In agreement with biochemical data, these structures demonstrate the functional organization of the PL into two structural domains: a large N-terminal domain, which contains the active s
Trang 1of pancreatic lipase-related protein 2
Ame´lie Berton, Corinne Sebban-Kreuzer and Isabelle Crenon
UMR, INSERM 476, INRA 1260, Universite´ de la Me´diterrane´e, Nutrition Humaine et Lipides, Faculte´ de Me´decine de la Timone,
Marseille, France
In 1992, Giller et al isolated mRNA coding for two
novel human pancreatic lipase-related proteins
(PLRPs) showing a high level of identity with the
human classic pancreatic lipase [1] On the basis of
amino acid sequence comparisons, Giller et al
pro-posed the classification of pancreatic lipases in three
subgroups: classic pancreatic lipase (PL), PLRP1 and
PLRP2 Numerous PLRP sequences have been
identi-fied in several species by isolating mRNA [2–11]
Furthermore, by using classic protein purification
procedures, the presence of PLRP1 and⁄ or PLRP2 has
been demonstrated in the pancreas or in the pancreatic
juice from different species and also in other secretions [8,11–15]
PLRP and PL differ in enzymatic properties such as substrate specificity, sensitivity to inhibition by bile salts and colipase dependence [16] Pancreatic lipases are highly active and selective for triglyceride sub-strates Under physiological conditions, the PL activity
is dependent on the presence of colipase, which able to overcome the inhibitory effect of bile salts [17,18] Despite extensive studies on a large variety of sub-strates, only very low lipolytic activity against triglyce-rides has been reported with PLRP1 [1,4,14,15] The
Keywords
chimera; colipase; domain; pancreatic lipase;
PLRP2
Correspondence
I Crenon, UMR, 476 INSERM ⁄ 1260 INRA,
Faculte´ de Me´decine, 27 Boulevard
Jean-Moulin, 13385 Marseille Cedex 5,
France
Fax: +33 4 91 78 21 01
Tel: +33 4 91 29 41 10
E-mail: Isabelle.Crenon@medecine.
univ-mrs.fr
(Received 7 August 2007, revised 10
September 2007, accepted 1 October 2007)
doi:10.1111/j.1742-4658.2007.06123.x
Although structurally similar, classic pancreatic lipase (PL) and pancreatic lipase-related protein (PLRP)2, expressed in the pancreas of several species, differ in substrate specificity, sensitivity to bile salts and colipase depen-dence In order to investigate the role of the two domains of PLRP2 in the function of the protein, two chimeric proteins were designed by swapping the N and C structural domains between the horse PL (Nc and Cc domains) and the horse PLRP2 (N2 and C2 domains) NcC2 and N2Cc proteins were expressed in insect cells, purified by one-step chromatogra-phy, and characterized NcC2 displays the same specific activity as PL, whereas N2Cc has the same as that PLRP2 In contrast to N2Cc, NcC2 is highly sensitive to interfacial denaturation The lipolytic activity of both chimeric proteins is inhibited by bile salts and is not restored by colipase Only N2Cc is found to be a strong inhibitor of PL activity, due to compe-tition for colipase binding Active site-directed inhibition experiments dem-onstrate that activation of N2Cc occurs in the presence of bile salt and does not require colipase, as does PLRP2 The inability of PLRP2 to form
a high-affinity complex with colipase is only due to the C-terminal domain Indeed, the N-terminal domain can interact with the colipase PLRP2 properties such as substrate selectivity, specific activity, bile salt-dependent activation and interfacial stability depend on the nature of the N-terminal domain
Abbreviations
E600, diethyl p-nitrophenyl phosphate; ho, horse; NaTDC, sodium taurodeoxycholate; PL, classic pancreatic lipase;
PLRP, pancreatic lipase-related protein.
