The identification of a phospholipase B precursor in human neutrophils Shengyuan Xu, Linshu Zhao, Anders Larsson and Per Venge Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden Keywords granulocytes; inflammation; neutrophils; phospholipase B; phospholipids Correspondence Shengyuan Xu, Department of Medical Sciences Clinical Chemistry, Uppsala University, SE-751 85, Uppsala, Sweden Fax: +46 18 611 3703 Tel: +46 18 611 4204 E-mail: shengyuan.xu@medsci.uu.se (Received 14 August 2008, revised 14 October 2008, accepted 30 October 2008) doi:10.1111/j.1742-4658.2008.06771.x A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns The molecular mass of the protein was estimated to be  130 kDa by gel filtration and 22 and 42 kDa by SDS ⁄ PAGE Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS ⁄ MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids Enzyme activity was associated with fragments derived from the 42 kDa fragment The enzyme revealed a PLB nature by removing fatty acids from both the sn-1 and sn-2 positions of phospholipids The enzyme is active at a broad pH range with an optimum of 7.4 Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation The neutrophil plays an important role in both innate immunity and in inflammatory reactions in human disease [1] The neutrophil eliminates invading microorganisms through phagocytosis, generation of reactive oxygen metabolites and release of microbicidal substances stored in different granules in the neutrophil In addition to secretory vesicles, neutrophils contain azurophil (primary), specific (secondary) and gelatinase-containing granules (tertiary) [2] formed in the bone marrow at subsequent stages of neutrophil maturation [3] During neutrophil-mediated inflammatory reactions, the secretory vesicles are mobilized first upon stimulation, followed by the tertiary, secondary and azurophil granules [4,5] Upon phagocytosis, the azurophil granules fuse with the phagosomes, which causes the release of proteolytic and bactericidal factors into the phagolysosome, where the invading microorganism is killed and digested [1] The identification and characterization of novel granule proteins in human neutrophils is important to understand the functions of human neutrophils In searching for novel granule proteins, we found a protein consisting of 22 and 42 kDa fragments in fractions from a separation of acid extracts of granulocytes by chromatographic procedures The amino acid sequence analysis identified this protein as a product of a gene FLJ22662 (Homo sapiens) which encodes an unknown protein of 63 kDa (cDNA accession no BC063561; protein accession no AAH63561) Comparison of this protein with the GenBank sequence database using the blast program revealed an amino acid sequence similarity with phospholipase B (PLB) expressed in the Abbreviations PLA, phospholipase A; PLB, phospholipase B; PtdCho, phosphatidylcholine; PtdE, phosphatidylethanolamine; PtdIns, phosphatidylinositol FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS 175 A phospholipase B precursor in human neutrophils S Xu et al amoeba Dictyostelium discoideum, suggesting a putative PLB PLBs [6] are a heterogeneous group of enzymes that can remove both the sn-1 and sn-2 fatty acids of glycerophospholipids, and thus display both phospholipase A1 (PLA1) or phospholipase A2 (PLA2) and lysophospholipase activities Several PLBs have been identified in various microorganisms [6,7], fungi [6], D discoideum [8] and in the brush border membrane of mature enterocytes from guinea pig [9], rat [10], rabbit [11] and human epidermis [12] PLBs are also important components of venoms from bees and snakes [13–17] Bacterial and fungal PLBs have been reported to be virulence factors that damage host cells, whereas the PLBs of enterocytes from mammals are involved in the digestion of dietary lipids, and PLB expressed in human epidermis probably plays a role in the differentiation process and is involved in the epidermal barrier function Alhough the human FLJ22662 protein has an amino acid sequence similarity with D discoideum PLB, its PLB activity has not been shown Therefore, in this study we report the purification and characterization of the human FLJ22662 protein from granulocytes, as well as its localization in neutrophils, aimed at elucidating its biological role Results For many years we have been working on the purification and characterization of novel proteins in granulocytes Acid extracts of granules from normal human granulocytes were first fractionized on a Sephadex G-75 column, resulting in several protein peaks Fractions in each peak were pooled and the proteins were further separated on ion-exchange chromatography to search for novel proteins During the course of this we found a 22 ⁄ 42 kDa doublet, which was identified as a product of the gene FLJ22662 and a putative PLB An attempt was made to purify the 22 ⁄ 42 kDa doublet based on deacylation activity However, the deacylation activity in the acid extracts of granules was low and the activity disappeared after the first separation step, i.e gel-filtration chromatography on the Sephadex G-75 column Therefore, the inactive protein was chosen for purification Granule acid extracts were first separated by gelfiltration chromatography As indicated in Fig 1A the 22 ⁄ 42 kDa doublet was eluted in the second peak on a Sephadex G-75 column equilibrated with 0.2 m NaAc pH 4.5 Fractions 58–69 were pooled and applied to a Mono-S cation-exchange column equilibrated with 0.1 m NaAc pH 4.0 Proteins were eluted with a linear 176 gradient