al. [28] examined the expression of LOs in myometrium, cervix, deciduas and chorion of pregnant baboons. Some reports describe the effects of LO-inhibitors in different monkey species, indicating the existence of a 5-LO [29,30]. None of these reports clarify the nature of the lipoxygenase involved in the formation of 12-hydroxyeicosa5E,8Z,11Z,14Z-tetraenoic acid (12-HETE) or 15-hydroxyeicosa5Z,8Z,11Z,13E-tetraenoic acid (15-HETE). Since the ortholog to human 15-LO-1 in animals, except rabbit reticulocytes, possesses mainly 12-LO activity we raised the question when the enzyme during evolution switched from primarily 12-LO activity to primarily 15-LO activity. Therefore, we set out to identify and characterize the 12/15-LO expressed in two non-human primate species.

2. Materials and methods 2.1. Materials TRIzolsLS reagent was bought from Invitrogen life technologies (Carlsbad, CA, USA). Recombinant human (rh)IL-4 was bought from In Vitro Sweden AB (Sweden). Transcriptor Reverse Transcriptase and Expand High Fidelity PCR System were obtained from Roche Diagnostics Scandinavia AB (Bromma, Sweden). All solvents used were of HPLC grade and bought from Skandinaviska Gentech AB (Stockholm, Sweden). Orang-utan (P. pygmaeus) cDNA clone DKFZp468J1413Q was bought from ¨ Deutsches Ressourcenzentrum fur Genomforschung GmbH (Berlin, Germany), primers were ordered from Cybergene AB (Huddinge, Sweden), BigDye Terminator Ready reaction kit reagents were from Applied Biosystems (Stockholm, Sweden). Monkey post-mortem tissue was kindly provided by the Astrid Fagreus Laboratory at the Karolinska Institutet. Rabbit anti-h15LO-1 antiserum was obtained after immunization of rabbits with purified human 15-LO-1 (Innovagen, Sweden). Synthetic 15-HETE-ethanolamine (15-HETE-EA) was kindly provided by Mats Hamberg, Karolinska Institutet, Sweden.

2.2. Cultivation of epithelial cells and incubation with IL-4 The rhesus lung epithelial cell line 4.MBr-5 was purchased from ATCC (LGC Promochem AB, Sweden). Cells were grown in Ham’s F12 medium with 2 mM L-glutamine supplemented with 30 ng/mL epidermal growth factor, penicillin and streptomycin and 10% fetal bovine serum. Cells were treated with IL-4 (10 ng/ mL) 5 days prior to harvest.

2.3. RNA isolation and cDNA synthesis Total RNA was prepared from either frozen post-mortem lung tissue or treated and untreated 4.MBr-5 cells using TRIzolsLS reagent according to manufacturer’s instructions. Total RNA was dissolved in DEPC-treated water containing 40 U of Protector RNase inhibitor. cDNA was synthesized using Transcriptor Reverse Transcriptase (Roche Diagnostics). Total RNA was combined with 320 pmol oligo(dT)15 in a total volume of 26 mL and heated to 65 1C for 5 min and transferred to ice. cDNA was synthesized using reverse transcription for 30 min at 55 1C in a 40 mL reaction containing 1  Transcriptor RT reaction buffer, 40 U of Protector RNase inhibitor, 1 mM dNTPs and 20 U Transcriptor Reverse Transcriptase. The obtained cDNA was subsequently used for PCR. 2.4. PCR amplification Two pairs of primers were used for separate reactions primed with cDNA from 4.MBr-5 cells treated with and without IL-4. For amplification of 15-LO-1 the following primers that introduced cleavage sites for BglII and XhoI restriction enzymes (Invitrogen) at the 50 and 30 end, respectively, were used; 15-LO-BglII 50 -ACC AGA TCT ATG GGT CTC TAC -30 and 15-LO-XhoI 50 -TAT TCT CGA GTT AGA TGG CCA CAC TGT T-30 . The PCR was programmed as follows: 94 1C for 2 min, 1 cycle; 94 1C for 15 s, 55 1C for 30 s, 72 1C for 2 min, 40 cycles; 72 1C for 7 min, 1 cycle; hold at 4 1C. The orang-utan (P. pygmaeus) cDNA clone DKFZp468J1413Q was PCR amplified using Platinum Pfx DNA Polymerase and the 15-LO1 primers described above using similar PCR conditions as above. PCR products were analysed by electrophoresis and products with the expected size were purified using QIAquick Gel Extraction Kit (Qiagen). 2.5. Degenerative-nested PCR A degenerative-nested PCR using primers encoding highly conserved sequences in mammalian lipoxygenases was performed according to Kawajiri et al. [31] (Fig. 1). Two different forward primers were used, encoding the amino acid sequences LFPCYQW and LFPCYRW, respectively. The reverse primer was combined with both of the forward primers and encoded the amino acid sequence WLLAKTWV. For the second round PCR a third forward primer was used encoding the amino acid sequence FFGYQFL. The first round PCRs were primed with cDNA, made either from total- or mRNA and primers as described above. PCR conditions were essentially as described above. 2.6. Cloning and sequence analysis The obtained PCR products were digested with Bgl II and Xho I and ligated directly into either pFastBacTM1 or pFastBacTMHTb donor vectors digested with BamHI and XhoI. The recombinant plasmids were transformed into DH5a subcloning efficiency cells (Invitrogen), plated on LB-plates containing 100 mg/mL of ampicillin and colonies were grown at 37 1C overnight. Plasmids from positive clones were sequenced using Applied Biosystems BigDye Terminator Ready reaction kit. Analysis of sequence data was performed by similarity comparison to known 12/15-LO cDNA sequences. Plasmids containing the correct insert in pFastBacTM1 or pFastBacTMHTb donor vectors were transformed into MAX Efficiency DH10BacTM Escherichia coli bacteria (Invitrogen). Recombinant bacmid DNA was then isolated using QIAprep Spin Miniprep Kit (Qiagen) and viral particles were obtained according to the manufacturer’s instruction.

