Báo cáo khoa học: Role of the structural domains in the functional properties of pancreatic lipase-related protein 2 pot

Role of the structural domains in the functional properties
of pancreatic lipase-related protein 2
´
Amelie Berton, Corinne Sebban-Kreuzer and Isabelle Crenon
´
´
´
´
´
UMR, INSERM 476, INRA 1260, Universite de la Mediterranee, Nutrition Humaine et Lipides, Faculte de Medecine de la Timone,
Marseille, France

Keywords
chimera; colipase; domain; pancreatic lipase;
PLRP2
Correspondence
I. Crenon, UMR, 476 INSERM ⁄ 1260 INRA,
´
´
Faculte de Medecine, 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 dependence. 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 chromatography, 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 competition for colipase binding. Active site-directed inhibition experiments demonstrate 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.

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. proposed the classification of pancreatic lipases in three
subgroups: classic pancreatic lipase (PL), PLRP1 and
PLRP2. Numerous PLRP sequences have been identified 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 substrates. 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 substrates, only very low lipolytic activity against triglycerides has been reported with PLRP1 [1,4,14,15]. The

Abbreviations
E600, diethyl p-nitrophenyl phosphate; ho, horse; NaTDC, sodium taurodeoxycholate; PL, classic pancreatic lipase;
PLRP, pancreatic lipase-related protein.

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PLRP2 and colipase ) no interaction

PLRP2s 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 solubilized [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 tributyrin substrate, but is strongly inhibited on trioctanoin substrate [3]. Concerning the rat PLRP2, because
of contradictory results, no conclusions can be drawn
[4,23].
The three-dimensional structure resolution of pancreatic 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 particular 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 (residues 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 questioned 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
6012

A. Berton et al.

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 pancreatic lipases allowed the development of the domainexchange 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 important 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 investigated 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 constructions in the chimeric proteins and compared with
the properties of the native hoPL and hoPLRP2 proteins. 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

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A. Berton et al.

to construct plasmids expressing the two chimeric
cDNAs was to exchange the cDNA fragment encoding
the C-terminal domain of the two lipases between plasmids pVLhoPL and pAcGP67hoPLRP2, which carry
the cDNA of hoPL and hoPLRP2, respectively. This
exchange could be done because an Eag1 site was engineered 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-terminal domain of hoPL and of the C-terminal 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-terminal 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 proteinỈL)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).

PLRP2 and colipase ) no interaction

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 fragments of 27.5 and 22.5 kDa (Fig. 2A, lane 4), in agreement 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. Microsequencing of purified NcCc and NcC2 yielded the
N-terminal sequence NEVCY, corresponding to the
N-terminal sequence of the mature hoPL. The N-terminal sequence of N2C2 and N2Cc was ADLKE,
corresponding to the three terminal amino acid extension 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.

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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 different 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.

A

Kinetic properties of chimeric proteins ) effects
of bile salts and colipase

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

hoPL
hoPLRP2
NcCc
N2C2
NcC2
N2Cc

NEVCY
ADLKE
NEVCY
ADLKE
NEVCY
ADLKE

49
50
49
50
49
50

449
455
449
455
451
453

5.19
5.50
5.19
5.62
5.80
5.10

710.47
362.16

696.45
387.23
559.32
524.36

NcCc (Fig. 2B,C). Concerning the chimera, NcC2
was recognized better by hoPL antibodies than by
hoPLRP2 antibodies, and conversely, N2Cc was recognized 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
6014

The lipolytic activity of the different chimeric proteins
was investigated by titrimetry using emulsified tributyrin as substrate. In a first experiment, the assays were
performed in the absence of bile salts [sodium taurodeoxycholate (NaTDC)], in the absence or in the presence of colipase. As shown in Fig. 3, the kinetic rate
for NcCc (3000 mg)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 mg)1) and prevented this inactivation. This well-known phenomenon, named interfacial
inactivation, has been extensively described with several pancreatic classic lipases, and in particular with
hoPL [37]. The kinetic rate for N2C2 in the absence of
bile salt was constant (560 mg)1), as it was in the
absence and in the presence of colipase (Fig. 3). Similar 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 mg)1) and N2Cc
(650 mg)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 possessing the PL C-terminal domain are able to bind colipase.

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A. Berton et al.