of 0–1.0 m NaCl in 0.1 m NaAc pH 4.0 The 22 ⁄ 42 kDa doublet was eluted at a NaCl concentration of  0.35 m in the second peak (in elution volume 19–22 mL), as shown in Fig 1B The 22 ⁄ 42 kDa doublet-containing elution volume 19–22 mL was loaded on the same Mono-S column, but equilibrated with 0.006 m NaCl ⁄ Pi pH 7.4 and proteins were eluted with a linear gradient from 0.006 to 0.5 m NaCl ⁄ Pi pH 7.4 The separation resulted in two peaks and, as shown in Fig 1C, the 22 ⁄ 42 kDa doublet was contained in the fractions of the second peak (elution volume 11–14 mL), whereas most contaminants passed through the column The proteins in elution volume 11–14 mL were further separated on a hydroxyapatite column as shown in Fig 1D with the 22 ⁄ 42 kDa doublet eluted in the last peak (elution volume 19–22 mL) Proteins from steps to of the purification were applied to SDS ⁄ PAGE and visualized by silver staining As shown in Fig 2, the protein from step of the purification showed only two bands at molecular masses of 22 and 42 kDa under nonreducing (lane 6) and reducing conditions (not shown) The molecular mass of the whole 22 ⁄ 42 kDa doublet was estimated to be 21 896 and 41 765 Da on MS, respectively However, these two fragments could not be separated by chromatographic means including Mono-P and reversed-phase chromatography On gel-filtration chromatography the purified native protein was eluted in one peak at a molecular mass of  130 kDa (not shown), and on Mono-P chromatography the protein was eluted in one peak at a pH around 8.6 (not shown) In order to identify the protein, the respective bands at 22 and 42 kDa on SDS ⁄ PAGE were digested by trypsin, followed by MALDI-TOF and MS ⁄ MS analyses The resulting spectrum was used to search for matching proteins in the NCBI database, using the mascot search program The search with the resulting spectrum from the bands at 22 and 42 kDa yielded top scores of 76 and 116, respectively, for the hypothetical protein FLJ22662 (H sapiens) with unknown function (a full-length protein of 63 kDa; protein scores > 67 are significant, P < 0.05; Fig 3A) The amino acid residues identified by MALDI-TOF and MS ⁄ MS are shown in Table The residues from the 22 kDa band were found towards the N-terminus of the full-length protein, whereas the residues from the 42 kDa band were found towards the C-terminus of the protein It appears that the 22 and 42 kDa bands on the SDS ⁄ PAGE are fragments of the full-length hypothetical protein Comparison of the hypothetical protein sequence with the GenBank sequence database by using the blast program revealed a number of similar mouse, rat and bovine proteins with unknown func- FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS S Xu et al A phospholipase B precursor in human neutrophils 10 Pool 60 80 100 120 140 160 Fraction number 10 1.0 0.5 19–22 0 12 18 24 30 Elution volume (mL) 36 1.6 0.50 11–14 0.8 0.25 0.0 10 15 20 Elution volume (mL) 0.00 25 D 0.6 0.4 0.3 0.2 19–22 0.0 10 15 20 Elution volume (mL) 0.0 25 Sodium phosphate (M) 40 B Absorbance at 280 nm Absorbance at 280 nm 58–69 12 Sodium phosphate (M) C 14 Sodium chloride (M) Absorbance at 280 nm Absorbance at 280 nm A Fig Chromatographic purification of the 22 ⁄ 42 kDa doublet (A) Acid extracts of granules obtained from human granulocytes were loaded on Sephadex G-75 column (2.5 · 90 cm) and eluted by 0.2 M NaAc, pH 4.5 as described in Materials and methods The majority of the 22 ⁄ 42 kDa doublet was contained in the second peak (fractions 58–69), as judged by SDS ⁄ PAGE after further separation of proteins in each pool on Mono-S column (not shown) (B) Ion-exchange chromatography was performed as described in Materials and methods The fractions of 58–69 from the gel-filtration chromatography were applied to the Mono-S column and eluted by a linear gradient from to 1.0 M NaCl in 0.1 M NaAc pH 4.0 The 22 ⁄ 42 kDa doublet was eluted in elution volume 19–22 mL in the second peak as indicated in the chromatogram (C) The elution volume 19–22 mL in the second peak from the Mono-S column was applied to the same column but equilibrated with 0.006 M sodium phosphate pH 7.4 and eluted by a linear gradient from 0.006 to 0.5 M sodium phosphate pH 7.4 The 22 ⁄ 42 kDa doublet was eluted in the second peak as indicated in the chromatogram (in elution volume 11–14 mL) (D) The 22 ⁄ 42 kDa doublet containing fractions from the second Mono-S column were applied to a hydroxyapatite column equilibrated with 0.02 M sodium phosphate buffer pH 7.2 and eluted with a linear gradient from 0.02 M NaCl ⁄ Pi pH 7.2 to 0.4 M sodium phosphate pH 6.8 The fractions, as indicated in the chromatogram (in elution volume 19–22 mL), were collected as pure protein tions, and a PLB from D discoideum (protein accession no AAN03644) The amino acid sequence of the hypothetical protein has 32% identity with that of PLB from D discoideum as shown in Fig 3B To determine a possible deacylation activity of the putative PLB, freshly purified protein and materials from different purification steps were incubated with one of several different substrates including didecanoyl-phosphatidylcholine (didecanoyl-PtdCho; Sigma Chemical Co St Louis, MO, USA), dipalmitoylphosphatidylcholine (dipalmitoyl-PtdCho; Sigma), phosphatidylinositol (PtdIns; Sigma), dipalmitoyl-phosphatidylethanolamine (PtdE; Sigma) and 1-palmitoyl-2hydroxylphosphatidylcholine (Lyso-PtdCho; Sigma) No activity was detected except for the activity found in acid extracts of granules (0.085 nmỈmin)1Ỉmg)1) However, the purified protein stored in a 0.3 m sodium