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human mouse human mouse mouse human mouse human mouse human mouse

1 94 110 5-LO DYIEFPCYRWITGDVEV 5-LO DYIEFPCYRWITGEGEI 15-LO DEVRFPCYRWVEGNGVL L12-LO SEYTFPCYRWVQGTSIL e12-LO EAF-FPCYSWVQGKETI 12-LO AEVAFPCYRWVQGEDIL 12-LO AEAVFPCYRWVQGEGIL 15-LO-2 GHLLFPCYQWLEGAGTL 8-LO AALHFPCYQWLEGAGEL 12R-LO RIYHFPAYQWMDGYETL 12R-LO RVYHFPAYQWMDGYETL

2 226 244 NHWQEDLMFGYQFLNGCNP NHWQEDLMFGYQFLNGCNP DSWKEDALFGYQFLNGANP NSWKEDAFFGYQFLNGANP GSWKEDALFGYQFLNGANP QCWQDDELFSYQFLNGANP QCWQEDELFGYQFLNGANP EHWQEDAFFASQFLNGLNP AHWQEDAFFASQFLNGINP EHWAEDTFFGYQYLNGVNP EHWTEDSFFGYQYLNGINP

123

3 339 364 FLPSDAKYDWLLAKIWVRSSDFHVHQ FLPTDSKYDWLLAKIWVRSSDFHVHQ FLPTDPPMAWLLAKCWVRSSDFQLHE FTPLDPPMDWLLAKCWVRSSDLQLHE FLPSDPPMAWLLAKIWVRSSDFQLHQ FLPSDPPLAWLLAKSWVRNSDFQLHE FLPSDPPLAWLLAKIWVRNSDFQLQE FLPTDDKWDWLLAKTWVRNAEFSFHE FLPSDDTWDWLLAKTWVRNSEFYIHE FLPSDSEWDWLLAKTWVRYAEFYSHE FLPNDSEWDWLLAKTWVRYAEFYSHE

Fig. 1. Amino acid alignment of different human and mouse lipoxygenases. The highly conserved sequences that were used to design primers for the degenerated nested PCR are shown in bold. Primer 1 was used during the first PCR and primer 2 was used during the second PCR as forward primers. Primer 3 was used as reverse primer in both the first and second PCR.

