Báo cáo Y học: Myristyl and palmityl acylation of pI 5.1 carboxylesterase from porcine intestine and liver Tissue and subcellular distribution potx

Myristyl and palmityl acylation of pI 5.1 carboxylesterase
from porcine intestine and liver
Tissue and subcellular distribution
Sylvie Smialowski-Fle
Â
ter, Andre
Â
Moulin, Josette Perrier and Antoine Puigserver
Institut Me
Â
diterrane
Â
en de Recherche en Nutrition, UMR-INRA, Faculte
Â
des Sciences et Techniques de St-Je
Â
ro
Ã
me, Marseille, France
Immunoblotting analyses revealed the presence o f carb-
oxylesterase in the porcine small intestine, liver, submaxillary
and parotid glands, kidney cortex, lungs and cerebral cortex.
In the intestinal mucosa, the pI 5.1 enzyme was d etected in
several subcellular fractions including the microvillar frac-
tion. Both fatty monoacylated and diacylated monomeric
(F1), trimeric (F3) and tetrameric (F4) forms of the intestinal
protein were puri®ed here for t he ®rst time by performing
hydrophobic chromatography and gel ®ltration. The
molecular mass of t hese three e nzymatic forms w as estimate d
to be 60, 180 and 240 kDa, respectively, based on size-
exclusion chromatography and SDS/PAGE analysis. The

existence of a covalent attachment linking palmitate and
myristate to porcine intestinal carboxylesterase (PICE),
which was suggested by the results of gas-liquid chroma-
tography (GLC) experiments in which the fatty acids
resulting from alkali treatment of the protein forms were
isolated, was con®rmed here by the fact that [
3
H]palmitic
and [
3
H]myristic acids were incorporated into porcine
enterocytes and hepatocytes in cell primary cultures. Besides
these two main fatty a cids, the p resence of oleic, ste aric, and
arachidonic acids was also detected by GLC and further
con®rmed by performing radioactivity counts on the
3
H-
labelled PICE forms after an immunoprecipitation proce-
dure using speci®c polyclonal antibodies, followed by a SDS/
PAGE separation step. Unlike the F1 and F4 forms, w hich
were both myristoylated and palmitoylated, the F3 form was
only palmitoylated. The monomeric, trimeric and tetrameric
forms o f PICE w ere all able to hydrolyse short chain f atty
acids containing glycerides, as well as phorbol esters. The
broad speci®city of fatty acylated carboxylesterase is dis-
cussed in terms of its possible involvement in the metabolism
of ester-containing xenobiotics and signal transduction.
Keywords: carboxylesterase; fatty acylation; gas-liquid
chromatography; porcine enterocytes; porcine hepatocytes.
Carboxylesterases (EC 3.1.1.1), which are f ound in many

vertebrates, insects, plants and mycobacteria, have been
reported to be involved in xenobiotic metabolism due to
their ability to hydrolyse a number o f substrates co ntaining
ester, thioester and amide bonds [1±3]. As some carboxy-
lesterase (CE) isoenzymes display lipase -like activity, it has
been suggested that they might play a part in lipid
metabolism [4]. Moreover, the t wo CE forms with p I values
of 5.2 and 5.6, which have be en isolated from rat liver [5],
are known to deacylate the s tructural analog of diacylglyc-
erol 4- b-phorbol-12-b-myristate-13-a-acetate (PMA). It has
therefore been suggested that these enzymes may have
activating ef fects on protein kinase C [5,6].
A porcine intestinal carboxylesterase (PICE) was re-
cently puri®ed to homogeneity and found to consist of a
single isoform with a pI of 5.1, based on isoelectric
focusing data [7]. The amino-acid sequence deduced from
the cloned cDNA consisted of 565 residues and showed
97% identity with that of porcine liver carboxyle sterase
(PLCE) [8], a protein which belongs to the GXSXG
family of serine proteases, and more than 50% identity
with those of other CE from various mammalian species
[9±11]. The molecular mass of the porcine intestinal
mucosa enzyme was estimated t o be 240 kDa by size-
exclusion chromatography, and 60 kDa using S DS/PAGE
under both reducing and nonreducing conditions [7],
which strongly suggests that the protein consisted of four
apparently identical and active polypeptide subunits,
unlike other mammalian CE which are known to be
monomeric or t rimeric e nzymes [12]. The two disul®de
bridges present in PICE were recently assigned to Cys70±

Cys99 ( loop A) and C ys256±Cys267 (loop B), whereas the
®fth Cys71 r esidu e was thought to be blocked rather t han
being p resent in the free f orm, from the lack o f a lkylation
with iodoacetamide [13].
In the present study, we report on the tissue and
subcellular distribution of PICE using speci®c polyclonal
antibodies and by purifying three active molecular f orms of
the enzyme, and show for the ®rst time that all these forms
are both m yristoylated and p almitoylated.
Correspondence to A. Puigserver, Institut Me
Â
diterrane
Â
en de Recherche
en Nutrition, UMR-INRA 1111, Faculte
Â
des Sciences et Techniques
de St-Je
Â
roà me, Av enue Escadrille Normandie Niemen, F-1 3397
Marseille cedex 20, Franc e. Fax: + 33 4 91 28 84 40,
Tel.:+33491288838,
E-mail:
Abbreviations: CE, carboxylesterase; DEAE, diethylaminoethyl; FA,
fatty acid; GLC, gas-liquid chromatography; KLH, keyhole limpet
haemocyanin; P ICE, porcine intestinal c arboxylesterase; PLCE,
porcine liver carboxylesterase; PMA, 4-b-phorbol-12-b-myristate-
13-a-acetate; pN PA, p-nitrophenylacetate; PVDF, poly(vinylidene
di¯uoride).
Enzyme: carboxylesterase (EC 3.1.1.1).