Trang 2PLRP2s are distinguishable from classical lipases by
their substrate specificity, because, besides triglycerides,
they are able to hydrolyze phospholipids, galactolipids
and esters of vitamins [3,8,9,19,20] Moreover, the
activities of PL and PLRP2 seem to be different
according to the vehicles in which the substrate is
solu-bilized [21]
Concerning the effect of bile salts and colipase on
the activity of PLRP2, there is no clear conclusion,
because the results appear to change according to the
different species and seem to depend on the triglyceride
substrate and the bile salt [9,16] Indeed, some of them,
such as horse PLRP2 and human PLRP2, are inhibited
on tributyrin substrate by the presence of bile salts,
and this inhibition is only slightly overcome or not
overcome by the presence of an excess of colipase
[8,13,22] Another PLRP2 group including guinea pig
and coypu PLRP2s is affected neither by the bile salt
concentration nor by the addition of colipase on
tri-butyrin substrate, but is strongly inhibited on
trioctan-oin substrate [3] Concerning the rat PLRP2, because
of contradictory results, no conclusions can be drawn
[4,23]
The three-dimensional structure resolution of
pancre-atic lipases provides important information concerning
the structure–function relationship of the PL [24–28]
In agreement with biochemical data, these structures
demonstrate the functional organization of the PL into
two structural domains: a large N-terminal domain,
which contains the active site with the catalytic triad
formed by Ser152, Asp176 and His263, and a smaller
C-terminal domain, which is important for colipase
binding In the inactivated state, the PL catalytic site is
inaccessible to substrate, being covered by a surface
loop called the lid domain (residues 237–261) In
partic-ular conditions, the lid must move to accommodate a
lipid substrate The closed PL conformation converts
into the open form upon interaction with lipid [26] The
principal elements that undergo space reorganization
during the activation of the enzyme are the lid
(resi-dues 238–262) and loop b5 of the N-terminal domain
(residues 77–86) The functional consequences of the
structural reorganization are as follows: (a) the active
site is accessible to the substrate; (b) the oxyanion hole
is created; (c) an important hydrophobic surface is
formed; and (d) a new binding site is generated between
the colipase and the open lid Some studies have
ques-tioned whether lipase activation is even interfacial in
the presence of bile salt and colipase, on the basis of
attaining an activated ternary complex of PL, colipase
and a small micelle in the absence of any interface [29]
Three-dimensional structures of canine PLRP1 and
rat PLRP2 have also been reported [30,31] These data
indicate that, as predicted by high primary structure homology, the three-dimensional structure of the PLRP members can be superimposed on that of PL, which cannot explain the particular features of the PLRPs Indeed, they possess an N-terminal domain with the same catalytic triad, a C-terminal domain in which the residues implicated in colipase binding are conserved, and a lid domain (except for guinea pig PLRP2, which has a naturally truncated lid [3]), which must move during the activation process Previous data indicate that the motion of the PLRP2 lid is dependent on the presence of bile salts and does not require the presence of colipase [32]
The two-domain structural organization of the pan-creatic lipases allowed the development of the domain-exchange strategy to provide further insights into the structure–function relationships of pancreatic lipases [14,33–36] These studies show that the lid domain alone is responsible neither for the substrate selectivity nor for the activation process They did not show whether the PLRP2 C-terminal domain could or could not bind colipase The differences in kinetic properties
of the various PLRP2s imply that these proteins should not be grouped together and that it is impor-tant to obtain new information about the properties of PLRP2 family members
In the present study, we produced, purified and characterized chimeric proteins designed by N-terminal and C-terminal domain exchange between horse PLRP2 (hoPLRP2) and horse PL (hoPL) in order to investigate the role of the two domains in the function
of hoPLRP2 The influence of bile salt and colipase on the lipase activity of the different chimeras was investi-gated using tributyrin as substrate Experiments were performed to investigate active site-directed inhibition and competition for colipase binding The properties
of the chimeras were compared to those of the original proteins bearing the modifications induced by the con-structions in the chimeric proteins and compared with the properties of the native hoPL and hoPLRP2 pro-teins This work provided new information about the ability of the hoPLRP2 C-terminal domain to bind colipase and the respective contribution of each PLRP2 domain to the activation process, the substrate specificity and the interfacial stability of this protein
Results
Expression and purification of chimeric proteins Chimeric proteins designed by domain exchange between hoPL and hoPLRP2 were constructed and expressed in insect cells The strategy that we followed
Trang 3to construct plasmids expressing the two chimeric
cDNAs was to exchange the cDNA fragment encoding
the C-terminal domain of the two lipases between
plas-mids pVLhoPL and pAcGP67hoPLRP2, which carry
the cDNA of hoPL and hoPLRP2, respectively This
exchange could be done because an Eag1 site was
engi-neered in each plasmid at the junction between the
N-terminal and C-terminal domain sequences of each
protein The chimeric protein composed of the
N-ter-minal domain of hoPL and of the C-terN-ter-minal domain
of hoPLRP2 was named NcC2 Conversely, the other
chimeric protein, bearing the N-terminal domain of