2.7. Protein expression, purification and HPLC analysis Sf9 cells in a total of 150 mL (2  106 cells/mL) were infected with 15 mL recombinant virus and incubated at 27 1C for 72 h. Cells were then pelleted by centrifugation at 1500 rpm for 5 min, washed twice with ice-cold PBS, resuspended in 20 mL binding buffer (see below) and sonicated 3 times for 5 s. The homogenate was then centrifuged at 4 1C, 23 000 rpm for 40 min. In order to reduce the ion strength in the supernatants they were diluted in one volume of binding buffer before loaded onto the purification column. Rhesus 12/15-LO was purified on a ReSource Q, 6 mL anion exchange column (Amersham). The binding buffer had the following composition: 25 mM Tris–HCl, pH 8, 1 mM TCEP–HCl, 10% glycerol and complete protease inhibitor. Proteins were eluted by step gradient with 1 M NaCl buffer. Fractions containing rhesus 12/15-LO was then concentrated by centrifugation through a Amicon Ultra 15 spin column and further purified on a HiTrap DEAE anion exchange column (GE Healthcare) using the same buffers as described above. Orang-utan 12/15-LO was expressed as a 6xHis tagged protein and purified on a HisTrap HP, 1 mL nickel sepharose column (Amersham). The binding buffer was composed by 500 mM NaCl, 20 mM Tris–HCl, pH 8, 1 mM TCEP–HCl, 10% glycerol, 5 mM imidazole, pH 8 and EDTA free complete protease inhibitor. Proteins were eluted by step gradient with 500 mM imidazole buffer. Fractions containing orang-utan 12/15-LO was then loaded onto HiTrap desalting columns (Amersham) and the buffer was exchanged to 25 mM Tris–HCl, pH 8, 1 mM TCEP–HCl, 10% glycerol and complete protease inhibitor. Active fractions were stored at  80 1C. Following purification an aliquot of the rhesus and orang-utan protein was diluted with ice-cold PBS and incubated with arachidonic acid, linoleic acid or anandamide at a final concentration of 12.5 mM at room temperature for 10 min. Reactions were then stopped by addition of 100 mL MeOH. The amounts of 12- and 15-HETE or 13-hydroxyoctadeca-9Z,11Edienoic acid (13-HODE) and 9-hydroxyoctadeca-9Z,11E-dienoic acid (9-HODE) were analysed by HPLC. Briefly, an aliquot of the incubation mixture was injected onto an Onyx monolithic C18 column (Phenomenex) and metabolites were eluted using a 1 min linear gradient from 50:50 (MeOH:H2O) to 72:28 MeOH:H2O and then an isocratic run for 6 min before washing the column with methanol for 1 min. The flow rate was 2.5 mL/min and 0.007% trifluoroacetic acid was added to all solvents. UV-absorbance was measured at 235 nm. The retention times were compared to authentic standards and qualitative measurements were performed using a 2996 photodiode-array detector (Waters) to verify the spectrum of peaks. 2.8. Western blot analysis Cells were resuspended in ice-cold 50 mM Tris (pH 8.0) buffer containing bacitracin (0.1 mg/mL), benzamid (1 mM), DTT

(2 mM), EDTA (2 mM), EGTA (4 mM), PMSF (1 mM) and leupeptin (0.01 mg/mL). Cells were sonicated 3 times (3 s each) and immediately put on ice. Proteins were mixed with Laemmli sample buffer (Bio-Rad Laboratories) and heated to 95 1C for 5 min. Equal amounts of protein (40 mg) were loaded in each lane and subjected to SDS-PAGE in a 4–12% Tris–Glycin gel (Invitrogen). Proteins were separated by electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Invitrogen). The membrane was blocked in TBS-T buffer (50 mM Tris–HCl, pH 7.5, 0.1 M NaCl, 0.1% Tween 20) supplemented with 5% milk solution and washed once in TBS-T. Subsequently, the membrane was incubated with rabbit anti-h15-LO-1 antiserum diluted 1:3000 in TBS-T with 0.5% milk solution in room temperature for 2 h. After washing three times in TBS-T, the blot was incubated for 60 min with donkey anti-rabbit IgG horseradish peroxidase linked secondary antibody (Amersham) in a dilution of 1:10 000 in TBS-T with 0.5% milk solution. The blot was then washed three times in TBS-T and bands were detected with ECL Plus western blotting detection system (Amersham Biosciences) according to manufacturer’s instructions.

3. Results 3.1. Identification of lipoxygenases in macaca lung tissue Preliminary studies in our laboratory using post-mortem material from the lungs of Macaca fascicularis (cynomolgus monkey) indicated that the tissue possessed lipoxygenase activity and converted linoleic acid to 13-HODE (data not shown). Since lipoxygenase activity also has been described previously in monkeys, our first approach was to unbiased investigate the molecular nature of the lipoxygenases that were expressed in the cynomolgus monkey. This was done by performing semi-nested PCR using degenerated primers located in highly conserved lipoxygenase regions (Fig. 1) essentially as described previously by Kawajiri et al. [31]. In successful PCRs, fragments of 350–400-bp were expected. When forward primer 1 (LFPCYQW) was used in the first round PCR, products of correct sizes where generated (data not shown). After subcloning into pCR4-TOPO, 30 colonies were analysed by PCR. Plasmid DNAs were isolated from 25 out of those clones and after restriction enzyme digestion with EcoRI, 12 clones were sequenced. Three of the clones corresponded to 5-LO and five to 12/15-LO. The remaining clones did not match with any lipoxygenase sequence. The obtained sequence for cynomolgus 5-LO showed more than 96% identity to the human sequences and the cynomolgus 12/15-LO sequence demonstrated more than 98% identity to the human 15-LO-1 sequence (data not shown). Products of correct sizes were also generated when forward primer 2 (LFPCYRW) was used in the first round PCR. After subcloning into the pCR4-TOPO, 36 of these clones were analysed