(Received 3 August 2001, revised 22 N ovember 2001 , accepted 2 7
November 2001)
Eur. J. Biochem. 269, 1109±1117 (2002) Ó FEBS 2002
MATERIALS AND METHODS
Tissues and reagents
All the pig organs used here w ere obtained from the local
slaughterhouse. EAH±Sepharose 4B, Octyl±Sepharose,
DEAE±Sepharose Fast Flow, Sephacryl S-200 (allyl
dextran and N,N-methylene-bisacrylamide matrix, 2.6 ´
60 cm column), Superdex 200 HR (dextran/agarose
matrix, 1.0 ´ 30 cm column), [9,10(n)-
3
H]myristic acid
(speci®c activity 54 Ciámmol
A1
) and [9,10(n)-
3
H]palmitic
acid (speci®c activity 54 Ciámmol
A1
) were purchased from
Pharmacia Biotech. Substrates, BSA, 5-bromo-4-chloro-3-
iodo-phosphate, Ponceau S , c arbodiimide, protein A±
Sepharose, prednisolone, glucagon, insulin and SDS were
from Sigma Chemical Co. Chloroform and butanol were
provided by SDS (Peypin, France), while methanol was
purchased from Carlo Erba. Williams E medium, fetal
bovine serum, penicillin and streptomycin were obtained
from Gibco-BRL. The nitrocellulose sheets (0.2 lm) were
from Schleicher and Schuell and the IgG fraction of goat

anti-(rabbit IgG) serum conjugated with peroxidase was
from Organon Teknika Corporation-Cappel.
Enzyme and protein assays
Enzyme activity determin ations were performed t itrimetri-
cally on tributyrin (65 m
M
), butyrylcholine (132 m
M
),
a-naphthylacetate (26 m
M
) and phorbol diester (1.3 m
M
)
at 37 °C using a Metrohm (Herisau, S witzerland) pH-stat
(718 STAT Titrino, Radiometer) and 0.01
M
NaOH as
described p reviously [7]. One unit of enzymatic activity
corresponds to 1 lmol fatty a cid released per min. All the
activities were measured at pH 8.0, including that towards
p-nitrophenylacetate [14]. In order to determine the activities
of aminopeptidase N (a microvillous membrane marker)
(Na
+
/K
+
)-ATPase (a basolateral plasma membrane
marker), NADPH-cytochrome c reductase (a microsomal
contamination marker), cytochrome c oxydase (a mito-

chondrial marker) and acid phosphatase (a lysosome
marker) during subcellular fractionation experiments, we
used methods which have been described in previous papers
[15±19]. Protein concentrations were determined as
described by B radford [20].
Peptide synthesis and puri®cation
The peptide KMKFLTLDLHGDPRE, corresponding to
the amino-acid sequence from positions 281±296 on the
PICE polypeptide chain, was s ynthesized by the M arseille
CNRS-CIML laboratory using an Applied Biosystem
Peptide Synthesizer 431 A. The peptide was puri®ed by
RP-HPLC on a Kontron apparatus equipped with an
ALLTIMA C18 column (4.6 mm ´ 25 cm), and its molec-
ular mass was determined on an Applied Biosystems
MALDI-TOF Voyager D E RP mass s pectrometer.
Preparation of polyclonal antiserum
The peptide was covalently a ttached to keyho le limpet
haemocyanin (KLH) from the mollusc Concholepas con-
cholepas with 2% glutaraldehyde. The resulting peptide±
KLH conjugate was dialyzed, lyophilized and further used
(1 mg dissolved in 0.5 m L N aCl/P
i
with complete Freund's
adjuvant) to immunize New Zealand INRA 1077 male
white rabbits by subcutaneous injection. Three weeks later,
the same a mount of peptide±KLH conjugate emulsi®ed
with incomplete F reund's adjuvant w as injected intramus-
cularly. After a further 10-day p eriod, 0.5 mg antigen was
injected subcutaneously, and the same amount of antigen
was then injected intravenously on the following day.