hoPLRP2 and the C-terminal domain of hoPL, was
named N2Cc This construction procedure induced
substitutions in the amino acid sequence of each
C-ter-minal domain, as shown in Fig 1 To ensure that these
substitutions did not influence the behavior of the
C-terminal domain as compared to the wild-type
proteins, we expressed NcCc and N2C2 as controls
The chimeric proteins were expressed in insect cells
using the Baculovirus Expression System The four
proteins were secreted into the culture medium with
yields reaching 10–40 mg of recombinant proteinÆL)1
After 5 days of culture, the secreted proteins were
purified from the dialyzed supernatant by a one-step
anionic exchange chromatography procedure, with a
recovery yield of 50% The four purified recombinant
proteins were analyzed and compared to native hoPL
and hoPLRP2 by SDS⁄ PAGE followed by Coomassie
blue staining (Fig 2A) or western blot (Fig 2B,C)
In the absence of dithiothreitol in the sample buffer, native hoPL and hoPLRP2 ran as a single band of about 50 kDa (Fig 2A, lanes 1 and 2) In the presence
of dithiothreitol in the sample buffer, in contrast to hoPL (Fig 2A, lane 3), hoPLRP2 ran as two frag-ments of 27.5 and 22.5 kDa (Fig 2A, lane 4), in agree-ment with previous results demonstrating the high sensitivity of the hoPLRP2 Ser244–Thr245 bond to proteolytic cleavage [32] The four chimeric proteins ran as a major single band with a molecular mass of about 50 kDa (Fig 2A, lanes 5–8) Nevertheless, the two chimeras bearing the N2 domain (N2C2, lane 6, and N2Cc, lane 8) had a slightly higher molecular mass than the chimera bearing the Nc domain (NcCc, lane 5, and NcC2, lane 7) according to the theoretical value, as indicated in Table 1 Micro-sequencing of purified NcCc and NcC2 yielded the N-terminal sequence NEVCY, corresponding to the N-terminal sequence of the mature hoPL The N-ter-minal sequence of N2C2 and N2Cc was ADLKE, corresponding to the three terminal amino acid exten-sion resulting from the construction strategy, followed
by the N-terminal sequence of the mature hoPLRP2 These results indicated that the cleavage of the signal sequence by the insect signal peptidase was correct Despite crossreactions due to the strong homology between the two proteins, hoPL antibodies recognized hoPL and NcCc better than hoPLRP2 and N2C2, and conversely, hoPLRP2 antibodies reacted better with hoPLRP2 and N2C2 than they did with hoPL and
Fig 1 Functional maps of the plasmids
expressing natural and chimeric isoforms of
hoPLRP2 and hoPL A novel EagI site was
engineered (see Experimental procedures).
Above each plasmid map, the nucleotide
and amino acid sequences of the region at
the junction between the two protein
domains are reported The EagI site is
underlined.
Trang 4NcCc (Fig 2B,C) Concerning the chimera, NcC2
was recognized better by hoPL antibodies than by
hoPLRP2 antibodies, and conversely, N2Cc was
recog-nized better by hoPLRP2 antibodies than by hoPL
antibodies This suggests that both antibodies were
preferentially raised against the N-terminal domain of
the respective proteins We observed slight sensitivity
of N2C2 and N2Cc to proteolytic cleavage generating one fragment detected by hoPLRP2 antibodies and corresponding to the larger proteolytic fragment of native hoPLRP2 (Fig 2C, lanes 6 and 8) Using differ-ent preparations of N2C2 and N2Cc, we checked that this proteolysis did not have an effect on either the activity or the behavior of the proteins
Kinetic properties of chimeric proteins) effects
of bile salts and colipase The lipolytic activity of the different chimeric proteins was investigated by titrimetry using emulsified tributy-rin as substrate In a first experiment, the assays were performed in the absence of bile salts [sodium tauro-deoxycholate (NaTDC)], in the absence or in the pres-ence of colipase As shown in Fig 3, the kinetic rate for NcCc (3000 UÆmg)1) rapidly decreased in the absence of colipase and bile salts NcCc was probably irreversibly inactivated at the surface of tributyrin droplets Prior addition of colipase enhanced the kinetic rate (7200 UÆmg)1) and prevented this inactiva-tion This well-known phenomenon, named interfacial inactivation, has been extensively described with sev-eral pancreatic classic lipases, and in particular with hoPL [37] The kinetic rate for N2C2 in the absence of bile salt was constant (560 UÆmg)1), as it was in the absence and in the presence of colipase (Fig 3) Simi-lar data were observed with hoPLRP2 [8] These results indicated that NcCc and N2C2 behaved like native hoPL and hoPLRP2, respectively [8,35,37] Thus, the modifications introduced at the junction between the N-terminal and C-terminal domains as compared to the native proteins influence neither their stability nor their activity
In the absence of bile salts and colipase, the kinetic rates of both NcC2 (6000 UÆmg)1) and N2Cc (650 UÆmg)1) decreased, and this decrease was even more rapid for NcC2 (Fig 3) These results indicated that both the N-terminal and C-terminal domains of hoPLRP2 contributed to the stability of the protein in the presence of the water–lipid interface Also, both the N-terminal and C-terminal domains of hoPL were involved in the inactivation of the protein at the water–triglyceride interface The inactivation of N2Cc
in the absence of bile salts was prevented by prior addition of colipase, suggesting that N2Cc was able to bind the colipase In contrast, the inactivation of NcC2 was not prevented by prior addition of colipase, suggesting that NcC2 was not able to bind colipase These results indicated that only the proteins possess-ing the PL C-terminal domain are able to bind coli-pase
A
B
C
Fig 2 Analysis of purified protein by SDS ⁄ PAGE 12% Coomassie
blue staining (A) and western blots using hoPL antibodies (B) and
hoPLRP2 antibodies (C) Lanes 1 and 2: protein migration without
dithiothreitol Lanes 3–8: protein migration with dithiothreitol.