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by PCR of which plasmids were isolated from 19. Digestion with EcoRI confirmed the presence of correct size of the inserts and all of the clones were sequenced. Thirteen clones corresponded to 5-LO whereas none of the clones were identified for other lipoxygenases. In conclusion, these results show that the transcript for both 5-LO and 12/15-LO could be detected in cynomolgus lung tissue. The methodology used herein is not quantitative so the absolute ratio between 5-LO and 12/15-LO transcript in cynomolgus lung remains to be determined.

cells were analysed by gel electrophoresis, the 12/15-LO protein could be detected in treated but not in untreated cells as judged by Western blot analysis (Fig. 2B), suggesting that the 4.MBr-5 cells indeed can express a 12/15-LO protein after IL-4 stimulation. The rabbit anti-human 15-LO-1 antibody did not demonstrate any crossreactivity towards purified human 12-LO or 5-LO when analysed by Western blot (data not shown).

3.2. 12/15-LO detection in rhesus lung epithelial cells

The 2 kb RT-PCR product obtained from IL-4-treated cells was gel purified, cloned into pFastBacTM1 and sequenced. Alignment with the human 15-LO-1 sequence showed 94% similarity at the level of amino acid sequence. Interestingly, the amino acid residue at positions 352 and 593 were conserved whereas the amino acids at positions 417 and 418 were different as compared to the human sequence (Fig. 3). The amino acids at positions 352, 417, 418 and 593 have previously been demonstrated as being important in determining positional specificity of 12/15lipoxygenases. In order to investigate the positional specificity of the rhesus 12/15-LO, it was expressed in Sf9 cells using a baculovirus based expression system. Following purification by anion exchange chromatography the specific activity and the positional specificity was tested by incubating the enzyme with arachidonic acid as a substrate. The metabolites were separated by RP-HPLC and as expected 12-HETE was formed to a higher degree than 15-HETE (Fig. 4 and Table 1). The deposition of a nucleotide sequence (accession number XM_001094627) containing a putative 12/15-LO from rhesus monkey was made during the progression of this work. This sequence show 100% identity to the sequence reported herein, thus confirming our results.

Due to the fact that the obtained partial sequence did not give any information about the positional specificity for the cynomolgus 12/15-LO, we sought to clone the full-length coding sequence. In order to investigate if 12/15-LO was expressed in the lung epithelial cell line 4.MBr-5 from the rhesus monkey (M. mulatta), RT-PCR was performed on RNA obtained from cells treated with or without IL-4 (10 ng/mL) for 5 days. The 12/15-LO mRNA could be amplified in reactions with cDNA from IL-4-treated cells, as judged by the appearance of an approximately 2 kb fragment, but not from untreated cells (Fig. 2A). When homogenate samples of

bp

3.3. Cloning and characterization of rhesus 12/15-LO

3.4. Cloning and characterization of orang-utan 12/15-LO

Fig. 2. Analysis of 15-LO-1 mRNA and protein in rhesus lung epithelial cells. (A) Cells were treated with or without IL-4 for 5 days and thereafter cDNA were prepared from RNA by reverse transcription. G3PDH mRNA was used for quality and loading control. The addition of IL-4 is indicated by (+ ) whereas the lack of IL4 is indicated by (  ). The left most lane contain molecular weight marker. (B) Cells were treated with or without IL-4 for 5 days. Equal amounts of protein (40 mg) were loaded into each lane and separated by SDS-PAGE. Immunodetection was performed using a rabbit anti-human 15-LO-1 antibody. The left most lane contain purified human 15-LO-1 as a positive control.

In order to identify other lipoxygenases from primates we performed a TBLASTN search of public databases and identified a cDNA clone containing a putative orang-utan (P. pygmaeus) 12/ 15-LO. This clone was obtained and subcloned into pFastBacTMHTb and sequenced to verify the reported sequence. Alignment with the human 15-LO-1 sequence showed 98% similarity at the amino acid level. Interestingly, amino acids in the orang-utan enzyme at positions 352, 417, 418 and 593 were identical to the ones in human 15-LO-1, suggesting a monkey enzyme with predominantly 15-lipoxygenase activity. Orangutan 12/15-LO was expressed in Sf9 cells, purified by immobilized metal ion affinity chromatography and tested for enzyme activity by incubating with arachidonic acid as a substrate. The metabolites were separated by RP-HPLC and the major metabolite was identified as 15-HETE (Fig. 4 and Table 1) thus demonstrating that the orang-utan enzyme indeed was a 15-LO. Table 1 shows a summary of the positional specificity for primate 12/15-LOs as well as for one rodent 12/15-LO used as a reference lipoxygenase.