Finally, 10 days later, blood was collected from the
marginal ear v e in, allowed to clot f or 1 h and s uccessively
centrifuged at 3000 g for 10 min and at 15 000 g for 15 min.
The immune serum was then c ollected , ®ltered and stored at
4 °C.
Puri®cation of polyclonal antibodies
and immunoprecipitation experiments
The PICE K281±E296 peptide (30 mg) was covalently
linked to EAH-Sepharose 4B (30 m gámL
A1
gel) using 0.1
M
carbodiimide according t o Pharmacia Biotech instru ctions.
The antibodies speci®cally bound to the immobilized
peptide w ere elute d w ith a 0.5
M
NaCl containing 0.1
M
acetate buffer ( pH 3.5), immediately neutralized with 1 .5
M
Tris/HCl buffer (pH 9.3) in the presence of 0 .5
M
NaCl to
prevent protein denaturation, and ®nally stored at 4 °C.
Antibody titration and speci®city determination were per-
formed using a conventional ELISA assay [21]. The puri®ed
anti-PICE Ig were then covalently linked to protein
A±Sepharose gel as previously described [22], and used to
precipitate the tritiated protein.
Molecular mass determination and immunoblot analyses

SDS/PAGE was carried out using Laemmli's m ethod [23].
Proteins were electrotransferred overnight o nto a nitrocel-
lulose sheet at 50 V in 20 m
M
Tris/HCl buffer (pH 8.5)
containing 150 m
M
glycine and 20% ethanol. The nitrocel-
lulose membrane was subsequently saturated with 10%
BSA before being incubated with 1: 100 (v/v) diluted rabbit
anti-PICE immune serum, and the reacting antibodies were
further detected with 800-fold diluted peroxidase-conjugat-
ed goat anti-(rabbit IgG) Ig. Both radiolabelled proteins and
immune protein p recipitates were separated by p erforming
electrophoresis on a 12% polyacrylamide gel in t he presence
of SDS, stained with Coomassie Blue for gel-slicing and
scintillation counting and/or subjected to immunoblot
analysis. Preparatory to the radioactivity assays, the sliced
gels and i mmunoblots were s olubilized in 0.5 mL of a 30%
(w/v) hydrogen peroxide solution for 5 h at 95 °C, and the
count was performed in 5 mL scintillation ¯uid on a
Packard-Tri-Carb Model 2100 TR liquid s cintillation
spectrometer as p reviously described [24]. Isoelectric focus-
ing (IEF) was p erformed as described b y R obertson et al .
[25].
Tissue distribution and intestinal mucosal subcellular
fractionation of carboxylesterase
Pig organs were dissected out and immediately homogen-
ized in 20 m
M

Tris/HCl buffer, pH 7.3, containing 0.25
M
sucrose, 10 m
M
KCl, 1 m
M
MgCl
2
,1l
M
phenylmethane-
sulfonyl ¯uoride and 1 m
M
benzamidine. The homogenates
were subsequently centrifuged at 10 000 g for 2 0 m in, the
1110 S. Smialowski-Fle
Â
ter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
supernatant w as again centrifuged a t 105 000 g and a t 4 °C
for an additional 45-min period, and the resulting superna-
tant was ®nally used for the anti-PICE Ig staining proce-
dure. Crude brush-border membrane preparations were
obtained from the subcellular fraction of the intestinal
mucosa as previously described [26,27]. Brie¯y, pig intestinal
mucosal scrapings were homogenized in four times their
mass of a 5-m
M
Tris/HCl buffer ( pH 7.3) containing 0.25
M
sucrose, 10 m

M
KCl and 1 m
M
MgCl
2
, ®ltered through a
gauze and further subje cted to d ifferential centrifugation to
obtain the membrane fraction.
PICE puri®cation
Fresh porcine intestine was s craped off and the muco sa was
either i mmediately used o r frozen at A80 °C until use. The
lipids w ere p artially extracted from about 200 g of the
mucosa by placing them in a chloroform/butanol mixture
(9 : 1, v /v). After a homogenization step in 500 mL of
20 m
M
Tris/HCl, 0.35
M
NaCl at pH 8.0, centrifugation
was performed at 10 000 g for1handproteinsfromthe
resulting s upernatant ( S1) w ere precipitated by adding solid
(NH
4
)
2
SO
4
to the solution (0.7
M
®nal concentration) under

gentle stirring at 4 °C for 2 h . A fter a ®rst centrifugation at
10 000 g for 30 min, the pellet was dissolved in 100 mL o f
20 m
M
Tris/HCl pH 8.0 c ontaining 0.7
M
(NH
4
)
2
SO
4
and
then dialysed against the buffer. A second centrifugation
took place under t he same experimental conditions and t he
resulting s upernatant was applie d to a n octyl±Sepharose gel
equilibrated with the same buffer and the proteins were
eluted with a 20-m
M
Tris/HCl buffer, pH 8.0, containing
0.4
M
NaCl (buffer A). The active proteins eluted were
successively precipitated with 80% (w/v) ammonium sulfate
at 4 °C, and after be ing centrifuged at 10 000 g for 30 m in,
theyweredissolvedin10mLofbufferAanddialysed
overnight against the same buffer. The dialysate was then
applied to a DEAE-Sepharose Fast Flow column
(1.5 ´ 14 cm) equilibrated w ith buffer A , an d the p roteins
were eluted with a linear 0.1±0.3

