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J Biol Chem, Vol. 273, Issue 48, 32121-32128, November 27, 1998
From the § Departments of Pediatrics and of Molecular
Biology and Pharmacology, Washington University School of Medicine, St.
Louis, Missouri 63110, The pancreas expresses several members of the
lipase gene family including pancreatic triglyceride lipase (PTL) and
two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Despite their similar amino acid sequences, PTL,
PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile
acid inhibition, colipase requirement, and interfacial activation. To
begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 Å structure of
rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a Lipases are ubiquitous enzymes expressed by diverse
organisms. They hydrolyze phospholipids and triglycerides to generate fatty acids for energy production or for storage and to release inositol phosphates that act as second messengers. The role of phospholipases in cellular signaling pathways has increased interest in
these lipases. Similarly, the central role of triglyceride lipases in
energy production and their potential industrial applications have
stimulated studies of these essential lipases. As a result, our
knowledge about lipases and of the molecular details underlying lipolysis has increased considerably.
Among these contributions was the cloning of cDNAs encoding various
lipases. Comparisons of the amino acid sequences predicted from these
cDNAs led to the hypothesis that a lipase gene family evolved from
a common ancestral hydrolase (1). At least three members of the lipase
gene family are synthesized and secreted by the pancreas. One, the
archetype of the family, colipase-dependent pancreatic
triglyceride lipase (PTL)1
has been studied for over 100 years. Giller et al. (2)
isolated human pancreatic cDNAs encoding the other two, named
pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2), 6 years
ago. The primary sequences of the human PLRP1 and PLRP2 have 68 and 65% identity to the primary sequence of PTL with conservation of the
catalytic triad and major determinants of tertiary structure. Subsequently, other groups reported the presence of related proteins in
the pancreas from several species (3-7).
The best studied of the PTL homologues is PLRP2. These studies provided
the first indications that PLRP2 has functional properties different
from those of PTL. Mouse PLRP2 was cloned from interleukin-4-stimulated cytotoxic T-lymphocytes, and rat (r)PLRP2 was cloned as a zymogen granule membrane protein, GP3 (4, 7). The presence of PLRP2 in
lymphocytes and on the zymogen granule membrane raised the possibility
that PLRP2 has functions other than hydrolyzing dietary fats. For
instance, lymphocyte PLRP2 may participate in cell killing, and the
PLRP2 on the zymogen granule membrane may mediate granule fusion with
the plasma membrane.
The expression and purification of PLRP2 lipases allowed the enzymatic
properties of these enzymes to be characterized. These studies revealed
that PLRP2 lipases have enzymatic properties that distinguish them from
PTL (4, 5, 7-9). First, PLRP2 has a broader substrate specificity and
will hydrolyze triglycerides, phospholipids, and galactolipids. PTL
hydrolyzes only triglycerides. Second, they have different behaviors in
the presence of bile salts and with colipase. Third, PLRP2 members
efficiently hydrolyze monomers of water-soluble, short chain
triglycerides, whereas PTL possesses low activity against monomeric
substrates. PTL activity increases dramatically against water-insoluble
substrates presenting an oil-water interface, a property known as
interfacial activation. Clearly, the explanation for these kinetic and
functional differences must lie in the structure of these proteins.
The first pancreatic lipase structure to be solved was that of the
human enzyme (hPTL) (10). Subsequently, the complex of hPTL with
porcine colipase was elucidated, as were the structures of hPTL-porcine
colipase in complex with phospholipid or phosphonate inhibitors
(10-13). The structures of horse PTL, hPTL-human colipase complex, and
porcine PTL-porcine colipase complex have also been determined
(14-16). The structures of hPTL-porcine colipase crystallized in the
presence of mixed phospholipid/bile salt micelles or of a C11
phosphonate inhibitor revealed that the lid domain (residues 237-261)
covering the active site of hPTL could move away from its closed
position (12, 13). The movement creates new contacts with colipase to
form the lipid-water interfacial binding site. Another structural
element, the Comparisons of PLRP2 and PTL family members requires high resolution
crystal structures, but no structure of a true PLRP2 is available.
