Báo cáo khoa học: The identification of a phospholipase B precursor in human neutrophils doc

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
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:
(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.
FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS 175
amoeba 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
A
1
(PLA
1
) or phospholipase A
2
(PLA
2
) 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 characterization 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 ⁄ P
i
pH 7.4 and proteins were eluted
with a linear gradient from 0.006 to 0.5 m NaCl ⁄ P
i
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 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-
A phospholipase B precursor in human neutrophils S. Xu et al.
176 FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS
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
)1
Æmg
)1
). How-
ever, the purified protein stored in a 0.3 m sodium
phosphate solution at pH 6.8 and 4 °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 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 4 °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 deacyla-
tion activity. Any bacterial contamination 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
40 60 80 100 120 140 160
0
2
4
6

8
10
12
14
Absorbance at 280 nm
Absorbance at 280 nm
Absorbance at 280 nm
Sodium phosphate (
M
)
Sodium chloride (
M
)
Sodium phosphate (
M
)
Absorbance at 280 nm
Pool 2
58–69
Elution volume (
mL)
0 6 12 18 24 30 36
0
2
4
6
8
10
1.0
0

0.5
19–22
Elution volume (mL)
0 5 10 15 20 25
0 5 10 15 20 25
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.
S. Xu et al. A phospholipase B precursor in human neutrophils
FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS 177
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 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 37 °C (not shown). From

the Hanes plots a k
m
of 1.1 mm and a V
max
of 21.4
nmÆmin
)1
Æmg
)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 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 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
deacylation activity (not shown). However, proteases
such as trypsin, elastase and cathepsin G or Ca
2+
,
Mg
2+
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 Ca
2+
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 4 °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 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 staining 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.
A phospholipase B precursor in human neutrophils S. Xu et al.
178 FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS
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 PLA
2
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.
S. Xu et al. A phospholipase B precursor in human neutrophils
FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS 179
theoretical 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 PLA
2
s present in neutrophil
primary and secondary granules [26,27], because these
are Ca
2+
-dependent enzymes and Ca
2+
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 Ca
2+
-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.
Calc. mass Obs. mass Delta residue no. Sequence Sequence coverage 12% (22 kDa)
1129.62 1130.60 )0.02 47–57 MPAEKTVQVK
1160.62 1161.63 0.01 52–61 TVQVKNVMDK
1144.62 1145.62 )0.01 75–84 TTGWGILEIR
895.42 896.42 0.01 134–140 VQDFMEK
832.42 833.42 )0.00 141–146 QDKWTR
1230.57 1231.57 0.00 151–159 EYKTDSFWR
810.37 811.37 )0.00 154–159 TDSFWR
1821.91 1822.88 )0.03 160–176 HTGYVMAQIDGLYVGAK
Sequence coverage 31% (42 kDa)
2685.34 2686.40 0.05 233–255 VLPGFENILFAHSSWYTYAAMLR
1819.85 1820.81 )0.04 259–273 HWDFNVIDKDTSSSR
1468.80 1469.70 )0.11 313–324 QVIPETLLSWQR
1921.89 1922.87 )0.03 345–360 YNSGTYNNQYMVLDLK
2715.36 2716.42 0.05 371–393 GTLYIVEQIPTYVEYSEQTDVLR
1750.85 1751.78 )0.07 394–407 KGYWPSYNVPFHEK
1622.75 1623.66 )0.10 395–407 GYWPSYNVPFHEK
1381.69 1382.58 )0.12 421–432 LGLDYSYDLAPR

1251.48 1252.37 )0.11 466–475 GDPtdChoNTICCR
2972.53 2973.61 0.07 493–520 VADIYLASQYTSYAISGPTVQGGLPVFR
2595.32 2596.35 0.02 527–548 TLHQGMPEVYNFDFITMKPILK
A phospholipase B precursor in human neutrophils S. Xu et al.
180 FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS
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 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
Time (weeks)
Control
Control
Enzyme
RT
37
o
C
Dideca-PC
Dipalmi-PC
PI PE Lyso-PC
*
*
Radioactivity (%)
A
B
D
C

Fatty acid release
(% of control)
Fatty acid release
(nm·min
–1
·mg
–1
)
Fatty ac
i

d release
(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).
S. Xu et al. A phospholipase B precursor in human neutrophils
FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS 181
speculate 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. Additional 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.
Calc. mass Obs. mass Delta residue no Sequence Sequence coverage 12% (20 kDa)
1129.62 1130.64 0.013 47–57 MPAEKTVQVK
1160.62 1161.66 )0.028 52–61 TVQVKNVMDK
1144.62 1145.65 0.020 75–84 TTGWGILEIR
895.41 896.42 0.003 134–140 VQDFMEK
832.42 833.42 )0.003 141–146 QDKWTR
810.37 811.37 )0.005 154–159 TDSFWR
1821.91 1822.92 0.005 160–176 HTGYVMAQIDGLYVGAK
Sequence coverage 30% (39–41 kDa)
1819.85 1820.88 0.026 259–273 HWDFNVIDKDTSSSR
1468.80 1469.84 0.032 313–324 QVIPETLLSWQR
865.43 866.44 )0.001 338–344 WADIFSK
2049.98 2051.01 0.019 345–361 YNSGTYNNQYMVLDLKK
825.43 826.44 )0.002 364–370 LNHSLDK
2715.36 2716.40 0.028 371–393 GTLYIVEQIPTYVEYSEQTDVLR
2843.46 2844.51 0.042 371–394 GTLYIVEQIPTYVEYSEQTDVLRK