phosphate solution at pH 6.8 and °C for some period removed fatty acid from didecanoyl-PtdCho, and the activity increased with storage time, as shown in Fig 4A As shown in Fig 4B, in addition to PtdCho deacylation, the enzyme also showed deacylation activity on PtdIns, PtdE and Lyso-PtdCho To investigate if a change in molecular mass was associated with the appearance of the deacylation activity, the purified protein stored at °C for 16 weeks was analysed by SDS ⁄ PAGE As shown in Fig 2, in addition to the major bands at 22 and 42 kDa, there appeared minor bands at molecular masses of around 20 and 39– 41 kDa, which partly shifted from the major bands, coinciding with the appearance of a significant deacylation activity Any bacterial contamination of our protein preparations that might be responsible for activation of the PLB precursor at prolonged storage was ruled out by the absence of bacterial DNA Possible protease contamination of the protein preparations was ruled out by the absence of protease activity when the commercially available universal protease substrate, casein (resorufin-labelled), was used as the substrate To confirm that the shifted bands were derived from the respective major bands the materials in the shifted bands were digested by trypsin, followed by MALDI-TOF and FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS 177 A phospholipase B precursor in human neutrophils S Xu et al kDa 188 62 49 38 28 18 14 3 Fig SDS ⁄ PAGE of materials from each step of the chromatographic purification procedure From each purification step,  0.5–20 lg of protein was applied to SDS ⁄ PAGE under nonreducing conditions, and analysed by silver staining as described in Materials and methods Marker proteins and their corresponding molecular masss are indicated in lane Lane 2, material from the acid extracts of granules; lane 3, material from fractions 58–69 of the Sephadex G-75 purification step; lane 4, material from the elution volume 19–22 mL of the first Mono-S purification step at pH 4.0; lane 5, material from the elution volume 11–14 mL of the second Mono-S purification step at pH 7.4; lane 6, material from the elution volume 19–22 mL of the hydroxyapatite purification step; lane 7, material from the purified protein stored for 15 weeks at °C MS ⁄ MS analyses As shown in Table 2, the shifted bands were the FLJ22662 gene products The shifted 20 kDa was derived from the 22 kDa fragment and the 39–41 kDa from the 42 kDa fragment Next we separated these fragments using preparative electrophoresis and measured the enzyme activity using didecanoyl-PtdCho as a substrate under the conditions described in Materials and methods As shown in Fig 5, the enzyme activity was detected in the fractions (41, 43, 46 and 48) containing the fragments derived from the 42 kDa fragment but not in other fractions (19, 22, 25, 51 and 53) The enzyme is active at a broad pH range with an optimum of 7.4, when didecanoyl-PtdCho was used as substrate and incubated at 37 °C (not shown) From the Hanes plots a km of 1.1 mm and a Vmax of 21.4 nmỈmin)1Ỉmg)1 were calculated when the protein (stored for 15 weeks) was used It is obvious that the native purified protein needed molecular modifications to acquire its activity To investigate if activating factors are present in granules of neutrophils, the purified protein (0.5 lg, stored for 19 weeks) was pre-incubated with materials (0.5 lg) released from neutrophils that had been incubated with 4b-phorbol 12-myristate 13acetate for 15 at room temperature or 37 °C As shown in Fig 4C the activity was increased slightly, but this slight increase was significant, whereas 0.5 lg of the released material did not by itself show any detectable 178 deacylation activity (not shown) However, proteases such as trypsin, elastase and cathepsin G or Ca2+, Mg2+ and EDTA (a calcium chelator) did not affect the enzyme activity (not shown) Having shown that the enzyme can remove fatty acid from the sn-1 position of Lyso-PtdCho we investigated whether it could also remove fatty acids from the sn-2 position We therefore incubated the enzyme with labeled PtdCho, 1-palmitoyl-2-[1-14C]palmitoylPtdCho (GM Healthcare, Uppsala, Sweden) and as shown in Fig 4D, fatty acid was removed from the sn-2 position Based on these results we conclude that we are dealing with a human PLB and the enzymatic activity of which is Ca2+ independent To reduce the likelihood of contaminating proteins further we included two more purification steps of another batch of the putative PLB, i.e chromatofocusing on Mono-P column and gel filtration on Superdex HR 200 column The newly purified protein did not show any deacylation activity It was stored at )70 °C for a few months before it was taken to °C and tested for protease activity It did not show any protease activity when the universal protease substrate casein–resorufin was used as substrate and incubated for > h at 37 °C However after a few days at °C the protein started to show deacylation activity The enzyme activity increased ± 0.6 and ± 1.2% (mean ± SE, n = 4) when kept at room temperature for and days, respectively, compared with the control (kept at °C) A simultaneous shift in molecular size was also seen (not shown) The results suggest spontaneous activation of the protein and the actual activation mechanism remains to be determined By the time of immunization, the 22 kDa fragment was not identified as part of the hypothetical protein (FLJ22662), therefore, the 22 and 42 kDa fragments were separated by preparative electrophoresis (not shown) and the antigens