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 e
Trang 1Myristyl and palmityl acylation of pI 5.1 carboxylesterase
from porcine intestine and liver
Tissue and subcellular distribution
Sylvie Smialowski-FleÂter, Andre Moulin, Josette Perrier and Antoine Puigserver
Institut MeÂditerraneÂen de Recherche en Nutrition, UMR-INRA, Faculte des Sciences et Techniques de St-JeÂroÃme, Marseille, France
Immunoblotting analyses revealed the presence of
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 detected 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 the ®rst time by performing
hydrophobic chromatography and gel ®ltration The
molecular mass of these three enzymatic forms was estimated
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 [3H]palmitic
and [3H]myristic acids were incorporated into porcine enterocytes and hepatocytes in cell primary cultures Besides these two main fatty acids, the presence of oleic, stearic, 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, which were both myristoylated and palmitoylated, the F3 form was only palmitoylated The monomeric, trimeric and tetrameric forms of PICE were all able to hydrolyse short chain fatty 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 found in many
vertebrates, insects, plants and mycobacteria, have been
reported to be involved in xenobiotic metabolism due to
their ability to hydrolyse a number of substrates containing
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 two CE forms with pI values
of 5.2 and 5.6, which have been isolated from rat liver [5],
are known to deacylate the structural 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 effects 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 carboxylesterase (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 to be 240 kDa by size-exclusion chromatography, and 60 kDa using SDS/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 trimeric enzymes [12] The two disul®de bridges present in PICE were recently assigned to Cys70± Cys99 (loop A) and Cys256±Cys267 (loop B), whereas the
®fth Cys71 residue was thought to be blocked rather than being present in the free form, from the lack of alkylation 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 forms of the enzyme, and show for the ®rst time that all these forms are both myristoylated and palmitoylated
Correspondence to A Puigserver, Institut MeÂditerraneÂen de Recherche
en Nutrition, UMR-INRA 1111, Faculte des Sciences et Techniques
de St-JeÂroÃme, Avenue Escadrille Normandie Niemen, F-13397
Marseille cedex 20, France Fax: + 33 4 91 28 84 40,
Tel.: + 33 4 91 28 88 38,
E-mail: antoine.puigserver@lbbn.u-3mrs.fr
Abbreviations: CE, carboxylesterase; DEAE, diethylaminoethyl; FA,
fatty acid; GLC, gas-liquid chromatography; KLH, keyhole limpet
haemocyanin; PICE, porcine intestinal carboxylesterase; PLCE,
porcine liver carboxylesterase; PMA,
4-b-phorbol-12-b-myristate-13-a-acetate; pNPA, p-nitrophenylacetate; PVDF, poly(vinylidene
di¯uoride).
Enzyme: carboxylesterase (EC 3.1.1.1).
(Received 3 August 2001, revised 22 November 2001, accepted 27
November 2001)
Trang 2M A T E R I A L S A N D M E T H O D S
Tissues and reagents
All the pig organs used here were 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)-3H]myristic acid
(speci®c activity 54 Ciámmol)1) and [9,10(n)-3H]palmitic
acid (speci®c activity 54 Ciámmol)1) were purchased from
Pharmacia Biotech Substrates, BSA,
5-bromo-4-chloro-3-iodo-phosphate, Ponceau S, carbodiimide, 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 determinations were performed
titrimetri-cally on tributyrin (65 mM), butyrylcholine (132 mM),
a-naphthylacetate (26 mM) and phorbol diester (1.3 mM)
at 37 °C using a Metrohm (Herisau, Switzerland) pH-stat
(718 STAT Titrino, Radiometer) and 0.01M NaOH as
described previously [7] One unit of enzymatic activity
corresponds to 1 lmol fatty acid 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 Bradford [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 synthesized by the Marseille
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 DE RP mass spectrometer
Preparation of polyclonal antiserum
The peptide was covalently attached to keyhole 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 mL NaCl/Piwith complete Freund's adjuvant) to immunize New Zealand INRA 1077 male white rabbits by subcutaneous injection Three weeks later, the same amount of peptide±KLH conjugate emulsi®ed with incomplete Freund's adjuvant was injected intramus-cularly After a further 10-day period, 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 vein, allowed to clot for 1 h and successively centrifuged at 3000 g for 10 min and at 15 000 g for 15 min The immune serum was then collected, ®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 mgámL)1gel) using 0.1M carbodiimide according to Pharmacia Biotech instructions The antibodies speci®cally bound to the immobilized peptide were eluted with a 0.5M NaCl containing 0.1M acetate buffer (pH 3.5), immediately neutralized with 1.5M Tris/HCl buffer (pH 9.3) in the presence of 0.5MNaCl 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 method [23] Proteins were electrotransferred overnight onto a nitrocel-lulose sheet at 50 V in 20 mM Tris/HCl buffer (pH 8.5) containing 150 mMglycine 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 radiolabellperoxidase-conjugat-ed proteins and immune protein precipitates were separated by performing electrophoresis on a 12% polyacrylamide gel in the 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 immunoblots were solubilized 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 scintillation spectrometer as previously described [24] Isoelectric focus-ing (IEF) was performed as described by Robertson et al [25]