Withers-Martinez et al. (38) reported the crystal structure
of a chimeric lipase with the C-terminal domain of human PTL and the
N-terminal domain of guinea pig PLRP2 (gPLRP2). Although inferences
about the conformation of PLRP2 can be made from the known PTL and
gPLRP2 structure, valid conclusions must be based on the actual PLRP2
structure. In this paper, we report the first crystal structure of a
PLRP2 family member that has a lid domain and further characterize the
enzymatic properties of this lipase.
Expression of Rat PLRP2 in Sf9 Cells--
We expressed
recombinant rat PLRP2 in baculovirus-infected Sf9 cells as
described previously with the following modifications (3, 8). The
protein was produced in 1-liter spinner flasks containing 350 ml of
serum-free medium, EX-CELL 400 (JRH Biosciences, Lenexa, KS), instead
of 1 liter of medium. We harvested the medium 3 days instead of 4 days
post-infection. The smaller medium volume and shorter culture times
gave higher protein yields as initially observed by Bezzine et
al. (17).
Purification of Rat PLRP2--
We removed cells and debris by
centrifuging the medium at 5000 rpm for 10 min in a Beckman J2-21
centrifuge with a JA-20 rotor. The medium was concentrated over a Pall
Filtron 10k Ultrasette membrane (Filtron Technology Corporation,
Northborough, MA) to about 50 ml and dialyzed by repeated dilution with
10 mM Tris-HCl, pH 8.0, and concentrating over the
Ultrasette membrane. We applied the sample to a 75-ml bed volume
DEAE-Blue-Sepharose (Bio-Rad) equilibrated in the Tris buffer. PLRP2
was eluted with a linear NaCl gradient from 0.0 to 0.6 M.
Assay with tributyrin in a pH-stat located the PLRP2. We pooled the
peak fractions and dialyzed against 10 mM MES, pH 6.2, buffer followed by concentration over an Amicon YM30 membrane (Amicon,
Inc., Beverly, MA). The sample was applied to a Pharmacia Mono-S column
(5-ml bed volume) (Amersham Pharmacia Biotech) attached to an Akta
Purifier (Amersham Pharmacia Biotech). The column was equilibrated in
50 mM MES, pH 6.2, and eluted with a linear NaCl gradient
from 0.0 to 1.0 M. rPLRP2 eluted from the column as a
symmetrical peak identified by activity against tributyrin. The peak
fractions were pooled, and the pH was adjusted to 8.0 with 1 M Tris-Cl, pH 8.0. The purified protein migrated as a
single band on 10% SDS-polyacrylamide gel electrophoresis and had
activity against tributyrin, trioctanoin, triolein, and
phosphotidylcholine as described previously (8). rPLRP2 concentrations
were determined by the BCA protein assay using purified bovine serum
albumin as the standard.
Lipase Assays--
1,2-rac-Didecanoyl glycerol (dicaprin) was
purchased from Sigma. 1,2-sn-Didodecanoyl
phosphatidylcholine, 1,2-sn-didodecanoyl phosphatidylethanolamine, and 1,2-sn-didodecanoyl
phosphatidylglycerol were purchased from Fluka (Paris, France).
3-Monogalactosyl-1,2-rac-didodecanoyl glycerol was prepared by chemical
synthesis and is a generous gift from Professor G. C. Ortaggi
(Roma). A rPLPR2 solution 0.5 mg/ml was used for kinetic experiments
using the monolayer technique as well as for the interfacial activation
experiments, using tripropionin as substrate. Bulk phase assays were
done by the pH-stat method as described (5, 8, 18). The conditions for
inhibitor assays are given in the figure legends. Tetrahydrolipstatin
was kindly provided by Dr. Hans Lengsfeld from Hoffmann-LaRoche.