PLRP2 and colipase ) no interaction

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)9 M), NcC2

(0.89 · 10)9 M), N2C2 (1.75 · 10)9 M) or
N2Cc (0.5 · 10)9 M) without (in black) and
with (in gray) colipase (5 · 10)9 M).

In a second experiment, the activities of the chimeras 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 inhibited the activity of NcCc (1500 mg)1 at 6 mm
NaTDC versus 3200 mg)1 at 0 mm NaTDC) and
N2C2 (300 mg)1 at 6 mm NaTDC versus
560 mg)1 at 0 mm NaTDC). In the presence of
colipase, only NcCc activity was increased, even in the

presence of bile salt (8000 mg)1 at 0.1 mm NaTDC
and 5600 mg)1 at 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 mg)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. 4. Bile salt and colipase dependence of
the chimeric protein activity. The assays
were done using 10)9 M 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.

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low NaTDC concentration (700 mg)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 mg)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 inactive 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)9 m) and
colipase (10)9 m) 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% inhibition 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 E600hoPL and N2Cc was abolished when an excess of colipase 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
6016

Fig. 5. Competition for colipase between PL and inactive or chimeric proteins. Colipase (10)9 M) was incubated with increasing
concentrations of inhibitor protein in the presence of a tributyrin

emulsion and bile salts at a final concentration of 4 mM. After
5 min, PL (10)9 M) was added. The activity was determined and
expressed as a percentage compared to the lipase activity measured in the absence of inhibitor protein. (A) E600-hoPL (d) and
E600-hoPLRP2 (.). (B) N2Cc (d) and NcC2 (.).

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 colipase 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

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PLRP2 and colipase ) no interaction

A. Berton et al.

Table 2. Influence of bile salt and colipase on the chimeric protein inhibition by E600. Chimeric proteins, at 2 · 10)6 M, were incubated in
the presence of E600 in the absence or in the presence of bile salt (NaTDC 0.5 mM or 4 mM) or colipase (10)5 M). T50% is the time needed
to reach 50% inhibition. ND, not determined.
T50% (min)
Without colipase

With colipase

Proteins

E600 (mM)

– NaTDC

0.5 mM NaTDC

4 mM NaTDC

– NaTDC

4 mM NaTDC

NcCc
N2C2
NcC2
N2Cc
NcCc
NcC2

0.05
0.05
0.05
0.05
2.5
2.5

> 1440

75
> 1440
90
> 1440
> 1440

> 1440
16
> 1440
30
ND
ND

> 1440
6
> 1440
3
> 1440
75

> 1440
70
> 1440
64
> 1440
> 1440

> 1440
7
> 1440

6
70
79

and 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 presence of bile salt alone (T50% ¼ 75 min), and the colipase 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 significant 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 concentrations 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 agreement with previous experiments 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 accessibility 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 inhibition of NcC2 and NcCc was 50 times higher than that
used for N2C2 and N2Cc. In conclusion, the accessibility of the active site was better in the protein bearing the N2 domain than in the protein bearing 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 contribution of the N-terminal and C-terminal domains to the
particular behavior of hoPLRP2. The structural organization of the pancreatic lipases is completely suitable

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PLRP2 and colipase ) no interaction

for the domain-exchange strategy, which has previously been used successfully in the study of the structure–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 contrast 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 Carriere 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 interface. 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 denaturation. The N-terminal domain of PLRP2 does not
possess this feature. PLRP2 is not sensitive to interfacial 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 smaller size [21]. Even if PL and PLRP2 hydrolyze triglycerides, 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
6018

A. Berton et al.

interfacial activation [3,9], and preferentially hydrolyzes triglycerides with short chains.
hoPLRP2 is characterized by a specific activity on
TC4 of about 600–700 mg)1 in the absence of bile
salts. The specific activity of hoPL is 8000 mg)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 mg)1 for N2 proteins
and 6000–8000 mg)1 for Nc proteins). In the presence 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 reactivated by colipase in the presence of bile salt, suggesting 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 residues 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 previously 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

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PLRP2 and colipase ) no interaction

A. Berton et al.

A

B

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.