M
NaCl gradient. The
active fractions on tributyrin were ®nally applied to a
Superdex 200 HR gel c olumn (1.0 ´ 30 cm) and eluted with
a20-m
M
Tris/HCl buffer pH 8 .0 containing 0.35
M
NaCl,
at a ¯ow rate of 0.5 mLámin
A1
.
Molecular mass determination
This was achieved by performing gel ®ltration on a
Sephacryl S -200 column (2.6 ´ 60 cm) and the proteins
were eluted with 0.35
M
NaCl in 20 m
M
Tris/HCl, pH 8.0,
and by SDS/PAGE on a 12% (w/v) gel as previously
described [23]. The electrophoretic molecular-mass markers
(14.4±97 k Da) and isoelectric focusing calibration kit
(pH 4 .5±9.6) were obtained from B io-Rad Laboratories.
The MW-GF 1 000 kit ( 29±2000 kDa) f rom Sigma Chem-
ical Co, was used for the g el ®ltration procedure.
Amino-acid composition and sequence determination
The amino-acid composition of t he puri®ed PICE was
determined using a Waters chromatography system as
previously described [13,28], after 24 h hydrolysis in 6

M
HCl at 110 °C. The amino-acid sequence of the Ponceau
red-stained proteins was determined by performing Edman
degradation on an Applied Biosystems Model 470 A
protein gas-phase sequencer [29].
Lipid extraction and fatty acid identi®cation
The lipids present in the puri®ed PICE and porcine serum
albumin u sed a s the control substance were completely
removed with chloroform/methanol/water (2 : 2 : 1 .8, v /v/
v) as described by Bligh & Dyer [30]. The covalently b ound
fatty acids were released from the protein under alkaline
conditions. E thanol containing 1
M
KOH w as used and t he
protein solution was i ncubated a t 8 0 °C for 1 h , a nd then
dried under a stream of nitrogen. After adding the same
amount of water, the aqueous layer was acidi®ed with HCl
and the free fatty acids were extracted with hexane and dried
before performing methanolysis at 100 °C for 1 h using 1 4%
BF
3
in methanol [31]. After the methylation, the fatty acids
were identi®ed on a PerkinElmer gas-liquid chromatogra-
phy autosystem XL equ ipped with P erkinElmer integrator
1022S, using n-heptadecanoic acid as an internal standard.
Cell cultures and labelling
Mature porcine enterocytes (16 ´ 10
6
cellsámL
A1

)and
hepatocytes (3.4 ´ 10
6
cellsámL
A1
)wereisolatedas
described by Bader et al. [32] and by Seglen [33], respec-
tively. Prior to the labelling experiments, enteroc ytes and
hepatocytes were i ncubated for 4 h at 37 °C in W illiams E
medium supplemented with 5% (v/v) fetal bovine serum,
prednisolone (5 lmoláL
A1
), glucagon (0.014 lgámL
A1
),
insulin (0.16 Uá mL
A1
), penic illin ( 200 U ámL
A1
), streptomy-
cin (200 lgámL
A1
)and63lCiámL
A1
of [9,10(n)-
3
H]myristic
acid or [9,10(n)-
3
H]palmitic acid (speci®c a ctivity

54 Ci ámmol
A1
). Prior to use, the fetal bovine serum was
delipidated using 1,2,2-trichloro-1,2,2-tri¯uoroethane [34].
At the e nd of the labelling period, cells were aspirated from
the dishes and centrifuged at 900 g for 5 min Cell pellets
were then washed extensively in NaCl/P
i
, homogenized,
centrifugated at 10 000 g for 10 min at 4 °C, and the
supernatant was sampled for analysis.
RESULTS
Tissue distribution of pI 5.1 carboxylesterase
The presence of the pI 5.1 CE isoform i n 1 1 homogenates
from various porcine tissues was checked by performing
immunoblot analysis on the soluble e xtracts u sing puri®ed
polyclonal antibodies directed against a synthetic amino-
acid peptide corresponding to the amino-acid sequence
located between residues 281 and 296 in the PICE
polypeptide chain. Figure 1 shows t hat these antibodies
speci®cally revealed a 60-kDa band corresponding to the
pI 5.1 CE in the soluble extracts from small intestine,
parotid and submaxillary glands, liver, kidney cortex, lung
and brain cortex. The highest level of expression of pI 5.1
CE was observed in the liver, followed by the small intestine,
but it is worth noting t hat the enzym e was not detected in
the soluble extracts of homogenates from colon, stomach,
pancreas and kidney medulla, or in those from skin,
bladder, tongue, trachea, b rain medulla and cerebellum,
heart, pharynx and suprarenals (data not shown). Esterase