Kinetic Experiments on Monolayers--
Before each utilization,
the Teflon trough used to form the monomolecular film was cleaned with
water, then gently brushed in the presence of distilled ethanol, washed
again with tap water, and finally rinsed with double-distilled water
(19, 20). The aqueous subphase was composed of 10 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 21 mM
CaCl2, and 1 mM EDTA for all lipases. The
buffer was prepared with double-distilled water and filtered through a
0.45-µm Millipore filter. Any residual surface-active impurities were removed before each assay by sweeping and suction of the surface. Kinetic experiments were performed with a KSV-2200 barostat
(KSV-Helsinki) and a "zero-order" Teflon trough (20). The trough
was equipped with a mobile Teflon barrier, which was used to compensate
for the substrate molecules removed from the film by enzyme hydrolysis (monodecanoyl glycerol, decanoic acid, lyso-dodecanoyl phospholipids, and dodecanoic acid are soluble in water), thereby keeping the surface
pressure constant. The latter was measured using a Wilhelmy plate
(perimeter 3.94 cm) attached to an electromicrobalance, which was
connected in turn to a microprocessor controlling the movement of the
mobile barrier. The reactions were performed at room temperature
(20 °C). The subphase of the reaction compartment was continuously
agitated with a 2.0-cm magnetic stirrer moving at 250 rpm. The rPLRP2
solution (10-60 µl at 0.5 mg/ml) was injected through the film over
the stirrer with a Hamilton syringe. The surface area of the reaction
compartment was 31 cm2, and the volume was 55 ml. The
length of the reservoir compartment was 30 cm, and the width was 17.6 cm.
Interfacial Activation of rPLRP2--
The TC3
solutions were systematically prepared by mixing three times 30 s
in a Waring blender, a given amount of TC3 in 15 ml of 1%
gum arabic in water (w/w) (21). Before each assay, 5 ml of the
TC3 gum arabic solution was added to 10 ml of pure water in
the thermostated (37 °C) pH-stat vessel. Deionized water, purified
with a Millipore Super Q system, was used throughout all the
experiments. Lipase activity was recorded with either a TTT 80 pH-stat
(Radiometer) equipped with a 250-µl syringe containing 0.1 N NaOH or a VIT 90 pH-stat (Radiometer) equipped with an
automatic burette containing 0.05 M NaOH. Activity was
measured potentiometrically at pH 7.0, because at pH 8.0 the
spontaneous hydrolysis of TC3 reaches relatively high
levels. The assay was carried out on a mechanically stirred solution of
substrate in the reaction vessel. Spontaneous hydrolysis was recorded
in the pH-stat mode for 2 min before lipase injection, and this
background value was subtracted from the activity measurement. One
international lipase unit is the amount of enzyme catalyzing the
release of 1 µmol fatty acid/min. Each assay contained a 5-fold molar
excess of pure colipase. We checked that bovine serum albumin (final
concentration, 1%) had no detectable catalytic activity on
a TC3 solution (7.7 mM) or on a TC3
emulsion (15.33 mM). Interfacial activation assays with p-nitrophenylbutyrate were done as described (22).
Crystallization, X-ray Data Collection, and
Processing--
Small crystals of rPLRP2 were obtained at room
temperature using the hanging drop vapor diffusion method, by mixing 2 µl of protein (18 mg/ml in 0.2 M NaCl, 0.1 M
Tris-HCl, pH 8.5) and 2 µl of the Hampton screen 1 solution 36 (Hampton Research, Laguna Hills, CA) containing 8%
polyethylene glycol 8000 and 0.1 M Tris-HCl, pH 8.4. Crystals were improved by diminishing the polyethylene glycol and
protein concentrations by a factor of two. Larger crystal were obtained
by macroseeding using a 4+4 µl mixture and 2% polyethylene glycol 8000.