were injected separately into different chickens The chicken given the 22 kDa fragment did not respond with antibody formation, whereas the chicken given the 42 kDa fragment produced specific antibodies that reacted with the 42 kDa band in an immunoblot (Fig 6) To investigate the origin of the protein in human granulocytes, neutrophil and eosinophil postnuclear supernatants were prepared and the proteins were separated on SDS ⁄ PAGE, followed by immunoblotting using the chicken anti-(42 kDa) IgY As shown in Fig 6, no band was detected in the postnuclear supernatant of eosinophils, but a band at a molecular mass of 42 kDa was detected in the postnuclear supernatant of neutrophils, indicating at least the neutrophil origin of the protein FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS S Xu et al A phospholipase B precursor in human neutrophils A B Fig Sequence of the full-length hypothetical protein (FLJ22662) and comparison with Dictyostelium PLB (A) Sequence of hypothetical protein (FLJ22662) The accession numbers for the full-length cDNA and the protein are BC063561 and AAH63561, respectively (B) Sequence comparison of hypothetical protein and Dictyostelium PLB Alignment of hypothetical protein (AAH63561) and D discoideum (AAN03644) sequences reveals amino acid identity as indicated with * The consensus lipase sequence GxSxG was not found in the sequences of FLJ22662 and Dictyostelium PLB Discussion This study has shown the identification, purification and characterization of a novel protein from acid extracts of granules of neutrophil granulocytes of healthy blood donors The protein was identified as a PLB precursor contained in the secretory organelles of human neutrophils PLBs are enzymes that can remove both the sn-1 and sn-2 fatty acids of glycerophospholipids, and thus display both PLA2 and lysophospholipase activities Several PLBs have been identified in bacteria [7], fungi [6], D discoideum [8], mammalian cells [9–12] and bee and snake venoms [13–17] Genes coding for these PLBs were cloned and three distinct gene families have been identified from bacteria [7], fungi [6] and mammals [11,12,18,19] However, the gene (FLJ22662) is not related to any of these gene families and the coded protein lacks a typical GxSxG motif [20] found in lipases and phospholipases towards the C-terminus Our findings suggest that the PLB precursor is a member of a new gene family of PLB as described for Dictyostelium PLB [21] Whether this protein is involved in arachidonic acid metabolism [22], atherosclerosis [23] and antibacterial defence [24,25], remains to be tested The human FLJ22662 protein, reported in the NCBI protein database is 552 amino acids long with a predicted signal peptide of 29 amino acids It has a FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS 179 A phospholipase B precursor in human neutrophils S Xu et al Table Peptide mass fingerprint of the 22 and 42 kDa fragments The 22 and 42 kDa bands on Coomassie Brilliant Blue-stained gel were excised, minced into small pieces and digested with trypsin The tryptic digest was analysed by MALDI-TOF The resulting spectra were used to search for matching proteins in the NCBI database using the MASCOT search program After the initial peptide scanning, four peptides were subjected to MS ⁄ MS analysis followed by search with the fragmentation spectra in the NCBI data using MASCOT The product of the gene (FLJ22662), reported in the NCBI protein database is 552 amino acids in length with a predicted signal peptide of 29 amino acids Amino acid sequences shown in bold were determined by MS ⁄ MS Calc mass Obs mass Delta residue no Sequence Sequence coverage 12% (22 kDa) 1129.62 1160.62 1144.62 895.42 832.42 1230.57 810.37 1821.91 1130.60 1161.63 1145.62 896.42 833.42 1231.57 811.37 1822.88 )0.02 0.01 )0.01 0.01 )0.00 0.00 )0.00 )0.03 47–57 52–61 75–84 134–140 141–146 151–159 154–159 160–176 MPAEKTVQVK TVQVKNVMDK TTGWGILEIR VQDFMEK QDKWTR EYKTDSFWR TDSFWR HTGYVMAQIDGLYVGAK Sequence coverage 31% (42 kDa) 2685.34 1819.85 1468.80 1921.89 2715.36 1750.85 1622.75 1381.69 1251.48 2972.53 2595.32 2686.40 1820.81 1469.70 1922.87 2716.42 1751.78 1623.66 1382.58 1252.37 2973.61 2596.35 0.05 )0.04 )0.11 )0.03 0.05 )0.07 )0.10 )0.12 )0.11 0.07 0.02 233–255 259–273 313–324 345–360 371–393 394–407 395–407 421–432 466–475 493–520 527–548 theoretical isoelectric point and molecular mass of 9.11 and 63 129 Da, respectively, before removal of the predicted signal peptide and 9.01 and 60 147 Da after removal of the predicted signal peptide However, the first peptide (MPAEKTVQVK, 47–57) detected by MALDI-TOF analyses in this study was not derived from a tryptic digest It is preceded by W, implying that the current proposal for the N-terminal end of FLJ22662 (H sapiens) may be wrong or that the peptide (MPAEKTVQVK, 47–57) was a product of nonspecific cleavage Our findings suggest that the molecular size of the native protein is  130 kDa On SDS ⁄ PAGE the protein fell apart in two fragments of 22 and 42 kDa, respectively, kept together by noncovalent forces These two fragments could also be dissociated by m guanidine hydrochloride treatment The fragmentation of PLB precursor was seen in the purified product and the neutrophil postnuclear supernatant This may indicate that fragmentation of the protein had taken place already in vivo and that only noncovalent bonds keep the fragments together within the cell From our results it became obvious that the enzyme activity was associated with the 42 kDa fragment of the PLB precursor and that it needed molecular