obtained 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 mechanism of action of lipase. The motion of the flap makes
the active site accessible to the substrate, simultaneously forming a functional oxyanion hole and generating lipase interfacial binding. It has been previously
proposed from active site-directed inhibition experiments 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 ternary lipase–colipase–micelle complex of biliary compounds [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 inclusion 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 soluble or included in micelles, as was proposed by Reboul 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 substrates, and probably allows the interaction of the
enzyme with the substrate structure. Whether the
motion of the lid promotes the recognition of the substrate structure, or the recognition of this structure promotes 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 structures 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 position of the opened lid, whether it can be stabilized by

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6019


PLRP2 and colipase ) no interaction

colipase, and what the nature is of the amino acids
that correspond to the exposed surface. The superposition 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 interesting, 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 electron 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 analysis of this domain will provide new information to

enable a better understanding of the role of the C-terminal domain in the function of PLRP2, mainly with
regard to the orientation of the residues essential for colipase 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). Antibiotics 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 Biolabs (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

6020

A. Berton et al.

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 hoPLRP2 (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¢ primer, 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 underlined 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 introduced 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 95 °C, 1 min at 50 °C and 1 min
at 72 °C 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 BamH1 ⁄ 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) plasmid purification protocol, the different constructions were
used with linearized genomic DNA from Autographa

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A. Berton et al.

californica virus (BaculoGold DNA from the BaculoGold
transfection kit) for cotransfection into Sf21 insect cells as
described in the Baculovirus Expression Vector System
Manual (Pharmingen). The SF21 cells were grown in a
monolayer at 27 °C in tissue culture flasks using X-press
medium containing 5% fetal bovine serum, 50 UIỈmL)1
penicillin and 50 mgỈmL)1 streptomycin. Recombinant

viruses were purified by plaque assay and amplified by an
additional SF21 cell infection cycle. For the production of
the chimera, six 162 cm2 tissue culture flasks were seeded
with 6 · 107 cells per flask in complete X-press medium.
When the cells were attached, the complete medium was
removed and replaced with 20 mL of serum-free X-press
medium. The high-titer stock solutions of recombinant
baculoviruses were added to the cells at a multiplicity of
infection close to 2. In all cases, the chimeric proteins were
secreted into the culture media, as observed by electrophoresis on SDS ⁄ PAGE. After 5 days of culture at 27 °C, the cells
were pelleted by centrifugation at 900 g for 5 min 4 °C, and
the supernatants were kept at 4 °C for further purification.

Purification of chimeric protein
All the chimeric proteins were purified following the onestep procedure reported previously for the purification of
horse recombinant PLRP2 expressed in insect cells [8]. The
culture supernatants were dialyzed overnight at 4 °C
against 20 mm Tris ⁄ HCl buffer (pH 8) containing 1 mm
benzamidine and loaded onto a Q-sepharose Fast Flow column equilibrated in the same buffer. Elution was performed
using a linear NaCl concentration gradient (from 0 to
200 mm NaCl). The fractions were analyzed by measuring
the lipase activity and by SDS ⁄ PAGE. The fractions containing the protein of interest were pooled, dialyzed overnight at 4 °C against distilled water, lyophilized or not, and
kept at ) 20 °C. Native hoPL and native hoPLRP2 were
purified as previously described [8].

N-terminal sequence analysis
The purified chimeric proteins were submitted to N-terminal microsequencing. Stepwise Edman degradation was performed using an automatic sequencer, model Procise 494,
from Applied Biosystems (Foster City, CA, USA).

Gel electrophoresis and western blotting

Electrophoresis on 12% polyacrylamide gels was carried
out in the presence of SDS as described by Laemmli [46].
Western blots were performed according to Burnette [47].
After electrophoresis, proteins were transferred to a
polyvinylidene difluoride membrane. Membranes were
incubated with rabbit polyclonal antibodies raised against
either hoPL or hoPLRP2. The rabbit sera were used at a

PLRP2 and colipase ) no interaction

1 : 5000 dilution, and the reacting antibodies were detected
with goat anti-rabbit IgG conjugated with alkaline phosphatase at a 1 : 5000 dilution.

Activity measurements and protein assays
The lipase activity was measured titrimetrically at 25 °C
using emulsified 0.11 m tributyrin in 1 mm Tris ⁄ HCl buffer
(pH 7.5) containing 0.1 m NaCl and 5 mm CaCl2. The
assays were performed either in the absence or in the presence of bile salt (NaTDC) 0.1–4 mm and ⁄ or a five-fold
molar excess of colipase. One unit corresponds to the
release of 1 lmol fatty acidỈmin)1.
Protein concentrations were determined with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL,
USA).