The crystals were soaked in a synthetic mother liquor containing 33%
ethylene glycol as cryoprotectant and were subsequently cryocooled
using the Oxford equipment (Oxford Cryosystems, Oxford, UK). X-ray
diffraction data were collected to 1.80 Å resolution on a 30-cm
Mar-research imaging plate at 0.970 Å wavelength on beamline DW32 in
LURE (Orsay, France). The data were processed using the DENZO software
package (23). The rPLRP2 crystallizes in the space group
P21 with cell dimensions a = 57.4 Å,
b = 79.1 Å, c = 60.9 Å, and Structure Determination--
The structure was solved with the
molecular replacement method using the AMoRe program (25).
The closed form of the classical human pancreatic lipase was used as
the search model. The rotation function of the two bodies, performed
with the N-terminal domain without the lid and the C-terminal domain,
yielded only one significant solution for the entire molecule
(correlation coefficient of 0.50 and R-factor of 0.39 between 10.0 and
3.5 Å resolution). The structure was refined using the X-PLOR
program (26). After performing 12 cycles of slow cooling
protocol starting at 2000 K and manual replacement and adjustments
using the Turbo-Frodo program, the R-factor had decreased from 45 to
24.6% (R-free 31.8%) (27). The water molecules located in the
(2Fo Overall Structure of rPLRP2--
The structure of rPLRP2 has been
refined at the highest resolution observed among the pancreatic
lipases. The current model consists of 3505 protein atoms, one GlcNAc
connected to Asn334, one calcium ion, seven molecules of
ethylene glycol, and 295 water molecules. One protein segment, located
between residues 405 and 411, was found to have no interpretable
electron density and was therefore removed from the model. In the
Ramachandran plot (Procheck software) of the final model, all of the
main chain dihedral angles but one (Ser152) fell within the
allowed regions (90.5% in the most favorable regions, and 9.3% in
additional allowed regions) (28). The active site Ser152
has the
The rPLRP2 structure belongs to the
The structure of rPLRP2 has been superimposed on all the known
pancreatic lipase structures: hPTL, hPTL bound to porcine colipase (hPTL-pCol), hPL-pCol in complex with a C11 phosphonate inhibitor (hPTL-pCol-C11), horse pancreatic lipase, gPLRP2, dog pancreatic PLRP1,
and porcine pancreatic lipase bound to porcine colipase (pPTL-pCol).
The N-terminal catalytic domains of the various molecules superimpose
well (within 1.0 Å), apart from the mobile loops (the lid and the Active Site Structure of rPLRP2--
We have proposed a model for
the putative binding of a triglyceride at the hPTL-pCol-C11 active site
crevice, based on the hPTL-pCol-C11 complex (13). One acyl chain of the
triglyceride, the leaving fatty acid, was assumed to bind at the
position of the C11 conformer 1, whereas a second acyl chain was taken
to bind at the position of the C11 conformer 2 (Fig.
3A). We superimposed and
compared the closed structure of rPLRP2 with the open structure of
hPTL-pCol-C11, to investigate, on the basis of our model, whether the
binding of a triglyceride to rPLRP2 was compatible with the active site
structure. To make these comparisons valid, a model of the open rPLRP2
has been built. The lid and the
hPTL has two colipase binding sites. One is located at the C-terminal
domain and was found to bind colipase when the catalytic domain is in
the closed or in the open conformation (11). A second site appears only
when lipase opens, yielding an interaction of colipase with the open
lid (12). We have investigated the likelihood of both sites with a
model of open PLRP2. The three lid domain residues involved in the
interaction with colipase are conserved. Among the 12 C-terminal
residues interacting with colipase, only four are substituted compared
with hPTL: Phe360
Finally, the biantennary saccharide is located at position 334, between
the N- and the C-terminal domains, on the enzyme face opposite to the
catalytic center. Despite this location, it should not interfere with
lid opening or with colipase binding.
Interfacial Activation--
Because it was previously reported
that rPLRP2 and cPLRP2 displayed no interfacial activation, we expected
structural differences between hPTL and rPLRP2, particularly in the lid
domain (5, 8). The results contradicted our expectations because the
lid domain was neither disordered nor in an open conformation. In fact,
the closed position of the lid in rPLRP2 closely resembled the position
of the lid domain in PTL (10). The good electron density and closed
conformation suggested that rPLRP2 should show interfacial activation.