1750.85 1751.87 0.019 394–407 KGYWPSYNVPFHEK
1622.75 1623.78 0.026 395–407 GYWPSYNVPFHEK
1579.84 1580.87 0.035 408–420 IYNWSGYPLLVQK
1381.69 1382.73 )0.012 421–432 LGLDYSYDLAPR
764.38 765.38 0.029 460–465 KDPYSR
1251.48 1252.52 0.030 466–475 GDPCNTICCR
1849.78 1850.81 0.030 476–492 EDLNSPNPSPGGCYDTK
4
2 kDa-
2
2 kDa-
1 23 45678910
–– –
––
+
+
++
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 deacylation 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
1234
Fig. 6. Detection of the 42 kDa fragment in neutrophils. Postnucle-
ar 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).
A phospholipase B precursor in human neutrophils S. Xu et al.
182 FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS
Preparation 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 ⁄ P
i
(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 ⁄ P
i
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 4 °C 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 4 °C. The supernatant
was centrifuged for 20 min at 10 000 g at 4 °C 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 4 ° C. 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 4 °C. 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
column (2.5 · 90 cm) (Amersham Biosciences, Uppsala,
Sweden) equilibrated with 0.2 m NaAc pH 4.5. 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 ⁄ P
i
pH 7.2. The bound proteins were eluted with a
linear gradient from 0.02 m NaCl ⁄ P
i
pH 7.2 to 0.4 m
NaCl ⁄ P
i
pH 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
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 () 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 ⁄ P
i
containing 3 mm sodium azide (NaN
3

) 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
S. Xu et al. A phospholipase B precursor in human neutrophils
FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS 183
was 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-
14
C]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-
14
C]palmitoyl-PtdCho, 1 · 10
5
cpm). The mixture was
dried under nitrogen gas and resuspended in reaction buffer

of 0.1 m sodium phosphate, 3 mm NaN
3
and 0.5% Triton
X-100 at pH 7.4 by sonication to form micelles of phospho-
lipids. 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% 1 m H
2
SO
4
,
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, K
m
and V
max
The purified protein (0.5 lg) was added to tubes containing
didecanoyl-PtdCho at varying pH (4.0–9.0). The K
m
and
V
max
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 ⁄ P
i
were 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
eggs was mixed with 4 mL of 0.9% (w ⁄ v) NaCl, 5.25%
(w ⁄ v) PEG 6000, 0.02% (w ⁄ v) NaN
3
. After incubation
overnight at 4 °C, 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% NaN
3

.
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 NH
4
Cl solution (155 mm NH
4
Cl, 10 mm
KHCO
3
, 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 ⁄ P
i
(with-

out Ca
2+
). To further separate neutrophils from eosinophils,
the isolated granulocytes were incubated for 1 h at 4 °C with
CD16 mAb-coated magnetic microbeads (at a proportion of
1 · 10
7
granulocytes in 30 lL NaCl ⁄ P
i
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 ⁄ P
i
. 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 · 10
8
cellsÆmL
)1
and stimulated with 4b-phorbol 12-myristate
13-acetate (Sigma-Aldrich; 4 · 10
)7
m) for 20 min at 37 °C.
After centrifugation the released material was aspirated.
Under these conditions,  6 and 60% of primary and sec-
ondary granules were released from activated neutrophils,
A phospholipase B precursor in human neutrophils S. Xu et al.
184 FEBS Journal 276 (2009) 175–186 ª 2008 Uppsala University. Journal compilation ª 2008 FEBS
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 man-
ufacturer’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 1 h. The blot was
incubated overnight with chicken antibodies against the
fragment of 42 kDa diluted 1 ⁄ 1000, followed by a 2 h incu-
bation with peroxidase-conjugated rabbit anti-chicken IgY
(Immuno-System, Uppsala, Sweden). Color was developed
with Immuno-Blot colorimetric assay kits (Bio-Rad).

Statistical analysis
Mann–Whitney rank sum test was used to test for signifi-
cant differences between groups. All statistical calculations
were performed on a personal computer by means of the
statistical package statistica for Windows v. 8 (Statsoft,
Tulsa, OK, USA).
Acknowledgements
This study was supported by grants from the Swedish
Medical Research Council. We are grateful to Dr A
˚
ke
Engstro
¨
m (Department of Medical Biochemistry and
Microbiology, Uppsala University, Sweden) for the
protein analysis and to Lena Moberg for skilful tech-
nical assistance.
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