Chimeric protein inhibition by E600
The inhibition experiments were performed in 50 mm
sodium acetate buffer (pH 6.0) containing 0.1 m NaCl. Proteins (2 · 10)6 m) were treated with E600 (0.05 or 2.5 mm
as indicated), either in the absence or in the presence of bile
salt and ⁄ or colipase (five-fold molar excess). The mixture
was incubated at 25 °C, and aliquots were withdrawn at
various time intervals and used to determine the remaining

lipase activity as described above. Control experiments were
also performed without E600 to check protein stability.

Competition experiments
The lipase activity was measured as described above in the
presence of 4 mm NaTDC, colipase and increasing concentrations of chimeric proteins. The lipase and colipase concentrations were 10)9 m. The colipase and the chimeric
proteins were added at the beginning of the test, and the
lipase was added during the test. Under these conditions, the
lipase activity was determined and expressed as a percentage
of the lipase activity measured in the absence of chimeric
protein. Native hoPLRP2 and hoPL previously inactivated
by E600 as described in [32] were used as controls.

Acknowledgements
This research was supported by grants from the Insti´
´
tut National de la Sante et de la Recherche Medicale
and from the Institut National de la Recherche Agron´
omique. The PhD work of Miss Amelie Berton was
supported by a grant from Institut National de la
Recherche Agronomique and ARILAIT RECHER´
CHE Industry. We thank Regine Lebrun and Danielle
Moinier for performing the sequence analyses, and
Mouhcine Louaste for helpful technical assistance. We

FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS. No claim to original French government works

6021

PLRP2 and colipase ) no interaction

thank Dr Catherine Chapus for helpful advice and
fruitful discussions. We are very grateful to Dr Franc¸
oise Guerlesquin for critical reading of the manuscript.

References
1 Giller T, Buchwald P, Blum-Kaelin D & Hunziker W
(1992) Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2. Differences in colipase
dependence and in lipase activity. J Biol Chem 267,
16509–16516.
2 Kerfelec B, LaForge KS, Puigserver A & Scheele G
(1986) Primary structures of canine pancreatic lipase
and phospholipase A2 messenger RNAs. Pancreas 1,
430–437.
3 Hjorth A, Carriere F, Cudrey C, Woldike H, Boel E,
Lawson DM, Ferrato F, Cambillau C, Dodson GG,
Thim L et al. (1993) A structural domain (the lid) found
in pancreatic lipases is absent in the guinea pig (phospho) lipase. Biochemistry 32, 4702–4707.
4 Payne RM, Sims HF, Jennens ML & Lowe ME (1994)
Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development. Am J Physiol 266, G914–G921.
5 Wicker-Planquart C & Puigserver A (1992) Primary
structure of rat pancreatic lipase mRNA. FEBS Lett
296, 61–66.
6 Remington SG, Lima PH & Nelson JD (1999) Pancreatic lipase-related protein 1 mRNA in female mouse lacrimal gland. Invest Ophthalmol Vis Sci 40, 1081–1090.
7 Wishart MJ, Andrews PC, Nichols R, Blevins GT Jr,
Logsdon CD & Williams JA (1993) Identification and
cloning of GP-3 from rat pancreatic acinar zymogen
granules as a glycosylated membrane-associated lipase.
J Biol Chem 268, 10303–10311.

8 Jayne S, Kerfelec B, Foglizzo E, Chapus C & Crenon I
(2002) High expression in adult horse of PLRP2 displaying a low phospholipase activity. Biochim Biophys
Acta 1594, 255–265.
9 Thirstrup K, Verger R & Carriere F (1994) Evidence
for a pancreatic lipase subfamily with new kinetic properties. Biochemistry 33, 2748–2756.
10 Grusby MJ, Nabavi N, Wong H, Dick RF, Bluestone
JA, Schotz MC & Glimcher LH (1990) Cloning of an
interleukin-4 inducible gene from cytotoxic T lymphocytes and its identification as a lipase. Cell 60, 451–459.
11 Sias B, Ferrato F, Pellicer-Rubio MT, Forgerit Y, Guillouet P, Leboeuf B & Carriere F (2005) Cloning and
seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma. Biochim Biophys
Acta 1686, 169–180.
12 Fauvel J, Bonnefis MJ, Sarda L, Chap H, Thouvenot
JP & Douste-Blazy L (1981) Purification of two lipases
with high phospholipase A1 activity from guinea-pig
pancreas. Biochim Biophys Acta 663, 446–456.