Lanes 1 and 3: hoPL Lanes 2 and 4: hoPLRP2 Lane 5: NcCc.
Lane 6: N2C2 Lane 7: NcC2 Lane 8: N2Cc.
Table 1 Theorical biochemical properties of the chimeric proteins.
Proteins
N-terminal
sequence
Molecular mass (Da)
Amino acids
Isoelectric point
Trang 5In a second experiment, the activities of the
chime-ras were tested on emulsified tributyrin in the presence
of increasing concentrations of bile salts (0–6 mm), in
the absence or in the presence of colipase As seen in
Fig 4, increasing the concentration of NaTDC
inhib-ited the activity of NcCc (1500 UÆmg)1 at 6 mm
NaTDC versus 3200 UÆmg)1 at 0 mm NaTDC) and
N2C2 (300 UÆmg)1 at 6 mm NaTDC versus
560 UÆmg)1 at 0 mm NaTDC) In the presence of
colipase, only NcCc activity was increased, even in the
presence of bile salt (8000 UÆmg)1 at 0.1 mm NaTDC and 5600 UÆmg)1at 6 mm NaTDC) These results were similar to those obtained for the native proteins [8,35,37] The activity of NcC2 was slightly increased
at a very low NaTDC concentration (8000 UÆmg)1 at 0.1 mm NaTDC) and inhibited when the NaTDC concentration increased The inhibitory effect was complete for NaTDC concentrations above 2 mm The colipase was not able to restore the NcC2 activity For N2Cc, a slight activator effect was observed at a very
Fig 3 Kinetics of hydrolysis of tributyrin by
chimeric proteins without bile salt and in
presence or absence of colipase Lipolytic
activity was measured titrimetrically at
pH 7.5 with NcCc (0.99 · 10)9M ), NcC2
(0.89 · 10)9M ), N2C2 (1.75 · 10)9M ) or
N2Cc (0.5 · 10)9M ) without (in black) and
with (in gray) colipase (5 · 10)9M ).
Fig 4 Bile salt and colipase dependence of
the chimeric protein activity The assays
were done using 10)9M each lipases in the
pH-stat at various concentrations of NaTDC
and in the absence (in black) or presence (in
gray) of a 5 M excess of colipase.
Trang 6low NaTDC concentration (700 UÆmg)1 at 0.1 mm
NaTDC) An inhibitory effect then appeared, increased
up to the NaTDC critical micellar concentration, and
stabilized at a plateau value corresponding to
400 UÆmg)1 Interestingly, the colipase failed to restore
the maximal activity for N2Cc The colipase effect on
the lipase activity in the presence of bile salt depended
not only on the presence of the classic C-terminal
domain, but also on the nature of the N-terminal
domain
Inhibition of the PL by the chimeras
In the presence of a supramicellar concentration of
NaTDC (4 mm), PL needs the colipase to develop its
full activity In the same conditions, the N2Cc and
NcC2 activities were inhibited and not restored in the
presence of colipase (see above) The influence of
increasing concentrations of NcC2, N2Cc, hoPL
inac-tive forms [diethyl p-nitrophenyl phosphate
(E600)-hoPL] and hoPLRP2 inactive forms (E600-hoPLRP2)
on the native PL activity was investigated In these
experiments, the concentrations of lipase (10)9m) and
colipase (10)9m) were constant Inactive forms of
hoPL and hoPLRP2 were prepared as previously
described [32] using high concentrations of E600,
which covalently binds to the active site serine As
shown in Fig 5A, E600-hoPL was found to be an
excellent inhibitor of the lipase activity, as 50%
inhibi-tion was obtained with an [E600-hoPL]⁄ [PL] molar
ratio of 0.5 Only 18% of residual activity remained
when E600-hoPL was used at a molar excess of 2
Interestingly, no inhibition of the lipase test activity
was observed when E600-hoPLRP2 was added, even at
a molar excess of 1800 As shown in Fig 5B, the
inhibitory effect with N2Cc was similar to that of
E600-hoPL Fifty per cent inhibition was obtained
with an [N2Cc]⁄ [PL] molar ratio of about 0.5, and
complete inhibition was observed when N2Cc was used
at a molar excess of 10 The inhibitory effect of
E600-hoPL and N2Cc was abolished when an excess of
coli-pase was added during the assay, and was observed
only in the presence of NaTDC (data not shown) In
the case of NcC2, no inhibition of the lipase activity
was observed, as 100% of the lipase activity still
remained even at a molar excess of 45 The effect of
NcC2 was similar to that of E600-hoPLRP2 The same
results were obtained using human or porcine lipase
and colipase
The proteins bearing the C-terminal domain of PL
were efficient inhibitors of the lipase activity, whereas
the proteins bearing the C-terminal domain of PLRP2
had no effect on the lipase activity The inhibitory
effect of E600-hoPL and N2Cc is probably due to competition for colipase binding These data suggested that the C-terminal domain of PL is able to bind coli-pase whatever the nature of the N-terminal domain Moreover, the C-terminal domain of hoPLRP2 was not able to bind colipase even in the presence of the
PL N-terminal domain
Influence of NaTDC and colipase on chimera inhibition by E600
The activation of the pancreatic lipase is a mechanism allowing accessibility of the active site to the substrate
Fig 5 Competition for colipase between PL and inactive or chime-ric proteins Colipase (10)9M ) was incubated with increasing concentrations of inhibitor protein in the presence of a tributyrin emulsion and bile salts at a final concentration of 4 m M After
5 min, PL (10)9M ) was added The activity was determined and expressed as a percentage compared to the lipase activity mea-sured in the absence of inhibitor protein (A) E600-hoPL (d) and E600-hoPLRP2 (.) (B) N2Cc (d) and NcC2 (.).