Human 15-LO-1 Rhesus 12/15-LO

351 DFQLHELQSHLLRGHLMAEVIVVATMRCLPSIHPIFKLIIPHLRYTLEIN DFQLHELQSHLLRGHLMAEVIAVATMRCLPSIHPIFKLIIPHLRYTLEIN

Human 15-LO-1 Rhesus 12/15-LO

401 VRARTGLVSDMGIFDQIMSTGGGGHVQLLKQAGAFLTYSSFCPPDDLADR VRARTGLVSDMGVFDQVVSTGGGGHVELLRRAGGFLTYSSFCPPDDLADR

Human 15-LO-1 Rhesus 12/15-LO

585 QASLQMSITWQLGRRQPVM QASLQMSITWQLGRRQPIM

Fig. 3. Partial sequence alignment of human and rhesus 12/15-LOs. The cloned cDNA from rhesus lung epithelial cells was used to compare the amino acid sequences of rhesus and human 12/15-LOs. The entire sequences are about 94% similar at the level of amino acids. Bolded and underlined amino acids are known to be of importance for positional specificity in 12/15-LO enzymes.

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125

0.0056

0.0070

B 0.0048

5.978

0.0060

0.0050

0.0040

0.0040

0.0032

0.0030

0.0024

12-HETE

0.0020

5.451

A

0.0016

15-HETE 12-HETE

0.0010

6,023

5.409

15-HETE 0.0008

0.000

0.000 4.0

4.5

5.0

5.5

6.0

6.5

Minutes

4.0

4.5

5.0 5.5

6.0

6.5

Minutes

Fig. 4. Activity of rhesus and orang-utan lipoxygenases. HPLC-UV chromatograms of the products formed by purified rhesus 12/15-LO (A) and orang-utan 12/15-LO (B) enzymes incubated with arachidonic acid at a final concentration of 12.5 mM. The major product formed was identified as 12- and 15-HETE, respectively.

Table 1 Specific activity of purified lipoxygenases and their positional specificity. Positional specificity

Specific activity

15-HETE (%) 12-HETE (%) (pmol HETE/lg protein/min) Human 15-LO-1 85 Orang-utan 15-LO 92 Rhesus 12/15-LO 6 Rat 12/15-LO 14

15 8 94 86

36 69 34 59

Both human, rhesus and orang-utan enzymes were approximately 90–95% pure as judged by SDS-PAGE analysis and Commassie staining (data not shown). 3.5. Functional comparison of primate 12/15-lipoxygenases In order to investigate if the substrate specificity was different between 12/15-LO enzyme from man, orang-utan or rhesus, purified enzyme was incubated with 3 different substrates, i.e. linoleic acid, arachidonic acid or anandamide. As depicted in Fig. 5A and B, incubations with 12.5 mM linoleic acid or arachidonic acid did not lead to any large differences in the formation of the monohydroxy acids between the three species. The LC-method that was used did not separate 9-HODE vs

13-HODE, thus we cannot make any assumptions of the ratio of this two metabolites after incubation with linoleic acid. However, incubations with anandamide led to less formation of the monohydroxy metabolites for the rhesus enzyme as compared to the human and orang-utan enzyme when anandamide was used as a substrate (Fig. 5C). This difference was investigated further by dose-response curves using arachidonic acid or anandamide. As depicted in Fig. 6A, arachidonic acid yielded bell-shaped dose-response curves and a Michaelis–Menten equation could not be fitted to the data points. Instead we tried to fit an equation for substrate inhibition to the data and as depicted in Fig 6A, the r2 for the human and the rhesus data were 0.78 and 0.70, respectively, indicating that the data points did not fit very well. Based upon the results in Fig. 6 and despite the fact that a Michaelis–Menten equation not could be fitted to the data, we draw the conclusion that both human and rhesus enzyme had similar apparent maximal velocity of product formation (Vmax(app)) although at different substrate concentrations. The bell-shaped dose-response curves could have several explanations including substrate inhibition, allosteric effects or biophysical properties of the substrate (aggregates, micells, etc.). It is interesting to note that, whatever the reason for the bell-shaped curve is, the rhesus enzyme utilized arachidonic acid significantly more effective at low substrate concentrations (0.78–6.25 mM) and less effective at higher substrate concentrations (25–100 mM) than the human enzyme. At 12.5 mM AA, there was no significant difference in the formation of HETEs between man and rhesus

