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.. Comparison of this pro
Trang 1Shengyuan Xu, Linshu Zhao, Anders Larsson and Per Venge
Department of Medical Sciences, Clinical Chemistry, Uppsala University, Sweden
The neutrophil plays an important role in both innate
immunity and in inflammatory reactions in human
dis-ease [1] The neutrophil eliminates invading
microor-ganisms through phagocytosis, generation of reactive
oxygen metabolites and release of microbicidal
sub-stances stored in different granules in the neutrophil
In addition to secretory vesicles, neutrophils contain
azurophil (primary), specific (secondary) and
gelati-nase-containing granules (tertiary) [2] formed in the
bone marrow at subsequent stages of neutrophil
matu-ration [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 pro-tein 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 simi-larity with phospholipase B (PLB) expressed in the
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 gran-ulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapa-tite 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 spec-trometry (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 mole-cular mass of 42 kDa, indicating a neutrophil origin of the novel PLB pre-cursor 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
Abbreviations
PLA, phospholipase A; PLB, phospholipase B; PtdCho, phosphatidylcholine; PtdE, phosphatidylethanolamine; PtdIns, phosphatidylinositol.
Trang 2amoeba Dictyostelium discoideum, suggesting a
puta-tive PLB
PLBs [6] are a heterogeneous group of enzymes that
can remove both the sn-1 and sn-2 fatty acids of
glyc-erophospholipids, and thus display both phospholipase
A1 (PLA1) or phospholipase A2 (PLA2) and
lyso-phospholipase activities Several PLBs have been
iden-tified 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],
rab-bit [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
eluci-dating its biological role
Results
For many years we have been working on the
purifica-tion and characterizapurifica-tion of novel proteins in
granulo-cytes Acid extracts of granules from normal human
granulocytes were first fractionized on a
Sepha-dex G-75 column, resulting in several protein peaks
Fractions in each peak were pooled and the proteins
were further separated on ion-exchange
chromatogra-phy to search for novel proteins During the course of
this we found a 22⁄ 42 kDa doublet, which was
identi-fied 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
sep-aration 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
gel-filtration 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
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⁄ PipH 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 1 to 4 of the purifica-tion were applied to SDS⁄ PAGE and visualized by silver staining As shown in Fig 2, the protein from step 4 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, respec-tively However, these two fragments could not be sepa-rated 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 analy-ses 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 1 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 hypotheti-cal protein Comparison of the hypothetihypotheti-cal protein sequence with the GenBank sequence database by using the blast program revealed a number of similar mouse, rat and bovine proteins with unknown
Trang 3func-tions, and a PLB from D discoideum (protein
acces-sion 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),
dipalmitoyl-phosphatidylcholine (dipalmitoyl-PtdCho; Sigma),
phosphatidylinositol (PtdIns; Sigma),
dipalmitoyl-phos-phatidylethanolamine (PtdE; Sigma) and
1-palmitoyl-2-hydroxylphosphatidylcholine (Lyso-PtdCho; Sigma)
No activity was detected except for the activity found in
acid extracts of granules (0.085 nmÆmin)1Æmg)1)
How-ever, the purified protein stored in a 0.3 m sodium
phosphate solution at pH 6.8 and 4C 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 activ-ity 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 pro-tein stored at 4C 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 deacyla-tion activity Any bacterial contaminadeacyla-tion of our protein preparations that might be responsible for acti-vation 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
Fraction number
0 2 4 6 8 10 12 14
Absorbance at 280 nm Absorbance at 280 nm
Pool 2 58–69
Elution volume (mL)
0 2 4 6 8
0
0.5
19–22
Elution volume (mL)
0.0 0.8 1.6
0.00
0.50
0.25
Elution volume (mL)
0.2 0.4
0.0 0.0
0.3 0.6
11–14
19–22
A
B
C
D
Fig 1 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 0 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 frac-tions 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 ⁄ P i pH 7.2 to 0.4 M sodium phosphate pH 6.8 The fractions, as indicated in the chromato-gram (in elution volume 19–22 mL), were collected as pure protein.