activity on tributyrin was observed only in t he so luble
Ó FEBS 2002 Carboxylesterase fatty acylation (Eur. J. Biochem. 269) 1111
fractions from small intestine, colon, liver and pancreas
homogenates. In the latter homogenates, the activity
observed was presumably that of lipase, although the
presence of some contaminating activity due to the presence
of microorganisms i n the colon c ould not be ruled out.
Subcellular distribution of porcine intestinal
carboxylesterase
The distribution o f PICE activity on t ributyrin and that o f
marker enzymes on their speci®c s ubstrates in a number of
subcellular f ractions from pig i ntestinal mucosa is g iven in
Table 1 . Esterase activity on t ributyrin was detected in four
subcellular f ractions, a nd in all these fractions, immunoblot
analysis using the puri®ed polyclonal a nti-PICE Ig yielded a
single band at 60 kDa. The highest level of activity was
observed in the microsomal and soluble fractions, which
yielded 41% and 32% of the total enzyme activity,
respectively. As the microvillar frac tion accounted for as
much as 18% of t he overall esterase a ctivity on t ributyrin, it
was suggested that some of the PICE might correspond to
an enterocytic b rush border membrane protein.
Puri®cation of the molecular forms
of porcine intestinal CE
Figure 2A shows the PICE elution pro®le systematically
obtained with a Sephacryl S-200 gel ®ltration column,
whether the puri®cation procedure was that used in the
present study or that described by David et al.[7].Asingle
molecular form (F4) was obtained, which showed the
presence of a single 60-kDa band with a pI value of 5.1 upon

SDS/PAGE analysis under reducing and nonreducing
conditions and isoelectric focusing. When the F4 molecular
form was further puri®ed using a Superdex 200 HR gel
®ltration c olumn, two distinct molecular forms (F3 and F1)
were separated (Fig. 2B ). Based on the elution pro®les of
standard proteins, the apparent molecular mass of t hese
forms w as found to be 180 k Da and 60 kDa, respectively.
Surprisingly, the dimeric molecular form F2 was not
observed. Again, SDS/PAGE and isoelectric focusing
analysis showed that F3 and F1 corresponded to a single
60-kDa band with a pI of 5 .1 (Fig. 3).
Our results and those obtained by David et al.[7]
strongly suggested the existence o f a single polypeptide
chain c orresponding to the monomeric form of P ICE (F1)
and giving r ise to t he noncovalent association of three and
four apparently identical subunits corresponding to the F3
and F4 molecular forms of PICE, r espectively.
PH- and substrate-dependent activity of PICE
molecular forms
At pH 8.0, which was used for running both the gel
®ltration experiments and esterase activity assays on trib-
utyrin, F4 a nd F1 were found to have similar speci®c
activity values on tributyrin as substrate (% 290 Uámg
A1
protein), whereas F3 was about threefold less active
(Table 2). At pH 6 .5, however, F4 and F3 were found to
have almost equal levels of esterase activity on tributyrin,
whereas F1 was slightly less active. Overall, at the more
acidic pH value, the three forms were 30±40% less active
than at the more basic pH value. A number of ester-

containing compounds including p-nitrophenylacetate,
a-naphthylacetate and butyrylcholine were also t ested as
possible substrates at pH 8.0 (Table 2 ). The tetrameric,
Fig. 1. Immunoblotting an d esterase a ctivity on tributyrin o f pI 5.1 CE
from porcine tissues. Esterase activity was measured as indicated in the
Materials and Methods section. Total proteins (30 lg) presen t in
homogenates from 11 p orcine tissues were electrop ho resed in a 12%
SDS/PAGE, transferred o nto a nitrocellulose memb rane, and reacted
with polyclonal antibodies raised against the syn thetic peptide corre-
sponding to the amino-acid sequence extending from K281 to E296 of
the PICE polypeptide chain. Lane 1, small intestine; lane 2, colon; lane
3, stomach; la ne 4, parotid; lane 5, submaxil lary; lane 6, liver; lane 7,
pancreas; lane 8, kidney cortex; lan e 9, kidney m edulla; lane 10, lung;
lane 11, brain cortex.
Table 1. Subcellular localization of CE activity in porcine intes tinal mucosa. At each st ep of the s ubcellular fractio nation procedure, t he enzyme
activities were measured in the pellet and the s up ernatant and expressed as a percentage of t he total activity. The values are means based on three
separate subcellular fractionations.
Fraction
Enzymatic marker
activities (%)
Subcellular
fraction
Esterase activity
on tributyrin (%)
Immunoblot
analysis
a
10 000 g pellet Cytochrome c oxidase (70  5) Mitochondrial 10  3 +
CaCl
2

pellet NADPH/H
+
Cytochrome c reductase (80  3); Microsomal and 40  5 +
Na
+
/K
+
ATPase (73  7) basolateral membranes
105 000 g pellet Aminopeptidase N (78  3) Microvillar 18  6 +
Final supernatant Acid phosphatase (81  7) Soluble 32  7 +
a
Presence (+) of immunoreactive PICE detected with polyclonal antibodies directed against the PICE K281±E296 peptide.
1112 S. Smialowski-Fle
Â
ter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
trimeric and monomeric forms of PICE were found to
display different enzymatic activities on these substrates.
Although F4 a nd F1 were equally active on tributyrin,
as already i ndicated i n Table 2, the latter form was roughly
2±3 times more active than the former on the other three
substrates, namely a-naphthylacetate, p-nitrophenylacetate
and butyrylcholine. It is worth noting that F 3 w as the most
active on butyrylcholine and the least active on tributyrin,
and that p-nitrophenylacetate is apparently the most
ef®cient substrate f or PICE molecular f o rms in general.
N-Terminal amino-acid sequence and fatty acid
content of PICE molecular forms
Table 3 gives t he fatty acid co ntent of t he F4, F3 and F1
molecular forms o f PICE, as well as the N-terminal amino-
acid sequence of F3, in addition to that of F4 previously