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250000 200000 150000 100000 50000 0

Area 15- or 12-HETE

Human

M. mulatta

120000 100000

* 40

ns ***

30

* *

**

20 10 0

80000

2.0×10-5 4.0×10-5 6.0×10-5 8.0×10-5 1.0×10-4 AA conc (M)

0

60000 40000 20000 0 Human

Area 15- or 12-HETE-EA

P. pygmaeus

pmol HETE/ min x µg protein

50

P. pygmaeus

M. mulatta

800000 600000 400000 200000

pmol HETE-EA/ min x µg protein

Area 9- and 13-HODE

126

0 Human

P. pygmaeus

enzyme. In contrast to when using AA as a substrate, the human enzyme displayed a significantly higher Vmax(app) than the macaca enzyme when anandamide (AEA) was used as a substrate (Fig. 6B). This difference is most likely not due to differences in the amount of active enzyme since both enzymes showed similar Vmax(app) with arachidonic acid. The bell-shaped-dose response was not so evident for the macaca enzyme when using anandamide as a substrate as compared to arachidonic acid. The human enzyme displayed similar bell-shaped for anandamide as observed for arachidonic acid (Fig. 6B) and both a Michaelis– Menten equation and an equation for substrate inhibition were fitted to the data for comparison. The difference in utilization of anandamide between these two primate enzymes is probably a reflection of different affinity for this particular substrate. As judged by the results in Fig. 5B and C, the orang-utan enzyme seems to behave as the human enzyme with respect to substrate specificity. Further analyses of substrate utilization of these enzymes are ongoing. During the characterization of human and macaca 12/15-LO, we observed that the macaca enzyme displayed an interesting shift in the positional specificity which was clearly evident when arachidonic acid was used as a substrate but is was also observed with anandamide (Table 2). At lower substrate concentrations, the rhesus enzyme was predominantly a 12-lipoxygenating enzyme whereas at higher concentrations of substrate the enzyme catalyzed the formation of both 12- and 15-HETE. Furthermore, one additional mono-hydroxylated product was observed at anandamide concentrations above 12.5 mM when using the rhesus enzyme (Fig. 7). The identity of this product eluting at

*** ***

*** 150

**

*

100 **

50

0 0

M. mulatta

Fig. 5. Comparison of 12/15-lipoxygenase activity. Purified enzyme was incubated with 12.5 mM of the indicated substrate and the enzyme products were quantified by RP-HPLC. (A) Linoleic acid. (B) Arachidonic acid (15- or 12-HETE is indicated by black or grey bars, respectively). (C) Anandamide (15- or 12-HETE-EA is indicated by black or grey bars, respectively). The results are expressed as the mean 7 S.D. of at least three separate experiments each performed in duplicates.

200

2.0×10-5 4.0×10-5 6.0×10-5 8.0×10-5 1.0×10-4 AEA conc (M)

Fig. 6. Dose-response curves of arachidonic acid or anandamide using human or rhesus enzyme. Purified enzyme was incubated with increasing concentrations of the indicated substrate and the enzyme products were quantified by RP-HPLC. (A) arachidonic acid (data points are sums of the formation of 15- and 12-HETE). A substrate-inhibition equation (Y =Vmax/(1+ (Km/X) + (X/Ki))) was fitted to each set of data as indicated by the solid line. (B) Anandamide, (data points are a sums of the formation of 15- and 12-HETE-EA). A Michaelis–Menten equation was fitted to each set of data as indicated by the dashed lines. A substrate-inhibition equation was also fitted to the data points as indicated by solid lines. The results are expressed as the mean 7 S.D. of at least three separate experiments each performed in duplicates. Data using human enzyme are indicated by open squares and data using rhesus enzyme are indicated by solid circles. Statistical differences are defined by; nnn, p o 0.001; nn, p o 0.01; n, p o0.05.

Table 2 Positional specificity at different substrate concentrations. Conc (lM)

0.78 1.56 3.12 6.25 12.5 25 50 100

Human. AA

Rhesus. AA

Human. AEA

Rhesus. AEA

Ratio. 15- vs 12-HETE

Ratio. 12- vs 15-HETE

Ratio. 15- vs 12- Mean ratio. 12- vs HETE-EA 15-HETE-EA

na na 5.6 7.3 8.9 8.2 7.2 8.0

na na na 82 16 7.9 3.7 2.9

5.4 9.1 11 17 15 13 12 11

14 16 8.1 5.1 4.3 3.9 2.8 3.2

The ratios for human enzyme is the amount of 15-HETE vs 12-HETE whereas the ratios for the rhesus enzyme is the amount of 12-HETE vs 15-HETE. The results are expressed as the mean ratio from at least three separate experiments each performed in duplicates. Na, not applicable (amount of metabolites below detection limit).