Because of our findings, we reexamined rPLRP2 for interfacial
activation using the recently validated method with tripropionin (21,
32). The use of tripropionin overcomes the difficulties that accompany
the poor water solubility of tributyrin, the substrate utilized in
previous studies of PLRP2 and interfacial activation (5, 6, 8). The
experiment was replicated independently in two different laboratories
under slightly different conditions (Fig. 3, A and
B). At concentrations below the solubility limit of
tripropionin, rPLRP2 had little activity. The activity increased considerably above the solubility limit of the substrate. Similar kinetics were found with another substrate,
p-nitrophenylbutyrate, that has been used to demonstrate
interfacial activation for other lipases (Fig. 3C). These
results clearly show that rPLRP2 possesses interfacial activation
preferring aggregated substrates over monomeric substrates as does PTL.
The observation of interfacial activation on tripropionin restores the
validity of the classical explanation: closed lid means interfacial
activation. Although open lids or disordered lid structure have been
observed in other lipases, even in the absence of inhibitor, and appear
to violate this principle, these structures were obtained in the
presence of less polar solvent or in detergent, which may simulate an
interface (33-36).
Inhibition of rPLRP2 by E600 and Tetrahydrolipstatin--
The
conformation of the rPLRP2 active site and the conserved Ser-His-Asp
catalytic triad suggested that it should be inhibited by lipase
inhibitors like E600 (diethyl p-nitrophenyl phosphate) and
tetrahydrolipstatin (37). To determine whether these compounds inhibit
rPLRP2, we incubated the lipase with both inhibitors for various
lengths of time and measured activity against tributyrin (Fig.
4). Both inhibitors effectively reduced
the activity of rPLRP2. The inhibition suggests that the catalytic
mechanism of rPLRP2 is similar to that of PTL. If PLRP2 participates in
fat digestion, its activity should be effectively decreased by
tetrahydrolipstatin. Finally, these results suggest that obtaining the
structure of rPLRP2 in the open form may be possible in the presence of
an inhibitor as previously done with PTL.
Substrate Specificity--
Bulk phase assays had previously
demonstrated that PLRP2 lipases have a broader substrate specificity
than does PTL (5, 6, 8). We extended these observations by measuring
activity against various substrates using the monolayer technique (Fig. 5). We compared the activities against
three different phospholipase substrates,
1,2-didodecanoyl-phosphatidylcholine,
1,2-didodecanoylphosphatidylethanolamine, and
1,2-didodecanoylphosphatidylglycerol; one lipase substrate, 1,2-dicaprin; and one galactolipase substrate,
monogalactosyldiglyceride. This is the first use of a galactolipid
substrate in the monolayer assay. Furthermore, we compared the activity
of rPLRP2 to those of gPLRP2, cPLRP2, and hPTL. All four lipases had
activity against 1,2-dicaprin. hPTL was not active against the
phospholipid or galactolipid substrates. In contrast, all three of
thePLRP2 lipases showed activity against phospholipids as previously
reported. Like cPLRP2, rPLRP2 shows a clear preference for
1,2-didodecanoylphosphatidylethanolamine and
1,2-didodecanoylphosphatidylglycerol over
1,2-didodecanoyl-phosphatidylcholine (5). The activity of rPLRP2 and
cPLRP2 against 1,2-didodecanoyl-phosphatidylcholine was quite low
compared with the other two phospholipid substrates. Both rPLRP2 and
gPLRP2 but not hPTL showed activity against monogalactosyldiglyceride. In addition to confirming the activity of PLRP2 lipases against galactolipids, this result demonstrates the utility of the monolayer assay for measuring galactolipase activity.