Trang 4MS⁄ 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
sepa-rated these fragments using preparative electrophoresis
and measured the enzyme activity using
didecanoyl-Ptd-Cho 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
frag-ment 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 37C (not shown) From
the Hanes plots a km of 1.1 mm and a Vmax of 21.4
nmÆmin)1Æmg)1were 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
pro-tein (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
13-acetate for 15 min at room temperature or 37C 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
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 investi-gated whether it could also remove fatty acids from the sn-2 position We therefore incubated the enzyme with labeled PtdCho, 1-palmitoyl-2-[1-14 C]palmitoyl-PtdCho (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 chromato-focusing 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 4 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 > 2 h at 37 C However after a few days at 4 C the protein started to show deacylation activity The enzyme activity increased 2 ± 0.6 and 9 ± 1.2% (mean ± SE, n = 4) when kept at room temperature for 1 and 3 days, respectively, compared with the con-trol (kept at 4C) 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 pro-duced specific antibodies that reacted with the
42 kDa band in an immunoblot (Fig 6) To investi-gate 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
188
62
49
38
28
18
14
6
3 kDa
1 2 3 4 5 6 7
Fig 2 SDS ⁄ PAGE of materials from each step of the
chroma-tographic purification procedure From each purification step,
0.5–20 lg of protein was applied to SDS ⁄ PAGE under
nonreduc-ing conditions, and analysed by silver stainnonreduc-ing as described in
Materi-als and methods Marker proteins and their corresponding molecular
masss are indicated in lane 1 Lane 2, material from the acid extracts
of granules; lane 3, material from fractions 58–69 of the
Sepha-dex 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 4 C.
Trang 5This 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 pro-tein lacks a typical GxSxG motif [20] found in lipases and phospholipases towards the C-terminus Our find-ings 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 arachi-donic 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
A
B
Fig 3 Sequence of the full-length
hypo-thetical protein (FLJ22662) and comparison
with Dictyostelium PLB (A) Sequence of
hypothetical protein (FLJ22662) The
acces-sion 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.
Trang 6theoretical isoelectric point and molecular mass of 9.11
and 63 129 Da, respectively, before removal of the
pre-dicted 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
nonco-valent forces These two fragments could also be
disso-ciated by 6 m guanidine hydrochloride treatment The
fragmentation of PLB precursor was seen in the
puri-fied product and the neutrophil postnuclear
super-natant 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
frag-ment of the PLB precursor and that it needed
molecu-lar modifications to acquire its deacylation activity A
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 gran-ules 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+ -indepen-dent 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 con-firmed 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 activa-tion Any bacterial contamination of our protein prep-arations that might be responsible for activation of the PLB precursor at prolonged storage was ruled out by the absence of bacterial DNA Characterization of
Table 1 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.
Sequence coverage 31% (42 kDa)
Trang 7substrate 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
cock-tail 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 nonre-ducing 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 filtra-tion 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 frag-ments 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
0
10
20
30
40
0
5
10
15
0
50
100
150
0
2
4
6
8
10
15 16 17 19
Dideca-PC Dipalmi-PC
* *
A
C
F (% of contr
F (nm·min
–1 ·mg
–1 )
Fig 4 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 4 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 4 C for 19 weeks) was preincubated at room temperature or 37 C for 15 min with released materials (0.5 lg) induced from neu-trophils 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 PLA 2 activity Radioactive phospholipid, 1-palmitoyl-2-[1- 14 C]palmitoyl-PtdCho was incubated without (Control)
or with 1 lg of the purified protein (stored at 4 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).
Trang 8speculate that to gain a deacylation activity the protein
should be truncated both from the N- and the
C-termi-nal ends of the 42 kDa fragment AdditioC-termi-nal
possibili-ties 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
pre-cursor from normal human granulocytes The
avail-ability of purified PLB precursor will enable us to
further define the functions of this enzyme in vivo and
in vitro
Materials and methods
Chemicals
All chemicals used were of analytical or the highest grade available, with most being purchased from Merck (Darms-tadt, Germany), unless otherwise indicated
Table 2 Peptide mass fingerprint of the 20 and 39–41 kDa fragments The amino acid sequences shown in bold were determined by
MS ⁄ MS.
Sequence coverage 30% (39–41 kDa)
42 kDa-
22 kDa-
Fig 5 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
bromophe-nol blue tracking dye, 0.3 mL fractions were collected Each
frac-tion was tested for the deacylafrac-tion activity and the representative
fractions were loaded on SDS ⁄ PAGE Lane 1, proteins before
sepa-ration; lanes 2–10, fractions, 19, 22, 25, 41, 43, 46, 48, 51 and 53.
+, enzyme activity detected; -, enzyme activity not detected.
188
62 49 38 28 14 kDa
1 2 3 4
Fig 6 Detection of the 42 kDa fragment in neutrophils
Postnucle-ar supernatants (25 lg) were sepPostnucle-arated 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).