4.7 min is currently unknown but the UV-spectrum indicates the presence of a conjugated diene since the absorbance maximum was 235 nm. Taken together, these results suggest that the

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3.596 min 245

3.915 min 245

280 nm

3.657 min 245

280 nm

237.6

280 nm

236.2

2.8

3.3

3.6

4.657

3.596

3.915

235.2

127

4.0

4.4

4.8

5.2

5.6

6.0

Time (min) Fig. 7. Chromatographic profile of anandamide metabolites. Anandamide metabolites were separated on an Onyx C18 monolithic column. The upper panel shows the UVspectrum of three peaks with the retention time 3.6, 3.9 and 4.7 min. Lower panel shows the chromatographic profile observed at 235 nm. The peaks at 3.6 and 3.9 min correspond to 15- and 12-HETE-EA, respectively.

12/15-LO enzyme from rhesus cannot utilize anandamide to the same extent as the human enzyme and that the concentration of the substrate can affect the positional specificity of the rhesus enzyme.

3.6. Phylogenetic relationship of various lipoxygenases The primary sequence for different lipoxygenases was used to investigate the phylogenetic relationship as well as conserved residues at position 352, 417, 418 and 593 for the known 12/15lipoxygenases (Fig. 8). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.1 [32]. Two predicted lipoxygenases were included in the alignment. First, a chimpanzee amino acid sequence was predicted from an annotated genomic sequence (XM_001160792) and it displays 99% identity to the human 15-LO protein. Second, by a TBLASN search we identified several genomic sequences from Callithrix jacchus (marmoset monkey) containing the 14 exons of a putative 12/15-LO. A predicted protein sequence of the marmoset enzyme was obtained by translating the exons in the three different reading frames and then forming a continuous sequence of the appropriate translated sequences. Although no activity for the chimpanzee or marmoset 12/15-LO has been determined, it is likely that the marmoset enzyme possesses primarily 12-LO activity whereas the chimpanzee enzyme posses 15-LO activity based upon the amino acid residues at position 353, 417, 418 and 593 (data not shown). An un-rooted tree demonstrating the phylogenetic relationship between the different lipoxygenase enzymes is presented in Fig. 8. The ultrametric tree was constructed using un-weighted pair group method with

arithmetic means (UPMGA) and the node between human and chimpanzee was used to calibrate the molecular clock using 5 million years as a divergence time for human and chimpanzees. The tree clearly shows that some 12/15-lipoxygenases, as exemplified by the porcine and bovine L12-LO are phylogenetically more related to the primate enzymes than the rodent forms of the enzyme. The rabbit 12/15-LOs were found to constitute an intermediate single branch in between the rodents and the porcine/bovine 12/15-LOs.

4. Discussion We have in this study used semi-nested degenerative PCR to identify the lipoxygenases expressed in the lung tissue from M. fascicularis and report on the identification of 5- and 12/15-LO transcripts from this tissue. Although we did not find transcripts encoding other lipoxygenases, we cannot rule out the possibility that such still are present, albeit as very low-copy number transcripts. To define the ratio between different transcripts a more quantitative method such as quantitative-PCR should be used. Nevertheless, our results indicate that the 5- and 12/15-LOs were the most abundantly expressed lipoxygenases in the investigated sample. The partial sequence obtained for 12/15-LO did not provide insight into the positional specificity of the lipoxygenase. Thus, we cloned the full-length coding sequence of the 12/15-LO enzyme from rhesus monkey. Some amino acid residues are known to play a key role in the positional specificity in 12/15-LO enzymes. In the human enzyme these are Phe 352, Ile 417, Met 418 and Ile592. All the known primate 12/15-lipoxygenases has a conserved phenylalanine at position

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H. sapiens 15-LO

0.00533 0.02138 0.01577

P. troglodytes 15-LO

0.00684

P. pygmaeus 15-LO

0.02822

0.02742

M. mulatta 12/15-LO

0.04399

0.01470

C. jacchus 12/15-LO

0.05927 0.02553

B. taurus 12/15-LO

0.05927

0.01214

S. scrofa 12/15-LO

0.08611

0.04736

C. familiaris 12/15-LO 0.00227

O. cuniculus 15-LO

0.10937

0.00227

O. cuniculus 12/15-LO

0.03036

100000000 0

R. norvegicus 12/15-LO

0.03036

0.12863

50000000 0

M. musculus 12/15-LO 0

0

years

0

Fig. 8. Phylogenetic relationship of 12/15-LOs. The amino acid sequence for different 12/15-lipoxygenases was used to investigate the phylogenetic relationship by the use of MEGA 3.1. The numbers on each branch indicate the distance from the nearest node. The scale at the bottom is calibrated using the node between human and chimpanzee with 5 million years as approximate time for divergence of the two species.