6022

A. Berton et al.

13 De Caro J, Sias B, Grandval P, Ferrato F, Halimi H,
Carriere F & De Caro A (2004) Characterization of
pancreatic lipase-related protein 2 isolated from human
pancreatic juice. Biochim Biophys Acta 1701, 89–99.
14 Crenon I, Foglizzo E, Kerfelec B, Verine A, Pignol D,
Hermoso J, Bonicel J & Chapus C (1998) Pancreatic
lipase-related protein type I: a specialized lipase or an
inactive enzyme. Protein Eng 11, 135–142.
15 De Caro J, Carriere F, Barboni P, Giller T, Verger R &
De Caro A (1998) Pancreatic lipase-related protein 1

(PLRP1) is present in the pancreatic juice of several
species. Biochim Biophys Acta 1387, 331–341.
16 Lowe ME (2002) The triglyceride lipases of the pancreas. J Lipid Res 43, 2007–2016.
17 Borgstrom B (1975) On the interactions between pancreatic lipase and colipase and the substrate, and the
importance of bile salts. J Lipid Res 16, 411–417.
18 Brockman H (2000) Kinetic behavior of the pancreatic
lipase–colipase–lipid system. Biochimie 82, 987–995.
19 Andersson L, Carriere F, Lowe ME, Nilsson A & Verger R (1996) Pancreatic lipase-related protein 2 but not
classical pancreatic lipase hydrolyzes galactolipids.
Biochim Biophys Acta 1302, 236–240.
20 Sias B, Ferrato F, Grandval P, Lafont D, Boullanger P,
De Caro A, Leboeuf B, Verger R & Carriere F (2004)
Human pancreatic lipase-related protein 2 is a galactolipase. Biochemistry 43, 10138–10148.
21 Reboul E, Berton A, Moussa M, Kreuzer C, Crenon I
& Borel P (2006) Pancreatic lipase and pancreatic
lipase-related protein 2, but not pancreatic lipase-related
protein 1, hydrolyze retinyl palmitate in physiological
conditions. Biochim Biophys Acta 1761, 4–10.
22 Sebban-Kreuzer C, Deprez-Beauclair P, Berton A &
Crenon I (2006) High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia
pastoris. Protein Expr Purif 49, 284–291.
23 Jennens ML & Lowe ME (1995) Rat GP-3 is a pancreatic lipase with kinetic properties that differ from colipase-dependent pancreatic lipase. J Lipid Res 36, 2374–
2382.
24 Winkler FK, D’Arcy A & Hunziker W (1990) Structure
of human pancreatic lipase. Nature 343, 771–774.
25 Bourne Y, Martinez C, Kerfelec B, Lombardo D, Chapus C & Cambillau C (1994) Horse pancreatic lipase.
The crystal structure refined at 2.3 A resolution. J Mol
Biol 238, 709–732.
26 van Tilbeurgh H, Egloff MP, Martinez C, Rugani N,
Verger R & Cambillau C (1993) Interfacial activation of

the lipase–procolipase complex by mixed micelles
revealed by X-ray crystallography. Nature 362, 814–820.
27 van Tilbeurgh H, Sarda L, Verger R & Cambillau C
(1992) Structure of the pancreatic lipase–procolipase
complex. Nature 359, 159–162.
28 Hermoso J, Pignol D, Kerfelec B, Crenon I, Chapus C
& Fontecilla-Camps JC (1996) Lipase activation by

FEBS Journal 274 (2007) 6011–6023 Journal compilation ª 2007 FEBS. No claim to original French government works