Tissue distribution and intestinal mucosal subcellular fractionation of carboxylesterase
Pig organs were dissected out and immediately homogen-ized in 20 mMTris/HCl buffer, pH 7.3, containing 0.25M sucrose, 10 mMKCl, 1 mMMgCl2, 1 lM phenylmethane-sulfonyl ¯uoride and 1 mMbenzamidine The homogenates were subsequently centrifuged at 10 000 g for 20 min, the
Trang 3supernatant was again centrifuged at 105 000 g and at 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-mMTris/HCl buffer (pH 7.3) containing 0.25M
sucrose, 10 mMKCl and 1 mM MgCl2, ®ltered through a
gauze and further subjected to differential centrifugation to
obtain the membrane fraction
PICE puri®cation
Fresh porcine intestine was scraped off and the mucosa was
either immediately used or frozen at )80 °C until use The
lipids were partially 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 mM Tris/HCl, 0.35M NaCl at pH 8.0, centrifugation
was performed at 10 000 g for 1 h and proteins from the
resulting supernatant (S1) were precipitated by adding solid
(NH4)2SO4to the solution (0.7M®nal concentration) under
gentle stirring at 4 °C for 2 h After a ®rst centrifugation at
10 000 g for 30 min, the pellet was dissolved in 100 mL of
20 mMTris/HCl pH 8.0 containing 0.7M(NH4)2SO4and
then dialysed against the buffer A second centrifugation
took place under the same experimental conditions and the
resulting supernatant was applied to an octyl±Sepharose gel
equilibrated with the same buffer and the proteins were
eluted with a 20-mM Tris/HCl buffer, pH 8.0, containing
0.4M NaCl (buffer A) The active proteins eluted were
successively precipitated with 80% (w/v) ammonium sulfate
at 4 °C, and after being centrifuged at 10 000 g for 30 min,
they were dissolved in 10 mL of buffer A and dialysed
overnight against the same buffer The dialysate was then
applied to a DEAE-Sepharose Fast Flow column
(1.5 ´ 14 cm) equilibrated with buffer A, and the proteins
were eluted with a linear 0.1±0.3M NaCl gradient The
active fractions on tributyrin were ®nally applied to a
Superdex 200 HR gel column (1.0 ´ 30 cm) and eluted with
a 20-mMTris/HCl buffer pH 8.0 containing 0.35MNaCl,
at a ¯ow rate of 0.5 mLámin)1
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 0.35MNaCl in 20 mMTris/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 kDa) and isoelectric focusing calibration kit
(pH 4.5±9.6) were obtained from Bio-Rad Laboratories
The MW-GF 1000 kit (29±2000 kDa) from Sigma
Chem-ical Co, was used for the gel ®ltration procedure
Amino-acid composition and sequence determination
The amino-acid composition of the puri®ed PICE was
determined using a Waters chromatography system as
previously described [13,28], after 24 h hydrolysis in 6M
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 used as 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 bound fatty acids were released from the protein under alkaline conditions Ethanol containing 1MKOH was used and the protein solution was incubated at 80 °C for 1 h, and 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 14%
BF3in methanol [31] After the methylation, the fatty acids were identi®ed on a PerkinElmer gas-liquid chromatogra-phy autosystem XL equipped with PerkinElmer integrator 1022S, using n-heptadecanoic acid as an internal standard Cell cultures and labelling
Mature porcine enterocytes (16 ´ 106cellsámL)1) and hepatocytes (3.4 ´ 106cellsámL)1) were isolated as described by Bader et al [32] and by Seglen [33], respec-tively Prior to the labelling experiments, enterocytes and hepatocytes were incubated for 4 h at 37 °C in Williams E medium supplemented with 5% (v/v) fetal bovine serum, prednisolone (5 lmoláL)1), glucagon (0.014 lgámL)1), insulin (0.16 UámL)1), penicillin (200 UámL)1), streptomy-cin (200 lgámL)1) and 63 lCiámL)1of [9,10(n)-3H]myristic acid or [9,10(n)-3H]palmitic acid (speci®c activity
54 Ciámmol)1) Prior to use, the fetal bovine serum was delipidated using 1,2,2-trichloro-1,2,2-tri¯uoroethane [34]
At the end 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/Pi, homogenized, centrifugated at 10 000 g for 10 min at 4 °C, and the supernatant was sampled for analysis
R E S U L T S
Tissue distribution of pI 5.1 carboxylesterase The presence of the pI 5.1 CE isoform in 11 homogenates from various porcine tissues was checked by performing immunoblot analysis on the soluble extracts using puri®ed polyclonal antibodies directed against a synthetic amino-acid peptide corresponding to the amino-amino-acid sequence located between residues 281 and 296 in the PICE polypeptide chain Figure 1 shows that 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 that the enzyme was not detected in the soluble extracts of homogenates from colon, stomach, pancreas and kidney medulla, or in those from skin, bladder, tongue, trachea, brain medulla and cerebellum, heart, pharynx and suprarenals (data not shown) Esterase activity on tributyrin was observed only in the soluble