Trang 7and resulting in the unmasking of the catalytic triad of
the enzyme induced by the motion of the flap The
accessibility of the active site can be tested using the
ability of an organophosphate, E600, to react with
the active site serine only when the enzyme adopts an
opened flap conformation E600 inhibition experiments
were carried out to investigate the influence of NaTDC
and colipase on the activation of the chimeric proteins
Table 2 shows T50%, corresponding to the time needed
to reach 50% inhibition
With 0.05 mm E600, no inhibition was observed for
NcCc and NcC2 At 2.5 mm E600, inhibition of NcCc
activity was observed after incubation in the presence
of bile salt and colipase (T50%¼ 70 min) In contrast,
inhibition of NcC2 activity was observed in the
pres-ence of bile salt alone (T50%¼ 75 min), and the
coli-pase addition had no effect on the rate of inhibition
In the absence of bile salt, noticeable inhibition
of N2C2 by 0.05 mm E600 was observed (T50%¼
75 min), and the addition of colipase had no
signifi-cant effect (T50%¼ 70 min) In contrast, the rate of
inhibition was significantly increased in the presence of
NaTDC monomers (T50%¼ 16 min) At NaTDC
con-centrations beyond the critical micellar concentration,
the rate of inhibition of N2C2 increased (T50%¼
6 min) The addition of colipase still had no significant
influence (T50%¼ 7 min) These results were in
agree-ment with previous experiagree-ments on inhibition by E600
performed on native hoPLRP2 [32]
Significant inhibition by E600 was observed for
N2Cc in the absence of colipase and bile salt (T50%¼
90 min) The rate of inhibition was increased in the
presence of NaTDC, concentrations beyond the critical
micellar concentration having a higher efficiency than
the monomer concentration (T50%¼ 3 min versus
T50%¼ 30 min) The addition of colipase alone had
a slight influence on N2Cc inhibition, as the rate of
inhibition was increased (T50%¼ 64 min versus
T50%¼ 90 min), in contrast to N2C2
Blank experiments performed in the absence of E600 showed that, in any case, proteins retained at least 85% of activity after 24 h, indicating that the enzymes were stable under the conditions used for the study These results indicated that the NcCc active site was accessible to high E600 concentrations only in the presence of colipase and bile salt, whereas the accessi-bility of the NcC2 active site depended only on the presence of bile salt The accessibility of the N2C2 and N2Cc active sites to E600 was possible even in the absence of colipase and bile salt, and was considerably increased by the presence of bile salt Therefore, the concentration of E600 needed to obtain clear inhibi-tion of NcC2 and NcCc was 50 times higher than that used for N2C2 and N2Cc In conclusion, the accessi-bility of the active site was better in the protein bear-ing the N2 domain than in the protein bearbear-ing the Nc domain Thus, the accessibility of the active site in the N2 proteins was independent of the nature of the C-terminal domain, in contrast to the situation with
Nc proteins Indeed, the C2 domain induced sensitivity
of the Nc active site to E600 inhibition in the presence
of bile salt
Discussion
Despite their structural similarities, the PLRP2s form
a subfamily that is clearly distinct from the classic lipase subfamily, notably concerning their functional properties Moreover, considerable variability is observed among the members of the PLRP2 subfamily The aim of our study was to investigate the contribu-tion of the N-terminal and C-terminal domains to the particular behavior of hoPLRP2 The structural orga-nization of the pancreatic lipases is completely suitable
Table 2 Influence of bile salt and colipase on the chimeric protein inhibition by E600 Chimeric proteins, at 2 · 10)6M , were incubated in the presence of E600 in the absence or in the presence of bile salt (NaTDC 0.5 m M or 4 m M ) or colipase (10)5M ) T 50% is the time needed
to reach 50% inhibition ND, not determined.