352 and an isoleucine at 592. Most likely, the amino acid residues at position 417 and 418 will thus govern the positional specificity of the primate 12/15-lipoxygenases. This is exemplified by the activity of the rhesus and the orang-utan enzymes characterized in this paper. Interestingly, the primate 12/15-LOs show a high degree of identity with the human enzyme. Despite of this, they can easily be distinguished as enzymes with mainly 12- or 15-lipoxygenase activity. In the rat and mouse 12/15-LO, the amino acid residue corresponding to the human residue 417 are Ala and Val, respectively, and the activities of these enzymes are predominately 12-lipoxygenase activities. Both Ala and Val are smaller amino acids than Ile found in the human enzyme which is thought to facilitate the entrance of arachidonic acid into the active site, leading to hydrogen abstraction at C-10 and consequently oxygenation at C-12. With this in mind and having both the rhesus and the orang-utan sequence, we assumed that the rhesus 12/15-LO enzyme had mostly 12-LO activity whereas the orang-utan enzyme should display mostly 15-LO activity. This assumption was confirmed and thus further lending support to the theory by Kuhn et al. [24] suggesting that the amino acid at position 352 is a primary sequence determinant. If this residue is rather bulky and space filling, positions 417 and 418 will be important for the positional specificity. Furthermore, our results suggest that the concentration of the substrate can affect the ratio between 12- and 15-HETE formed by the rhesus enzyme. This is most likely due to the fact that the fatty acid is presented to the enzyme in different biophysical forms, i.e. as monomers at low concentrations, as polymers or microaggregates at intermediate concentrations or as larger secondary structures such as aggregates, micelles or lipid bilayers at higher concentrations. Indeed, the length of the fatty acid chain and the degree, type and position of unsaturation has been shown to affect the pKa of lipids by the formation of secondary structures due to van der Waals forces and interactions between the polar carboxylate groups [33,34]. Presumably, when the substrate is in a more rigid physical state than monomers, it may not align deep enough in the active site to allow for 12-lipoxygenation. Thus, caution should be taken when interpreting positional specificity for 12/15-lipoxygenases since it can be affected by the substrate concentration. The phylogenetic relationship demonstrates that both bovine and porcine 12/15-LO is more related to the human enzyme than

the rabbit enzyme, despite the fact that the rabbit reticulocyte enzyme is a 15-lipoxygenase. The rabbit is one single species that expresses a 12/15 LO with 15-LO activity while in its most related species the corresponding enzyme has 12-LO activity. In fact, not even all primates express an enzyme with 15-LO activity. This seems to be restricted to the hominids which are the members of the biological family Hominidae including humans, chimpanzees, gorillas and orang-utans. If the restricted expression of a 15-LO-1 enzyme in hominids can be coupled to formation of eoxins or certain airway inflammatory diseases, such as asthma, in these species remains to be investigated. The expression of the second form of 15-LO, i.e. 15-LO-2, in rhesus and orang-utan has in contrast to human cells [35,36], so far not been described. The relevance for the difference between man and macaca in using anandamide as a substrate is presently unclear but it might indicate differences in metabolism of endocannabinoids in these two species. In conclusion, we identify in this report the molecular nature of the 12- and 15-lipoxygenases in M. mulatta and P. pygmaeus and also that the expression of the rhesus enzyme in lung epithelial cells is regulated by interleukin-4. The switch from a mainly 12-LO enzyme to a mainly 15-LO enzyme during evolution indicates that the 15-lipoxygenating activity of the enzyme in higher primates is of importance for the function of this enzyme. Also, our results show that 12/15-LO from primates can have distinct differences in using endocannabinoids as substrate.

Acknowledgement The report reflects only the author’s views and the European Commission is not liable for any use that may be made of the information herein. References [1] O. Radmark, Arachidonate 5-lipoxygenase, Prostaglandins Other Lipid Mediators 68–69 (2002) 211–234. [2] T. Yoshimoto, Y. Takahashi, Arachidonate 12-lipoxygenases, Prostaglandins Other Lipid Mediators 68–69 (2002) 245–262. [3] H. Kuhn, M. Walther, R. Kuban, Mammalian arachidonate 15-lipoxygenases structure, function, and biological implications, Prostaglandins Other Lipid Mediators 68–69 (2002) 263–290.

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