reported by David et al. [7]. The nine ®rst amino acids of the
F4 and F3 polypeptide chains were found to be identical,
whereas in F1, no N-terminal amino acid could be d etected,
which strongly suggests that the polypeptide chain was
Fig. 3. Polyacrylamide gel electrophoresis of PICE molecular forms.
(A) The electro phoresis was carried out on a 12% polyacrylamide gel
in the presence of SDS under reducing conditions. (B) IEF was per-
formed at pH 4±9 with a calibration kit (pH 4.46±9.6) and silver
staining.
Fig. 2. Gel ®ltration of porcine intestinal CE molecular forms. (A)
Porcine i ntestinal CE, puri® ed as indicated in Materials and methods
or as described by David et al. [ 7], was applied to a Sephacryl-S200
column (2.6 ´ 60 cm) and eluted with 20 m
M
Tris/HCl buer con-
taining 0.35
M
NaCl, pH 8.0. (B) The F4 molecular form w as then
applied to a Superdex 200-HR column (1.0 ´ 30 cm) and eluted with
the above-mentioned buer. Esterase activity on tributyrin was
assayed as indicated in Materials and methods. Solid and d otted lines
represent the protein absorban ce at 280 nm and the esterase activity on
tributyrin, respectively.
Table 2. Substrate-dependent activity of PICE molecular forms. 100 % speci®c activity on tributyrin at pH 8.0 corresponds to 290 U ámg protein
A1
for both the F4 and F1 f orms. All the results are m eans on three enzymatic determinations.
Substrate
Activity
determination pH
Relative speci®c activity (%)

F4 F3 F1
Tributyrin 8.0 100 30 100
6.5 40 40 27.5
a-Naphthylacetate 8.0 42 74 85
p-Nitrophenylacetate 8.0 103 123 170
Butyrylcholine 8.0 15 60 52
Ó FEBS 2002 Carboxylesterase fatty acylation (Eur. J. Biochem. 269) 1113
blocked. As the amino-acid composition of the three
molecular f orms of PICE was found to have remained
unchanged, these data are not shown.
As far as fatty acylation is concerned, both the F4 and F1
forms of P ICE were found to have fairly similar FA p ro®les
in sharp c ontrast with the F3 form ( Table 3). Myristic and
palmitic acids were the predominantly linked FA, while a
number of minor fatty acids including stearic, oleic and
arachidonic acids could also be detected. It is worth noting
that myristic acid was not released from the F3 form after
alkaline hydrolysis, contrary to what was observed in the
case of the F4 and F1 forms. The quantitative determination
of fatty acids released from a given molecular form of P ICE
relative to the amount of protein deduced from its amino-
acid composition indicated t hat s toichiometric amounts of
myristic and palmitic acids were present in F1 (1 mol FA per
mol of F1). B y contrast, less m yristic acid than palmitic acid
was detected in F4 (0.2±0.4 mol of myristic acid as
compared to 1 mol palmitic acid per mol of F4).
PICE acylation in enterocyte and hepatocyte cell
cultures
Figure 4A shows the SDS/PAGE protein bands and the
immunoblot pro®le obtained u sing the polyclonal antibod-

ies raised against the K281±E296 amino-acid sequence of
PICE, with the soluble proteins from enterocyte primary cell
cultures in the presence of labelled [
3
H]palmitic acid and
[
3
H]myristic acid. A single band corresponding to a protein
with a m olecular m ass o f % 60 kDa was observed in b oth
cases in t he enterocytic c ells. The patte rn o f r adioactivity in
the gel slices showed a good correlation with the relative
mobility of the immunoreactive PICE (Fig. 4A). About a
four-fold higher level of
3
H radioactivity was counted in the
Table 3. N-terminal amino-acid sequence and fatty acid c ontent o f PICE molecular forms.
Molecular
forms
N-Terminal
sequence
Fatty acid content
Major
c
Minor
d
F4 NH2-GQPASPPVV
a
C14:0 ; C16:0 C18:0 ; C18:1 ; C20:4(n-6)
F3 NH2-GQPASPPVV
b