A. Berton et al.

29

30

31

32

33

34

35

36
37

nonionic detergents. The crystal structure of the porcine
lipase–colipase–tetraethylene glycol monooctyl ether
complex. J Biol Chem 271, 18007–18016.
Pignol D, Hermoso J, Kerfelec B, Crenon I, Chapus C
& Fontecilla-Camps JC (1998) The lipase ⁄ colipase complex is activated by a micelle: neutron crystallographic
evidence. Chem Phys Lipids 93, 123–129.
Roussel A, de Caro J, Bezzine S, Gastinel L, de Caro
A, Carriere F, Leydier S, Verger R & Cambillau C
(1998) Reactivation of the totally inactive pancreatic
lipase RP1 by structure-predicted point mutations.
Proteins 32, 523–531.
Roussel A, Yang Y, Ferrato F, Verger R, Cambillau C
& Lowe M (1998) Structure and activity of rat pancreatic lipase-related protein 2. J Biol Chem 273, 32121–
32128.
Jayne S, Kerfelec B, Foglizzo E, Granon S, Hermoso J,
Chapus C & Crenon I (2002) Activation of horse
PLRP2 by bile salts does not require colipase.
Biochemistry 41, 8422–8428.
Crenon I, Jayne S, Kerfelec B, Hermoso J, Pignol D &
Chapus C (1998) Pancreatic lipase-related protein type
1: a double mutation restores a significant lipase activity. Biochem Biophys Res Commun 246, 513–517.
Bezzine S, Roussel A, de Caro J, Gastinel L, de Caro
A, Carriere F, Leydier S, Verger R & Cambillau C
(1998) An inactive pancreatic lipase-related protein is
activated into a triglyceride-lipase by mutagenesis
based on the 3-D structure. Chem Phys Lipids 93,
103–114.
Carriere F, Thirstrup K, Hjorth S, Ferrato F, Nielsen
PF, Withers-Martinez C, Cambillau C, Boel E, Thim L
& Verger R (1997) Pancreatic lipase structure–function

relationships by domain exchange. Biochemistry 36,
239–248.
Yang Y & Lowe ME (2000) The open lid mediates pancreatic lipase function. J Lipid Res 41, 48–57.
Gargouri Y, Bensalah A, Douchet I & Verger R (1995)
Kinetic behaviour of pancreatic lipase in five species
using emulsions and monomolecular films of synthetic
glycerides. Biochim Biophys Acta 1257, 223–229.

PLRP2 and colipase ) no interaction

38 Wong H, Davis RC, Nikazy J, Seebart KE & Schotz
MC (1991) Domain exchange: characterization of a
chimeric lipase of hepatic lipase and lipoprotein lipase.
Proc Natl Acad Sci USA 88, 11290–11294.
39 Rietsch J, Pattus F, Desnuelle P & Verger R (1977)
Further studies of mode of action of lipolytic enzymes.
J Biol Chem 252, 4313–4318.
40 Chahinian H, Bezzine S, Ferrato F, Ivanova MG, Perez
B, Lowe ME & Carriere F (2002) The beta 5¢ loop of
the pancreatic lipase C2-like domain plays a critical role
in the lipase–lipid interactions. Biochemistry 41, 13725–
13735.
41 Miled N, Berti-Dupuis L, Riviere M, Carriere F &
Verger R (2003) In vitro lipolysis by human pancreatic
lipase is specifically abolished by its inactive forms.
Biochim Biophys Acta 1645, 241–246.
42 Ayvazian L, Kerfelec B, Granon S, Foglizzo E, Crenon
I, Dubois C & Chapus C (2001) The lipase C-terminal
domain. A novel unusual inhibitor of pancreatic lipase
activity. J Biol Chem 276, 14014–14018.

43 Rouard M, Sari H, Nurit S, Entressangles B & Desnuelle P (1978) Inhibition of pancreatic lipase by mixed
micelles of diethyl p-nitrophenyl phosphate and bile
salts. Biochim Biophys Acta 530, 227–235.
44 Mancheno JM, Jayne S, Kerfelec B, Chapus C, Crenon
I & Hermoso JA (2004) Crystallization of a proteolyzed
form of the horse pancreatic lipase-related protein 2:
structural basis for the specific detergent requirement.
Acta Crystallogr D Biol Crystallogr 60, 2107–2109.
45 Freie AB, Ferrato F, Carriere F & Lowe ME (2006)
Val-407 and Ile-408 in the beta5¢-loop of pancreatic
lipase mediate lipase–colipase interactions in the presence of bile salt micelles. J Biol Chem 281, 7793–7800.
46 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
47 Burnette WN (1981) ‘Western blotting’: electrophoretic
transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated
protein A. Anal Biochem 112, 195–203.

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