Trang 4fractions 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 in the colon could not be ruled out
Subcellular distribution of porcine intestinal
carboxylesterase
The distribution of PICE activity on tributyrin and that of
marker enzymes on their speci®c substrates in a number of
subcellular fractions from pig intestinal mucosa is given in
Table 1 Esterase activity on tributyrin was detected in four
subcellular fractions, and in all these fractions, immunoblot
analysis using the puri®ed polyclonal anti-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 fraction accounted for as much as 18% of the overall esterase activity on tributyrin, it was suggested that some of the PICE might correspond to
an enterocytic brush 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] A single 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 column, 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 these forms was found to be 180 kDa 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 of a single polypeptide chain corresponding to the monomeric form of PICE (F1) and giving rise to the noncovalent association of three and four apparently identical subunits corresponding to the F3 and F4 molecular forms of PICE, respectively
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 and F1 were found to have similar speci®c activity values on tributyrin as substrate ( 290 Uámg)1 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 tested as possible substrates at pH 8.0 (Table 2) The tetrameric,
Fig 1 Immunoblotting and esterase activity on tributyrin of pI 5.1 CE
from porcine tissues Esterase activity was measured as indicated in the
Materials and Methods section Total proteins (30 lg) present in
homogenates from 11 porcine tissues were electrophoresed in a 12%
SDS/PAGE, transferred onto a nitrocellulose membrane, and reacted
with polyclonal antibodies raised against the synthetic 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; lane 4, parotid; lane 5, submaxillary; lane 6, liver; lane 7,
pancreas; lane 8, kidney cortex; lane 9, kidney medulla; lane 10, lung;
lane 11, brain cortex.
Table 1 Subcellular localization of CE activity in porcine intestinal mucosa At each step of the subcellular fractionation procedure, the enzyme activities were measured in the pellet and the supernatant and expressed as a percentage of the total activity The values are means based on three separate subcellular fractionations.
Fraction Enzymatic markeractivities (%) Subcellularfraction Esterase activityon tributyrin (%) Immunoblotanalysis 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.
Trang 5trimeric and monomeric forms of PICE were found to display different enzymatic activities on these substrates Although F4 and F1 were equally active on tributyrin,
as already indicated in 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 F3 was the most active on butyrylcholine and the least active on tributyrin, and that p-nitrophenylacetate is apparently the most ef®cient substrate for PICE molecular forms in general N-Terminal amino-acid sequence and fatty acid content of PICE molecular forms
Table 3 gives the fatty acid content of the F4, F3 and F1 molecular forms of 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 detected, which strongly suggests that the polypeptide chain was
Fig 3 Polyacrylamide gel electrophoresis of PICE molecular forms (A) The electrophoresis 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 intestinal 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 buer
con-taining 0.35 M NaCl, pH 8.0 (B) The F4 molecular form was then
applied to a Superdex 200-HR column (1.0 ´ 30 cm) and eluted with
the above-mentioned buer Esterase activity on tributyrin was
assayed as indicated in Materials and methods Solid and dotted lines
represent the protein absorbance 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 Uámg protein )1 for both the F4 and F1 forms All the results are means on three enzymatic determinations.
Substrate Activitydetermination pH
Relative speci®c activity (%)
Trang 6blocked As the amino-acid composition of the three
molecular forms 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 PICE were found to have fairly similar FA pro®les
in sharp contrast 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 PICE
relative to the amount of protein deduced from its
amino-acid composition indicated that stoichiometric amounts of
myristic and palmitic acids were present in F1 (1 mol FA per
mol of F1) By contrast, less myristic 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 using 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 [3H]palmitic acid and [3H]myristic acid A single band corresponding to a protein with a molecular mass of 60 kDa was observed in both cases in the enterocytic cells The pattern of radioactivity in the gel slices showed a good correlation with the relative mobility of the immunoreactive PICE (Fig 4A) About a four-fold higher level of3H radioactivity was counted in the
Table 3 N-terminal amino-acid sequence and fatty acid content of PICE molecular forms.