modifications to acquire its deacylation activity A 180 VLPGFENILFAHSSWYTYAAMLR HWDFNVIDKDTSSSR QVIPETLLSWQR YNSGTYNNQYMVLDLK GTLYIVEQIPTYVEYSEQTDVLR KGYWPSYNVPFHEK GYWPSYNVPFHEK LGLDYSYDLAPR GDPtdChoNTICCR VADIYLASQYTSYAISGPTVQGGLPVFR TLHQGMPEVYNFDFITMKPILK similar observation was made in guinea-pig intestinal PLB, which is produced as a pro-enzyme and which was activated upon shifting the molecular mass from 170 to 140 kDa by trypsin treatment [18] Materials from the different steps of purification showed no deacylation activity except for the acid extracts of granules The activity seen in the acid extracts of granules was not likely due to PLA2s present in neutrophil primary and secondary granules [26,27], because these are Ca2+-dependent enzymes and Ca2+ was not added to our incubation mixture Moreover calcium chelators such as EDTA did not affect the activity Although we cannot rule out the presence of other Ca2+-independent enzymes in the acid extracts of granules, we believe that there might be factors in the granules that lead to activation of the PLB precursor This was confirmed by the pre-incubation of the purified protein (0.5 lg, stored for 19 weeks) with released materials from neutrophils This preincubation further activated the enzyme However, the proteases trypsin, elastase and cathepsin G had no effects on the enzyme activation Any bacterial contamination of our protein preparations that might be responsible for activation of the PLB precursor at prolonged storage was ruled out by the absence of bacterial DNA Characterization of FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS S Xu et al A phospholipase B precursor in human neutrophils A C 30 20 10 150 * Fatty acid release (% of control) Fatty acid release (nm·min–1·mg–1) 40 15 16 17 Control Radioactivity (%) (nm·min–1·mg–1) Fatty acid release D 10 10 0 Dideca-PC PI Dipalmi-PC 37 oC 50 19 15 RT 100 Time (weeks) B * PE Lyso-PC Control Enzyme Fig Deacylation activity (A) Didecanoyl-PtdCho was incubated with the purified protein at different time of storage and free fatty acid release was measured Enzymatic reactions were carried out with 0.5 lg of the purified protein for 20 h at 37 °C Values are means ± SE from triplicate assays representative of at least three independent experiments around each time point (B) Free fatty acid release from phospholipids, didecanoyl-PtdCho (Dideca-PC), dipalmitoyl-PtdCho (Dipalmi-PC), PtdIns (PI), PtdE (PE) and lysophosphatidylcholine (Lyso-PC) was measured Enzymatic reactions were carried out with 0.5 lg of the purified protein (stored at °C for 15 weeks) for 18–20 h at 37 °C Values are means ± SE from triplicate assays representative of at least three experiments around the time indicated (C) The purified protein (stored at °C for 19 weeks) was preincubated at room temperature or 37 °C for 15 with released materials (0.5 lg) induced from neutrophils by 4b-phorbol 12-myristate 13-acetate before incubation with didecanoyl-PtdCho (The 0.5 lg of the released materials did not show any deacylation activity.) Enzymatic reactions were carried out with 0.5 lg of the protein for 20 h at 37 °C and free fatty acid release was measured Values are means ± SE from triplicate assays of five experiments Asterisks indicate statistical significance (P < 0.05, compared with control) (D) Detection of PLA2 activity Radioactive phospholipid, 1-palmitoyl-2-[1-14C]palmitoyl-PtdCho was incubated without (Control) or with lg of the purified protein (stored at °C 16 weeks) for 20 h at 37 °C and radioactivity was counted as described in Materials and methods Data correspond to percentages of total radioactivity recovered in free fatty acid after subtraction of counts from control (means of duplicate assays) substrate specificity indicates that the activated PLB precursor is not limited to hydrolysing PtdCho, as PtdIns and PtdE also serve as substrates The enzyme is active at a broad pH range with an optimum of 7.4, suggesting an extracellular deacylation role However, the activity of the activated PLB precursor looked not that strong as compared to other known mammal PLBs [9–12] The immunoblotting indicated a neutrophil origin of PLB precursor However, the immunoblotting only showed one band at the molecular mass of 42 kDa, whereas no band of the expected size of the entire gene product of  60 kDa was seen The explanation for this could be that apart from the predicted signal peptide the protein of 60 kDa is cleaved by proteases present in the preparation of the postnuclear supernatant However, we find this unlikely, because a protease inhibitor cocktail was included in the preparation Another explana- tion, as discussed above, could be the fact that the protein already had been processed into fragments of 22 and 42 kDa in vivo The fragments 22 and 42 kDa were seen on SDS ⁄ PAGE under both reducing and nonreducing conditions However, the two fragments were not separated by chromatographic procedures applied in this study including chromatofocusing and reversed phase chromatography (not shown) These findings and the apparent molecular mass of 130 kDa by gel filtration suggest that the protein in fact is an oligomeric protein comprising at least two 22 kDa and two 42 kDa fragments associated noncovalently In our attempts to determine the position of the cleavage site between the 22 and 42 kDa fragments and the N- and C-terminal ends of the shifted fragments it became obvious that the residues 233–255, 493–520 and 527–548 were not detected in the shifted fragments of 39–41 kDa Therefore, it is tempting to FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS 181 A phospholipase B precursor in human neutrophils S Xu et al Table Peptide mass fingerprint of the 20 and 39–41 kDa fragments The amino acid sequences shown in bold were determined by MS ⁄ MS Calc mass Obs mass Delta residue no Sequence Sequence coverage 12% (20 kDa) 1129.62 1160.62 1144.62 895.41 832.42 810.37 1821.91 1130.64 1161.66 1145.65 896.42 833.42 811.37 1822.92 0.013 )0.028 0.020 0.003 )0.003 )0.005 0.005 47–57 52–61 75–84 134–140 141–146 154–159 160–176 MPAEKTVQVK TVQVKNVMDK TTGWGILEIR VQDFMEK QDKWTR TDSFWR HTGYVMAQIDGLYVGAK Sequence coverage 30% (39–41 kDa) 1819.85 1468.80 865.43 2049.98 825.43 2715.36 2843.46 1750.85 1622.75 1579.84 1381.69 764.38 1251.48 1849.78 1820.88 1469.84 866.44 2051.01 826.44 2716.40 2844.51 1751.87 1623.78 1580.87 1382.73 765.38 1252.52 1850.81 0.026 0.032 )0.001 0.019 )0.002 0.028 0.042 0.019 0.026 0.035 )0.012 0.029 0.030 0.030 259–273 313–324 338–344 345–361 364–370 371–393 371–394 394–407 395–407 408–420 421–432 460–465 466–475 476–492 speculate that to gain a deacylation activity the protein should be truncated both from the N- and the C-terminal ends of the 42 kDa fragment Additional possibilities could be post-translational modifications of the protein by, e.g lipids In summary, we have described for the first time the purification and characterization of a human PLB precursor from normal human granulocytes The availability of purified PLB precursor will enable us to further define the functions of this enzyme in vivo and in vitro – – – + + + + – – 42 kDa- HWDFNVIDKDTSSSR QVIPETLLSWQR WADIFSK YNSGTYNNQYMVLDLKK LNHSLDK GTLYIVEQIPTYVEYSEQTDVLR GTLYIVEQIPTYVEYSEQTDVLRK KGYWPSYNVPFHEK GYWPSYNVPFHEK IYNWSGYPLLVQK LGLDYSYDLAPR KDPYSR GDPCNTICCR EDLNSPNPSPGGCYDTK Materials and methods Chemicals All chemicals used were of analytical or the highest grade available, with most being purchased from Merck (Darmstadt, Germany), unless otherwise indicated kDa 188 62 49 38 28 22 kDa- 14 10 Fig SDS ⁄ PAGE of the partly modified and unmodified proteins separated by preparative electrophoresis The partly modified and unmodified proteins were separated by preparative electrophoresis on a 12% polyacrylamide separation gel After elution of bromophenol blue tracking dye, 0.3 mL fractions were collected Each fraction was tested for the deacylation activity and the representative fractions were loaded on SDS ⁄ PAGE Lane 1, proteins before separation; lanes 2–10, fractions, 19, 22, 25, 41, 43, 46, 48, 51 and 53 +, enzyme activity detected; -, enzyme activity not detected 182 Fig Detection of the 42 kDa fragment in neutrophils Postnuclear supernatants (25 lg) were separated on SDS ⁄ PAGE which was immunoblotted using chicken anti-42 kDa IgY Lane 1, molecular mass standard; lane 2, the 42 kDa fragment (0.1 lg) obtained by preparative electrophoresis as described in Materials and methods; lane 3, postnuclear supernatant from neutrophils (25 lg); lane 4, postnuclear supernatant from eosinophils (25 lg) FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS S Xu et al A phospholipase B precursor in human neutrophils Preparation of granule protein Electrophoretic analysis Granules were isolated from buffy coats of normal human blood by a modification of the method described previously [28] The pooled buffy coats,  L, originating from 96 healthy blood donors, were mixed with an equal volume of 2% Dextran T-500 in NaCl ⁄ Pi (Dulbecco, without calcium and magnesium) The granulocyte-rich plasma was collected after sedimentation of the red cells for h at room temperature Granulocytes were washed twice in NaCl ⁄ Pi and once in 0.34 m sucrose by centrifugation at 400 g for 10 The granulocyte pellet was resuspended in vol of 0.34 m sucrose Isolated cells were then disrupted by nitrogen cavitation Cell suspension was mixed with an equal volume of 0.34 m sucrose and the cells were pressurized at °C for 30 under nitrogen at 52 bar with constant stirring in a nitrogen bomb (Parr Instrument Company, Moline, IL, USA) The cavitate was collected into an equal volume of 0.34 m sucrose, 0.3 m NaCl and centrifuged for 20 at 450 g at °C The supernatant was centrifuged for 20 at 10 000 g at °C to sediment the granules After one cycle of freezing and thawing the granules were extracted with vol of 50 mm acetic acid for h at °C An equal volume of 0.4 m sodium acetate pH 4.0 was added and the extraction procedure was continued with magnetic stirring for h at °C The granule extract was then concentrated to approximately mL using YM-2 filter (Amicon Corporation, Lexington, KY, USA) Proteins were analysed with SDS ⁄ PAGE under reducing and nonreducing conditions using precast NuPAGE gel (Novex, Carlsbad, CA, USA), according to manufacturer’s instructions Proteins were visualized by silver staining Chromatographic procedures Gel filtration was performed on a Sephadex G-75 superfine column (2.5 · 90 cm) (Amersham Biosciences, Uppsala, Sweden) equilibrated with 0.2 m NaAc pH 4.5 Ionexchange chromatography was performed using the FPLCsystem (Amersham Biosciences) on a strong cationic exchanger Mono-S prepacked column (Amersham Biosciences) equilibrated with 0.1 m NaAc pH 4.0 The bound proteins were eluted with a linear gradient from to 1.0 m NaCl in 0.1 m NaAc pH 4.0 The proteins eluted to the third peak were applied to the same column equilibrated with 0.006 m phosphate buffer pH 7.4 The bound proteins were eluted with a linear gradient from 0.006 to 0.5 m phosphate buffer pH 7.4 Hydroxyapatite chromatography was performed on a column of hydroxyapatite (Bio-Rad, Laboratories, Hercules, CA, USA) equilibrated with 0.02 m NaCl ⁄ Pi pH 7.2 The bound proteins were eluted with a linear gradient from 0.02 m NaCl ⁄ Pi pH 7.2 to 0.4 m NaCl ⁄ Pi pH 6.8 The chromatographic runs were monitored at 280 nm of absorbance Ultrafiltration of pooled fractions was performed on an YM-10 filter (Millipore Corp., Bedford, MA, USA) Buffer change was performed on PD-10 columns (Amersham Biosciences) Identification and analysis of proteins by MS The 22 and 42 kDa bands from one lane in a Coomassie Brilliant Blue-stained SDS ⁄ PAGE were excised and the protein content was alkylated with jodoacetamide The proteins in the bands were digested with trypsin (Promega modified porcine trypsin; Promega, Madison, WI, USA) and the tryptic peptides were extracted and analysed in a Bruker Ultraflex MALDI TOF ⁄ TOF instrument using alpha-cyano-4-hydroxycinnaminic acid (Sigma) as matrix The instrument was calibrated with a mixture of peptides and each spectrum was internally calibrated using auto digestion products of trypsin m ⁄ z values in the spectra were used for searches in the NCBInr database using the Mascot search engine (http://www.MatrixScience.com) for identification of proteins Selected signals in spectra were used for MS ⁄ MS fragmentation and search for matching peptides using the same database and search engine Mass determination of proteins was done with the same instrument operated in linear mode and externally calibrated with a mixture of proteins Protein determination and bacterial, protease contamination Protein concentration was determined with a Bio-Rad protein assay kit using BSA as a standard according to the manufacturer’s protocol Any bacterial and protease contamination of the purified protein preparations was ruled out by the absence of bacterial DNA and the absence of protease activity The former analyses were performed at the routine Department of Clinical Microbiology, University Hospital, Uppsala, Sweden The latter analyses were performed using a universal protease substrate, casein (resorufin-labeled) (Boehringer, Mannheim, Germany), according to manufacturer’s instructions Enzyme assay The reaction mixture (40 lL final volume) contained 10 mm substrates (unless otherwise indicated), 100 mm NaCl ⁄ Pi containing mm sodium azide (NaN3) and 0.5% Triton X-100, pH 7.4, and 0.5 lg of enzyme or as indicated Since the formation of the product (fatty acid) was linear with time for at least 24 h under the standard conditions, the reaction mixture was incubated for 18–20 h and the reaction was stopped by cooling on ice Free fatty acid FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS 183 A phospholipase B precursor in human neutrophils S Xu et al was determined by means of the NEFA-C kit (WAKO Chemicals, Neuss, Germany) according to the manufacturer’s instructions Positional specificity of the purified enzyme was determined using 1-palmitoyl-2-hydroxyl-PtdCho and 1-palmitoyl-2-[1-14C]palmitoyl-PtdCho (Amersham Biosciences) as substrates The hydrolysing activity of the enzyme at the position of sn-1 acyl ester bonds of glycerophospholipids was determined as described above using 1-palmitoyl-2hydroxyl-PtdCho as substrate The hydrolysing activity of the enzyme at the position of sn-2 was determined by a modification of the methods described previously [29,30] Briefly, carrier dipalmitoyl-PtdCho (final concentration 50 nm) was mixed with radiolabeled PtdCho (1-palmitoyl-2[1-14C]palmitoyl-PtdCho, · 105 cpm) The mixture was dried under nitrogen gas and resuspended in reaction buffer of 0.1 m sodium phosphate, mm NaN3 and 0.5% Triton X-100 at pH 7.4 by sonication to form micelles of phospholipids Incubation was carried out at 37 °C for 20 h, the reaction was stopped by mixing with 0.8 mL Dole’s reagent (32% isopropyl alcohol ⁄ 67% heptane ⁄ 1% m H2SO4, 20 : : v ⁄ v ⁄ v) and vortexed After centrifugation for at 1000 g, the upper phase containing free fatty acids was further purified by extraction with 50 mg silica gel (Bio-Rad) suspended in heptane as described Radiolabeled fatty acids were quantified by scintillation counting Analyses of the pH optimum, Km and Vmax The purified protein (0.5 lg) was added to tubes containing didecanoyl-PtdCho at varying pH (4.0–9.0) The Km and Vmax were calculated from Hanes plots of s ⁄ vi on didecanoyl-PtdCho concentration(s) Preparative electrophoresis Preparative gel electrophoresis was performed in the PrepCell system (Bio-Rad), following the supplier’s instructions The acrylamide concentration of the cylindrical separation gel was 10 or 12%, and the gel was about cm long The stacking gel had an acrylamide concentration of 4% and was 2.5 cm long Antibody production Laying hens were immunized with the purified protein For the immunization 0.5 mL antigens in NaCl ⁄ Pi were emulsified with an equal volume of Freund’s adjuvant The first immunization was performed with Freund’s complete adjuvant and the booster immunization was with Freund’s incomplete adjuvant The amounts of antigen used for each immunization were lg White Leghorn hens were immunized intramuscularly in the breast muscle with the emulsified antigens After the initial immunization, animals 184 received three booster injections at 2-week intervals and eggs were collected continuously after the initial immunization period of weeks Egg-yolk (2 mL) from individual eggs was mixed with mL of 0.9% (w ⁄ v) NaCl, 5.25% (w ⁄ v) PEG 6000, 0.02% (w ⁄ v) NaN3 After incubation overnight at °C, the mixture was centrifuged at 2000 g for 30 The supernatant was