Proteins E600 (m M )
T50%(min)
Trang 8for the domain-exchange strategy, which has
previ-ously been used successfully in the study of the
struc-ture–function relationships of different lipases [35,38]
Chimeric proteins, named NcC2 and N2Cc, were
designed by N and C structural domain exchange
between hoPL (Nc and Cc domains) and hoPLRP2
(N2 and C2 domains) NcC2 and N2Cc were produced
as secreted proteins and purified Their properties were
compared to those of NcCc and N2C2, corresponding
to the original proteins PL and PLRP2, respectively,
bearing the modifications induced by the construction
in the chimeric proteins These modifications have no
effects on the behavior of the proteins [8,35,37]
The kinetic characterizations of proteins in the
absence of bile salts and colipase show that, in
con-trast to N2C2, the NcCc, NcC2 and N2Cc proteins
undergo irreversible inactivation, which is thought to
result from denaturation of these enzymes in the lipid–
water interface [39] These observations underline the
fact that the proteins possessing at least one of the two
domains of hoPL are more sensitive to interfacial
denaturation The involvement of the C-terminal
domain in the interfacial denaturation of the PL was
already proposed by Carrie`re et al [35] Indeed, these
authors showed that, in the absence of bile salts, a
chimeric protein composed of the N-terminal domain
of guinea pig PLRP2 and of the C-terminal domain of
human PL (gpN2⁄ huCc) was inactivated at the
inter-face Moreover, it was reported that the C-terminal
domain of PL bound efficiently to a triglyceride–water
interface and was an absolute requirement for possible
interfacial binding of PL [40] In the case of the
gpN2⁄ huCc chimera, the rate of denaturation was
higher, indicating that the C-terminal domain of hoPL
is less sensitive to the interface than that of huPL In
the present work, the similarities between NcCc and
NcC2 with regard to the rate of inactivation show for
the first time the dominant role of the N-terminal
domain of PL in the phenomenon of interfacial
dena-turation The N-terminal domain of PLRP2 does not
possess this feature PLRP2 is not sensitive to
interfa-cial denaturation; either the two domains confer high
stability on the lipid–water interface, or PLRP2 has no
affinity for the lipid–water interface We recently
showed that PL and PLRP2 hydrolyzed retinyl esters
Moreover, PL preferentially hydrolyzed the substrate
when it was included in droplets, and PLRP2 was
more efficient when it was included in micelles of
smal-ler size [21] Even if PL and PLRP2 hydrolyze
triglyce-rides, it is probable that the physical property of the
substrate is specific for each enzyme: droplet for PL,
and a water-soluble structure for PLRP2 It has also
been previously reported that PLRP2 does not display
interfacial activation [3,9], and preferentially hydro-lyzes triglycerides with short chains
hoPLRP2 is characterized by a specific activity on TC4 of about 600–700 UÆmg)1 in the absence of bile salts The specific activity of hoPL is 8000 UÆmg)1 (in the presence of colipase) In the absence of bile salts, the proteins containing the same N-terminal domain show a similar specific activity on tributyrin (in the presence of colipase) (500–700 UÆmg)1for N2 proteins and 6000–8000 UÆmg)1 for Nc proteins) In the pres-ence of bile salts, we observed that the behavior of chimeric proteins is also related to the nature of the N-terminal domain Indeed, the specific activities of NcCc and NcC2 are very strongly decreased, whereas those of N2C2 and N2Cc are much less sensitive to the inhibitory effect of bile salts This indicates that in the absence or in the presence of bile salts, the specific activity of hoPL and hoPLRP2 depends on the nature
of their N-terminal domain
In contrast to NcCc, NcC2 was not protected from the interfacial denaturation by colipase and not reacti-vated by colipase in the presence of bile salt, suggest-ing that NcC2 is not able to form a stable complex with colipase Competition experiments on colipase binding reveal that NcC2, like hoPLRP2, is a very bad competitor This observation indicates that NcC2 and N2C2 do not bind well to colipase, probably due to the C2 domain However, as shown in Fig 6, the resi-dues of the C-terminal domain involved in the primary interaction of PL with colipase are preserved in the C-terminal domain of hoPLRP2 (Asn366, Gln369, Lys400) It is possible that these residues are not in an ideal conformation to allow either binding to colipase
or correct orientation of colipase, in particular for stabilizing the lid Although N2Cc activity was not restored by colipase in the presence of bile salt, there are substantial arguments in favor of N2Cc–colipase complex formation N2Cc is protected from interfacial denaturation by colipase and behaves as an excellent inhibitor of colipase binding Indeed, the [N2Cc]⁄ [lipase] molar ratio needed to obtain 50% inhibition is the same as that found with E600-hoPL competitor or with other inactive forms of PL used as competitors