Molecular
forms N-Terminalsequence
Fatty acid content
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 soluble proteins from enterocytes (A) and hepatocytes (B) incubated with [ 3 H]fatty acid 1, Ponceau red protein staining; 2, immunoblotting with polyclonal antibodies directed against the PICE peptide K 281 to E 296 (the arrow indi-cates the position of immunoreactive PICE) The relative mobilities of molecular mass markers in SDS/PAGE are indicated below the blot: (a) phosphorylase 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).
Trang 7PICE labelled with [3H]myristic acid than in that labelled
with [3H]palmitic acid, in agreement with the above ®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 [3H]palmitic
(2.6 ´ 103 d.p.m.) or [3H]myristic acids (2.1 ´ 103 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 cross-react with
PLCE, we extended the fatty acylation analysis to
hepato-cytes As shown in Fig 4B, a single band with an apparent
molecular mass of 60 kDa, corresponding to PLCE, was
revealed by the speci®c polyclonal antibodies and the
protein was labelled by myristic or palmitic acids Analysis
of the radioactivity patterns in the blot showed the
presence of a similar 3H content in PLCE, whether the
protein was labelled with [3H]palmitic acid or [3H]myristic
acid
D I S C U S S I O N
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 the digestive tract, namely the stomach 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 the analysis
However, the peptide extending from K281to E296in 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 brain [38] CE As esterase
activity on tributyrin was detected only in the small
intestine, liver, pancreas, where lipase activity is known to
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 corresponding to mitochondria, microsomes,
microvilli and cytosol Although the enzyme has previously
been found to contain the tetrapeptide HAEL at the
C-terminus of the polypeptide chain [7], which is thought to
serve as a retention signal for proteins on the luminal side of
the ER, it is apparently not retained in the ER Both the
immunoblot analysis and esterase activity on 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 therefore be isolated using a Superdex column, while the tetrameric form (240 kDa) which was isolated by gel ®ltration on Sephacryl S-200 column corresponded to that previously described by David et al [7] Hydrophobic interactions may contribute signi®cantly to the polymerization of PICE monomers, due
to the presence of covalently bound fatty acids, 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 from the Sephacryl S-200 column, in contrast to the pro®le of the Superdex column (Fig 2) The possibility that there may have been a difference in af®nity between the molecular 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 to have lipolytic activities [39,40] Whatever arguments may be put forward
to explain the existence of several molecular forms in PICE, the behaviour of this protein on Sephacryl S-200 is comparable to that of liver CE [8] but different from that
of rat intestinal 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 state of protonation of the histidine residue from the catalytic triad In addition, most of the F4 esterase activity on tributyrin at pH 8.0 was due to F1, and
as F3 was found to be threefold less active than both F4 and F1, the interactions between monomers in F4 and F3 were probably different, leading to distinct conformational states that did not display the same catalytic activity on tributyrin
Fig 5 A possible scheme for explaining the existence of PICE mono-meric and polymono-meric forms M and P stand for activated myristic acid and palmitic acid, respectively NMT, N-myritoyltransferase; PAT, palmitoylacyltransferase; and MPT, myritoylproteinthioesterase.
Trang 8Covalent changes in proteins with myristate have been
observed in several 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 consensus sequence, 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, no fatty acylation of CE has been reported to
occur so far The results of the present study clearly indicate
that PICE contained covalently bound fatty acids, and the
fact that acylation of the enzyme occurred was further
con®rmed using 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 about a quarter of that recorded
in palmitic acid The resistance of F1 to Edman degradation
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 fatty acid As we recently established
that Cys71 in the PICE amino-acid sequence could not be
alkylated with iodoacetamide, except in the presence of
100 mM 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 assumption
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 free amino group
containing an N-terminal glycine residue To answer the
question as to 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
experi-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 palmitoylated, the functional signi®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, anchoring, and crossing of the cellular
mem-branes, as suggested by the subcellular pattern of
distribu-tion of the enzyme observed here Moreover, PICE was
found in the present study to deacylate PMA (data not
shown), a structural analog of diacylglycerol, and to be
variably active on a number of 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]
A C K N O W L E D G E M E N T S
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 mass spectrometry determinations, respec-tively, Dr G Pieroni for fatty acids analysis, Dr V Girod for dissection
of pigs, and Dr J Blanc for revising the English manuscript.
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