precipitated by adding solid PEG 6000 to a final concentration of 12% After centrifugation at 2000 g for 30 min, the precipitate was dissolved in and dialysed against 0.9% NaCl, 0.02% NaN3 The clear supernatant was used for the detection of antibody response All animal experiments were approved by the local animal ethical committee (Uppsala Djurforsokă ă setiska Namnd), Tierps district court, Sweden ¨ Cell separation and postnuclear supernatant preparation Blood cells were separated by density gradient centrifugation over 67% (v ⁄ v) of isotonic Percoll (Amersham Biosciences) The interphase, containing the mononuclear cells and lymphocytes, was removed The pellet fraction, containing erythrocytes and granulocytes, was treated for 15 with ice-cold isotonic NH4Cl solution (155 mm NH4Cl, 10 mm KHCO3, 0.1 mm EDTA, pH 7.4) to lyse the erythrocytes, followed by hypotonic lysis of residual erythrocytes The remaining granulocytes were washed twice in NaCl ⁄ Pi (without Ca2+) To further separate neutrophils from eosinophils, the isolated granulocytes were incubated for h at °C with CD16 mAb-coated magnetic microbeads (at a proportion of · 107 granulocytes in 30 lL NaCl ⁄ Pi with 2% v ⁄ v newborn calf serum to 15 lL microbeads; Miltenyi Biotec, Bergisch Gladbach, Gemany) The cells were subsequently allowed to pass through a steel matrix column in a magnetic field Thereafter, the eosinophils that passed through were collected The purity and viability of the eosinophils were > 96 and 99%, respectively After removing the magnetic field, the neutrophils were eluted with NaCl ⁄ Pi The purity of the neutrophils was > 98% Isolated eosinophils and neutrophils were resuspended respectively in 6% (w ⁄ v) of sucrose solution containing 10 lLỈmL)1 of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) Ultrasonication was performed to disrupt the eosinophils and neutrophils Ultrasonicates were adjusted to 9% (w ⁄ v) of sucrose before centrifugation at 450 g for 20 to eliminate the nuclei and intact cells The postnuclear supernatants (25 lg) were loaded onto SDS ⁄ PAGE gels for immunoblotting To obtain released materials, isolated neutrophils were resuspended in Hanks balanced salt solution at  · 108 cellsỈmL)1 and stimulated with 4b-phorbol 12-myristate 13-acetate (Sigma-Aldrich; · 10)7 m) for 20 at 37 °C After centrifugation the released material was aspirated Under these conditions,  and 60% of primary and secondary granules were released from activated neutrophils, FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS S Xu et al A phospholipase B precursor in human neutrophils judged by the measurement of myeloperoxidase and human neutrophil lipocalin releases [31] Immunoblotting SDS ⁄ PAGE was performed under nonreducing conditions using precast NuPAGE gels (Novex, CA, USA), according to the manufacturer’s instructions For the immunoblotting, the proteins on the NuPAGE gel were transferred to a nitrocellulose membrane (0.2 lm), as described in the manufacturer’s instructions Additional binding sites were blocked by incubation of the nitrocellulose blot in 2% skim milk in 20 mm Tris ⁄ HCl, pH 7.4 for h The blot was incubated overnight with chicken antibodies against the fragment of 42 kDa diluted ⁄ 1000, followed by a h incubation with peroxidase-conjugated rabbit anti-chicken IgY (Immuno-System, Uppsala, Sweden) Color was developed with Immuno-Blot colorimetric assay kits (Bio-Rad) 10 Statistical analysis Mann–Whitney rank sum test was used to test for significant differences between groups All statistical calculations were performed on a personal computer by means of the statistical package statistica for Windows v (Statsoft, Tulsa, OK, USA) 11 Acknowledgements This study was supported by grants from the Swedish ˚ Medical Research Council We are grateful to Dr Ake Engstrom (Department of Medical Biochemistry and ¨ Microbiology, Uppsala University, Sweden) for the protein analysis and to Lena Moberg for skilful technical assistance 12 13 14 References Burg ND & Pillinger MH (2001) The neutrophil: function and regulation in innate and humoral immunity Clin Immunol 99, 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Peterson CGB, Jornvall H & Venge P (1988) Puricaă tion and characterization of eosinophil cationic protein from normal human eosinophils Eur J Haematol 40, 415–423 Sapirstein A, Spech RA, Witzgall R & Bonventre JV (1996) Cytosolic phospholipase A2 (PLA2), but not secretory PLA2, potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells J Biol Chem 271, 21505–21513 Dole VP & Meinertz H (1960) Microdetermination of long-chain fatty acids in plasma and tissues J Biol Chem 235, 2595–2599 Xu S, Zhao L, Larsson A, Smeds E, Kusche-Gullberg M & Venge P (2005) Purification of a 75 kDa protein from the organelle matrix of human neutrophils and identification as N-acetylglucosamine-6-sulphatase Biochem J 387, 841–847 FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University Journal compilation ª 2008 FEBS ... using BSA as a standard according to the manufacturer’s protocol Any bacterial and protease contamination of the purified protein preparations was ruled out by the absence of bacterial DNA and the. .. contamination of our protein preparations that might be responsible for activation of the PLB precursor at prolonged storage was ruled out by the absence of bacterial DNA Characterization of FEBS... that might be responsible for activation of the PLB precursor at prolonged storage was ruled out by the absence of bacterial DNA Possible protease contamination of the protein preparations was