Trang 9Preparation of granule protein
Granules were isolated from buffy coats of normal human
blood by a modification of the method described
previ-ously [28] The pooled buffy coats, 5 L, originating from
96 healthy blood donors, were mixed with an equal
vol-ume 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 1 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 min The granulocyte pellet was resuspended
in 5 vol of 0.34 m sucrose Isolated cells were then
dis-rupted by nitrogen cavitation Cell suspension was mixed
with an equal volume of 0.34 m sucrose and the cells were
pressurized at 4C for 30 min 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 min at 450 g at 4C The supernatant
was centrifuged for 20 min at 10 000 g at 4C to sediment
the granules After one cycle of freezing and thawing the
granules were extracted with 5 vol of 50 mm acetic acid
for 1 h at 4C An equal volume of 0.4 m sodium acetate
pH 4.0 was added and the extraction procedure was
con-tinued with magnetic stirring for 4 h at 4C The granule
extract was then concentrated to approximately 5 mL
using YM-2 filter (Amicon Corporation, Lexington, KY,
USA)
Chromatographic procedures
Gel filtration was performed on a Sephadex G-75 superfine
Ion-exchange chromatography was performed using the
FPLC-system (Amersham Biosciences) on a strong cationic
exchanger Mono-S prepacked column (Amersham
Bio-sciences) equilibrated with 0.1 m NaAc pH 4.0 The bound
proteins were eluted with a linear gradient from 0 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⁄ PipH 6.8
The chromatographic runs were monitored at 280 nm of
absorbance Ultrafiltration of pooled fractions was
per-formed on an YM-10 filter (Millipore Corp., Bedford, MA,
USA) Buffer change was performed on PD-10 columns
(Amersham Biosciences)
Electrophoretic analysis
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
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 pro-teins 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
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 instru-ment 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 pro-tein assay kit using BSA as a standard according to the manufacturer’s protocol Any bacterial and protease con-tamination 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, Univer-sity Hospital, Uppsala, Sweden The latter analyses were performed using a universal protease substrate, casein (res-orufin-labeled) (Boehringer, Mannheim, Germany), accord-ing to manufacturer’s instructions
Enzyme assay
The reaction mixture (40 lL final volume) contained
10 mm substrates (unless otherwise indicated), 100 mm NaCl⁄ Picontaining 3 mm sodium azide (NaN3) and 0.5% Triton X-100, pH 7.4, and 0.5 lg of enzyme or as indi-cated Since the formation of the product (fatty acid) was linear with time for at least 24 h under the standard condi-tions, the reaction mixture was incubated for 18–20 h and the reaction was stopped by cooling on ice Free fatty acid
Trang 10was determined by means of the NEFA-C kit (WAKO
Chemicals, Neuss, Germany) according to the
manufac-turer’s instructions
Positional specificity of the purified enzyme was
deter-mined using 1-palmitoyl-2-hydroxyl-PtdCho and
1-palmi-toyl-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-2-hydroxyl-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, 1· 105cpm) The mixture was
dried under nitrogen gas and resuspended in reaction buffer
of 0.1 m sodium phosphate, 3 mm NaN3 and 0.5% Triton
X-100 at pH 7.4 by sonication to form micelles of
phospho-lipids Incubation was carried out at 37C for 20 h, the
reaction was stopped by mixing with 0.8 mL Dole’s reagent
(32% isopropyl alcohol⁄ 67% heptane ⁄ 1% 1 m H2SO4,
20 : 5 : 1 v⁄ v ⁄ v) and vortexed After centrifugation for
2 min 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, Kmand 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
dideca-noyl-PtdCho concentration(s)
Preparative electrophoresis
Preparative gel electrophoresis was performed in the
Prep-Cell 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 6 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⁄ Piwere
emulsi-fied with an equal volume of Freund’s adjuvant The first
immunization was performed with Freund’s complete
adju-vant and the booster immunization was with Freund’s
incomplete adjuvant The amounts of antigen used for each
immunization were 5 lg White Leghorn hens were
immu-nized intramuscularly in the breast muscle with the
emulsi-fied antigens After the initial immunization, animals
received three booster injections at 2-week intervals and eggs were collected continuously after the initial immuniza-tion period of 6 weeks Egg-yolk (2 mL) from individual
(w⁄ v) PEG 6000, 0.02% (w ⁄ v) NaN3 After incubation overnight at 4C, the mixture was centrifuged at 2000 g for 30 min 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 Djurfo¨rso¨k-setiska Na¨mnd), 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 min 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 (with-out Ca2+) To further separate neutrophils from eosinophils, the isolated granulocytes were incubated for 1 h at 4C with CD16 mAb-coated magnetic microbeads (at a proportion of
1· 107 granulocytes in 30 lL NaCl⁄ Pi with 2% v⁄ v new-born 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 lLÆmL)1 of protease inhibi-tor 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 min to elimi-nate the nuclei and intact cells The postnuclear supernatants (25 lg) were loaded onto SDS⁄ PAGE gels for immunoblot-ting To obtain released materials, isolated neutrophils were resuspended in Hanks balanced salt solution at 1 · 108
cellsÆmL)1 and stimulated with 4b-phorbol 12-myristate 13-acetate (Sigma-Aldrich; 4· 10)7m) for 20 min at 37C After centrifugation the released material was aspirated Under these conditions, 6 and 60% of primary and sec-ondary granules were released from activated neutrophils,