by Miled et al [41] This result indicates that N2Cc binds to colipase as well as hoPL Experiments previ-ously carried out with the C-terminal domain of PL as inhibitor showed that a [C-terminal domain]⁄ [lipase] molar ratio of 1000 was needed to give 50% inhibition [42] It was assumed that the whole lipase is a better inhibitor than the C-terminal domain alone, because new interactions, which stabilize the lipase–colipase complex, were created between colipase and the lid
of lipase in the opened conformation The results
Trang 9obtained with N2Cc as a competitor mean that the
C-terminal domain in this context is able to bind
colipase, but especially that the complex formed would
probably be stabilized by the open lid of the N2 domain
The movement of the lid making it possible to adopt
an open conformation is a crucial stage in the
mecha-nism of action of lipase The motion of the flap makes
the active site accessible to the substrate,
simulta-neously forming a functional oxyanion hole and
gener-ating lipase interfacial binding It has been previously
proposed from active site-directed inhibition
experi-ments with an organophosphate that the accessibility of
the active site of pancreatic lipase, in the absence of
interface, could be obtained in the presence of colipase
and bile salts, probably through the formation of a
ter-nary lipase–colipase–micelle complex of biliary
com-pounds [29] The same type of experiment indicates
that the lid of PLRP2 is already more mobile than that
of PL, and especially that it moves and uncovers the
active site in the presence only of the bile compounds
[32] The accessibility of E600 to the N2 active site is
considerably increased in the presence of bile salts,
which masks the slight influence of colipase observed
with N2Cc in the absence of bile salt The unmasking
of the active site of the Nc domain absolutely requires
colipase and bile salts in micellar concentrations in the
presence of the Cc domain and bile salt only in the
presence of the C2 domain The inhibition of the active
site serine by E600 needs both the motion of the flap
and the recognition of the vehicle in which E600 was
solubilized It was clearly established that soluble E600
can be included in bile salt micelles, and that this
inclu-sion is a prerequisite for inhibition of PL [43] Our
work indicates that E600 included in bile salt micelles is
a better inhibitor of the N2 active site than of the Nc
active site This observation supports the idea that PLRP2 preferentially hydrolyzes substrates that are sol-uble or included in micelles, as was proposed by Re-boul et al [21] Nevertheless, the mechanism of activation of PLRP2, which involves the C2 domain, is different from that of PL, which involves the Cc domain and colipase The C-terminal domain alone is involved in the affinity of the PLRP2 for micellar sub-strates, and probably allows the interaction of the enzyme with the substrate structure Whether the motion of the lid promotes the recognition of the sub-strate structure, or the recognition of this structure pro-motes the displacement of the lid, is still questionable Three structures of PLRP2 are now available in the Protein Data Bank: rat PLRP2 (Protein Data Bank code 1BU8 [31], human PLRP2 (Protein Data Bank code 2OXE, to be published), and hoPLRP2 (Protein Data Bank code 1W52 [44]) These three PLRP2 struc-tures are comparable to the hoPL structure in the closed conformation, or to the porcine PL structure in the opened conformation [25,26] With respect to the conformation of loop b5 of the N-terminal domain, hoPLRP2 and human PLRP2 are probably in the opened conformation, in contrast to rat PLRP2, which
is in the closed conformation For the human and rat proteins, it is not possible to draw conclusions about the exact position of the lid, because a sequence of approximately 20 amino acids is missing In the case
of hoPLRP2, the lid is partially opened (Fig 6) A comparison of the exposed surface between PLRP2 and PL in the opened conformation would explain the difference in behavior between PL and PLRP2 with respect to the interface The resolution of the structure
of N2Cc would be very useful to determine the posi-tion of the opened lid, whether it can be stabilized by
Fig 6 (A) Amino acid sequence comparison between hoPLRP2 and hoPL The residues of the catalytic triad are in red, the lid sequence is
in blue, and the amino acids of the C-terminal domain involved in colipase binding are in green (B) Superimposition of hoPLRP2 (1W52) and hoPL (1HPL) Ca traces are displayed in red and blue, respectively.