C16:0 C18:0 ; C18:1 ; C20:4(n-6)
F1 Blocked C14:0 ; C16:0 C18:0 ; C18:1 ; C20:4(n-6)
a
From David et al. [7], and with an amino-acid sequence yield of about 0.5 mol glycine per mol of protein.
b
Yield of about 0.9 mol glycine
per mol of protein.
c
About 1 mol fatty acid per mol of protein, except for F4 (0.2±0.4 mol myristic acid per mol of protein).
d
Less than
0.1 mol fatty acid per mol of protein.
Fig. 4. SDS/PAGE, immunoblotting and
pattern of radioactivity obtained with s oluble
proteins from enterocytes (A) and hepatocytes
(B) incubated with [
3
H]fatty acid. 1, Ponceau
red protein staining; 2, immunoblotting with
polyclonal antibodies dire cted against the
PICE peptide K
281
to E
296
(the arrow indi-
cates the position of immunoreactive PICE).
The r elative m obilities o f molecular mass
markersinSDS/PAGEareindicatedbelow
the b lot: (a) phospho rylase b (97 kDa); ( b )
albumin (66 kDa); (c) ovalbumin ( 45 kDa);

(d) carbonic anhydrase (30 kDa); (e) trypsin
inhibitor ( 20.1 kDa); and (f) a-lactalbumin
(14.4 kDa).
1114 S. Smialowski-Fle
Â
ter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
PICE labelled with [
3
H]myristic acid than in that labelled
with [
3
H]palmitic acid, in agreement with the a bove ®nding.
In order to check whether the labelling was really due to
PICE and not to another protein with the same molecular
mass, the enzyme from enterocyte homogenates was
immunoprecipitated with the puri®ed polyclonal antibodies.
A single band at % 60 kDa which contained [
3
H]palmitic
(2.6 ´ 10
3
d.p.m.) or [
3
H]myristic acids (2.1 ´ 10
3
d.p.m.)
was revealed in the blot (data not shown).
As the CE from porcine intestine and liver show 97%
amino-acid sequence identity, and as the puri®ed speci®c
polyclonal antibodies raised against PICE c ross-react w ith

PLCE, w e e xtended the fatty acylation analysis to hepato-
cytes.AsshowninFig.4B,asinglebandwithanapparent
molecular mass of 60 kDa, corresponding to PLCE, was
revealed by the speci®c polyclonal antibodies and the
protein was l abe lled by m yristic or palmitic acids. An alysis
of the radioactivity patterns in the blot showed the
presence of a similar
3
H content in PLCE, whether the
protein was l abelled with [
3
H]palmitic acid or [
3
H]myristic
acid.
DISCUSSION
The results of immunoblot analysis performed on soluble
extracts from porcine tissue homogenates showed that the
pI 5.1 CE was present mainly in the liver, but also to a lesser
extent in the small intestine, submaxillary and parotid
glands, kidney cortex, lungs, and brain cortex. This CE
isoform was not detected, however, in the other two main
parts of t he digestive tract, namely the s tomach and colon,
or in the pancreas and kidney medulla. The fact that the
highest expression of the protein isoform was recorded in
the liver might be due to the presence of several CE
isoenzymes in this tissue [35,36] and to some lack of
speci®city of the polyclonal antibodies used for t he analysis.
However, the peptide extending from K
281

to E
296
in PICE
was chosen as a speci®c antigen site because it showed more
than 86% sequence identity with those from porcine liver
[8], human liver [37] and human b rain [38] CE. A s esterase
activity on tributyrin was detected only in the small
intestine, liver, p ancreas, where lipase activity is known t o
exist, and in the colon, it is suggested that there was no direct
relationship between the presence of esterase activity on
tributyrin in a given tissue and that of the pI 5.1 CE.
Subcellular fractionation of the intestinal mucosa showed
that PICE was unevenly distributed among the various
fractions co rresponding to mitochondria, microsomes,
microvilli and c ytosol. Although t he enzyme has previously
been found to contain the tetrapeptide HAEL at the
C-terminus of the polypeptide chain [7], w hich is thought to
serve as a retention signal f or proteins on the luminal side of
the ER, it is apparently not retained in the ER. Both the
immunoblot analysis and e sterase activity o n tributyrin
determinations showed that PICE was present in the cytosol
fraction as well as in the mitochondrial and microvillar
fractions, although the possible occurrence of some non-
speci®c adsorption of PICE to subcellular membranes
during the fractionation procedure cannot be ruled out.
A tetrameric form of the porcine intestinal CE was
recently puri®ed from a soluble protein fraction (105 000 g
supernatant) and characterized [7,13]. In the present study, a
separate puri®cation procedure was carried out from the
total protein fraction (10 000 g supernatant) in order to