Trang 10colipase, and what the nature is of the amino acids
that correspond to the exposed surface The
superposi-tion of hoPL and hoPLRP2 (Fig 6) shows that
loop b5¢ of the C-terminal domain (residues 405–414)
is oriented differently This observation is very
interest-ing, because this loop was shown to play an important
role in lipase function and could influence the binding
of colipase [45] No conclusion is possible about the
orientation of this b5¢ loop in the other PLRP2s, as
this fragment was found to have no interpretable
elec-tron density
In conclusion, the studies on the functional properties
of the two structural N-terminal and C-terminal
domains of hoPLRP2 show that the enzyme stability in
the presence of the lipid–water interface, the motion of
the lid and the substrate specificity are properties that
are mainly related to the nature of the N-terminal
domain On the other hand, PLRP2 is not able to form
a stable complex with colipase, and its C-terminal
domain is responsible for this feature Structural
analy-sis of this domain will provide new information to
enable a better understanding of the role of the
C-termi-nal domain in the function of PLRP2, mainly with
regard to the orientation of the residues essential for
col-ipase binding and the behavior of PLRP2 towards the
lipid–water interface or water-soluble micelles These
structural data will be very important to determine the
real contribution of PLRP2 to intestinal lipolysis
Experimental procedures
Reagents
The BaculoGold Starter Package pVL1393 and pAcGP67
transfer vectors were purchased from Pharmingen (San
Diego, CA) X-Press medium and fetal bovine serum were
supplied by BioWhittaker (Walkersville, MD, USA)
Anti-biotics were obtained from Invitrogen (Carlsbad, CA, USA)
Alkaline phosphatase-labeled goat anti-(rabbit IgG), E600,
tributyrin and NaTDC were purchased from Sigma-Aldrich
(St Louis, MO, USA) Taq polymerase, restriction enzymes
and T4 DNA ligase were purchased from New England
Bio-labs (Ipswich, MA, USA) or Eurogentec (Seraing, Belgium)
Construction of chimeric proteins
The constructions encoding the chimeric proteins composed
of a PL domain and a PLRP2 domain are described in
Fig 1 First, pVLhoPL, previously described, resulted in
the integration of the nucleotide sequence encoding hoPL,
including the peptide signal, at the EcoR1 restriction site of
pVL1393 transfer vector Thereafter, an Eag1 restriction
site was introduced by site-directed mutagenesis at the
junction between the N-terminal and C-terminal domain sequences (named Nc and Cc, respectively) that induced the substitution A337G [14] The resulting vector pVLNcCc encoded the protein named NcCc
The N-terminal and C-terminal domain sequences of hoP-LRP2 (named N2 and C2, respectively) were amplified by PCR using pAcGP67hoPLRP2, previously described [8], as template This plasmid resulted in the insertion of the mature hoPLRP2 sequence into the BamH1⁄ EcoR1 restriction site
of the pAcGP67 transfer vector, downstream of the signal sequence of the baculovirus glycoprotein GP67 The two oligonucleotides 5¢-N2 (5¢-GGAATTCAGATCTCAAAGA GGTTTGCTATACCCC-3¢) and 3¢-N2 (5¢-CCCGGCCG TAGTCACCACTTTCTCC-3¢) were used as 5¢ and 3¢ pri-mer, respectively, to amplify the N2 sequence The sequences
in bold correspond to the Bgl2 restriction site for primer 5¢-N2 and Eag1 restriction site for primer 3¢-N2 The under-lined sequences in the primers correspond to the sequences encoding the first and the last residues of N2, respectively
To amplify the C2 sequence, the two oligonucleotides (5¢-C2) 5¢-CCCGGCCGTTGGAGATATAGAGTATC-3¢ and (3¢-C2) 5¢-GGTTCTTGCCGGGTCCCCAGG-3¢ were used The sequence in bold corresponds to the Eag1 restriction site The sequence in italic corresponds to the sequence encoding the first residue of C2 The 3¢-C2 primer corresponds to the end of the multiple cloning site of the pAcGP67 vector The underlined sequence corresponds to the substitutions intro-duced in the C2 domain as compared to the wild-type PLRP2 The PCR reactions were carried out under standard conditions, with 0.5 min at 95C, 1 min at 50 C and 1 min
at 72C for 25 cycles After the PCR reaction, the N2 and C2 PCR fragments were purified and digested by Bgl2–Eag1 and by Eag1, respectively, and introduced into the Bam-H1⁄ Eag1 restriction site of the pAcGP67 transfer vector The resulting construction pAcN2C2 encoded the protein named N2C2 NcCc and N2C2 were used as controls
The pVLNcCc and pAcN2C2 vectors were digested by Eag1 and subjected to electrophoresis on polyacrylamide gel, and the Eag1 fragments were electroeluted The small Eag1 fragments corresponding to the Cc and C2 domains were interchanged and cloned in the Eag1 pAcN2 and pVLNc fragments, respectively The resulting constructions pVLNcC2 and pAcN2Cc encoded the chimeric proteins NcC2 and N2Cc All the constructions were propagated in the JM101 Escherichia coli strain and checked by DNA sequencing carried out by Genome Express (Grenoble, France)
Expression of chimeric proteins using the Baculovirus Expression System
After purification using the Qiagen (Hilden, Germany) plas-mid purification protocol, the different constructions were used with linearized genomic DNA from Autographa