isolate the membrane-bound enzyme. Both monomeric
(60 kDa) and trimeric forms (180 kDa) could t herefore be
isolated using a Superdex column, while the tetrameric form
(240 kDa) which was isolated b y g el ®ltration on Sephacryl
S-200 column corresponded to t hat previously described by
David et al. [7]. Hydrophobic interactions may contribute
signi®cantly to the polymerization of PICE monomers, due
to the p resence of covalently bound fatty a cids, as suggested
in Fig. 5. The interactions between the monomers in the
tetrameric form F4 were apparently stronger than those
occurring in the trimeric form F3, as no monomeric form F1
was observed in the elution pro®le f rom the Sephacryl S-200
column, in con trast to the pro®le of t he Superdex column
(Fig. 2 ). The possibility that there may have been a
difference in af®nity between the molecu lar forms depend-
ing on the type of polysaccharide matrix used for gel
®ltration purposes cannot be ruled out. Similar results have
been observed, for example, in the case of galectins and
ricins, two groups of proteins known t o have lipolytic
activities [39,40]. Whatever arguments may be put forward
to explain the existence of several mo lecular forms in PICE,
the behaviour of this protein on Sephacryl S-200 is
comparable to that o f liver CE [8] but different from that
of rat i ntestinal CE [41].
PICE was found to be more active on tributyrin at pH 8.0
than at pH 6.5, which is not surprising for a serine enzyme
on account of the s tate of protonation of th e histidine
residue from the catalytic triad. In addition, most of the F4
esterase activity on tributyrin at pH 8.0 was due to F 1, and
as F3 was found to be threefold less active than both F 4 and

F1, the interactions between monomers in F4 and F3 were
probably d ifferent, l eading to distinct conformational states
that did not display t he same catalytic a ctivity on tributyrin.
Fig. 5. A possible scheme for explaining the existence of PICE mono-
meric and pol ymeric f orms. M and P s tand for activated myristic acid
and palmitic acid, respectively. NMT, N-myritoyltransferase; PAT,
palmitoylacyltransferase; a nd MPT, myritoylproteinth ioesterase.
Ó FEBS 2002 Carboxylesterase fatty acylation (Eur. J. Biochem. 269) 1115
Covalent changes in proteins with myristate have been
observed i n s everal eukaryotic proteins [ 42,43]. The preva-
lent type, myristoylation, which has been thoroughly
characterized, seems to occur cotranslationally at the
a-amino group of the N-terminal glycine, included in the
Gly-XXX-Ser/Thr c onsensus s equence, whereas palmitic
acid is thought to be added post-translationally at the
sulfhydryl group of cystein via a thioester bond [43]. As far
as we know, n o fatty acylation of CE h as been reported to
occur so f ar. The results of the present study clearly indicate
that PICE contained covalently bound fatty acids, and the
fact that acylation o f t he enzyme occurred was f urther
con®rmed u sing enterocyte and hepatocyte cell primary
cultures in the presence of the two main corresponding
radiolabelled fatty acids. F1 contained the same amount of
myristic acid and palmitic acid, close to stoichiometry, while
F3 contained only palmitic acid. The amount of myristic
acid present in F4 was only a bout a quarter of that recorded
in palmitic acid. The resistance of F1 to Edman d egradation
might therefore be due to the myristoylation of the
N-terminal G-Q-P-A-S- consensus sequence [7], as the
monomeric form of PICE was found to contain a stoichio-

metric amount of the f atty acid. As we r ecen tly established
that Cys71 in t he PICE amino-acid sequence could not be
alkylated with iodoacetamide, except in t he presence o f
100 m
M
dithiothreitol in the medium, this residue was
thought to be a good candidate for palmitoylation of the
PICE F1 form via a thioester linkage [13]. This a ssumption
is consistent with the well-known sensitivity of thioester-
type fatty acid linkages to alkaline methanolysis and the
effects of reducing agents [44]. A question therefore arises
about the ®nding that F1 apparently has an N-terminal
myristoylated glycine, whereas F3 has a f ree amino group
containing an N-terminal glycine residue. To answer the
question as t o whether F1 is cotranslationally myristoylated
and then deacylated before undergoing trimerization, or
whether the formation of the trimer occurs competitively
with N-terminal blocking of the monomer, further e xperi-
ments are certainly required. As mentioned above, Fig. 5
gives a possible scheme for the formation of PICE
multimers.
As the F1 and F4 molecular forms of PICE are both
myristoylated and palm itoylated, the functional s igni®cance
of this twofold fatty acylation of the intestinal CE is still
unclear. The increase in the af®nity with membranes
resulting from the presence of covalently linked palmitic
and myristic acids in PICE should facilitate the possible
targeting, anc horing, and crossing of the cellular m em-
branes, a s s uggested by the subcellular pattern of distribu-
tion of the enzyme observed here. Moreover, PICE was

found in the present study to deacylate PMA (data n ot
shown), a structural analog of diacylglycerol, and to be
variably active on a number o f ester containing xenobiotics.
The high speci®city of PICE towards exogenous ester
containing substrates along with the presence of the enzyme
in the microsomal and cytosolic fractions suggests that it
may be involved in the xenobiotic metabolism. This
hypothesis needs to be con®rmed by further experimental
data, as does the suggestion that the enzyme may be
involved in cell signal transduction via diacylglycerol and
protein kinase C [5,6].
ACKNOWLEDGEMENTS
We are grateful to Dr E. H. Ajandouz for his helpful advice. We thank
Mrs D. Moinier and Mr J. Bonicell for their contribution to the
automatic sequencing and m ass spectrometry determinations, r espec-
tively, Dr G. Pieroni for fatty acids analysis, Dr V. Girod for dissection
of pigs, and Dr J. Blanc f or revising the English manuscript.
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