Although rPLRP2 activity against these various substrates could be
easily measured in the monolayer assay, rPLRP2 had lower activity than
did the other lipases. This finding is consistent with the results of
the bulk phase assay with galactolipids where rPLRP2 had decreased
activity compared with gPLRP2 (9). The lower activity of rPLRP2 against
1,2-dicaprin was surprising. In bulk phase assays, the specific
activity of rPLRP2 compares favorably with that of hPTL (Table
II). The explanation for this difference
was not examined, but the finding may indicate that rPLRP2 is more
sensitive to denaturation by the monolayer than are the other lipases
or that rPLRP2 may partition itself less favorably in the monolayer
system than other lipases. Direct comparisons of bulk phase
phospholipase activity of rPLRP2 and the other PLRP2 lipases have not
been done. The monolayer data indicate that rPLRP2 has lower activity
against phospholipids then do other members of the PLRP2 family.
The current rPLRP2 structure does not explain the different activities
of rPLRP2 and hPTL against phospholipids and galactolipids. There were
no differences in the residues or positions of the residues around the
active sites of rPLRP2 and hPTL to explain the substrate preferences.
We observed differences in the lid domain and in the Concluding Remarks--
In this paper, we report the first
structure for a member of the PLRP2 family and demonstrate that rat
PLRP2 does show interfacial activation. Additionally, we confirm and
extend the observations that PLRP2 lipases possess broader substrate
specificities than do the closely homologous pancreatic triglyceride
lipases. These studies represent the beginning of investigations that
will contribute to understanding the molecular mechanisms underlying lipolysis.
We thank Frédéric Carriére
for critically reading the manuscript and for the generous gift of
coypu PLRP2 and Josiane de Caro for performing the inhibition studies
with tetrahydrolipstatin and E600.
*
This work was supported by National Institutes of Health
Grant HD3306002.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1bu8) have
been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
The abbreviations used are:
PTL, pancreatic
triglyceride lipase; Col, colipase; c, coypu; g, guinea pig; h, human; PLRP1, pancreatic lipase-related protein 1; PLRP2, pancreatic
lipase-related protein 2; p, porcine; r, rat; MES, 4-morpholineethanesulfonic acid.
Structure and Activity of Rat Pancreatic Lipase-related Protein
2*
,, and
Architecture et Fonction des
Macromolécules Biologiques, CNRS-IFR1 UPR 9039, 31 Chemin Joseph
Aiguier, 13402 Marseille cedex 20, France, and the ¶ Laboratoire
de Lipolyse Enzymatique, CNRS-IFR1 UPR 9025, 31 Chemin Joseph Aiguier,
13402 Marseille cedex 20, France
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-sandwich C-terminal domain. The lid domain
occupied the closed position, suggesting that rPLRP2 should show
interfacial activation. When we reexamined this issue with tripropionin
as substrate, rPLRP2 exhibited interfacial activation. Because the
active site topology of rPLRP2 resembled that of human PTL, we
predicted and demonstrated that the lipase inhibitors E600 and
tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have
similar active sites, rPLRP2 has a broader substrate specificity that
we confirmed using a monolayer technique. With this assay, we showed
for the first time that rPLRP2 prefers phosphatidylglycerol and
ethanolamine over phosphatidylcholine. In summary, we confirmed and
extended the observation that PLRP2 lipases have a broader substrate
specificity than PTL, we demonstrated that PLRP2 lipases show
interfacial activation, and we solved the first crystal structure of a
PLRP2 lipase that contains a lid domain.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
5 loop, undergoes a spatial reorganization and folds
back on the core of the protein. These drastic conformational changes,
leading to the open conformation, give substrate free access to the
catalytic triad.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
= 102.1°. Specific volume calculations yielded one molecule/asymmetric
unit, with a Vm of 2.7 Å3/Da and a
solvent content of approximately 54% (24). A total number of 36,060 unique reflections were indexed using the SCALEPACK program with an
R-factor on intensities of 6.3%, a data set multiplicity of 2.4 and a
completeness of 97.5%, between 10.0 and 1.8 Å (Table I) (25).
Data collection and final refinement statistics
Fc) and
(Fo Fc) maps numbered
295, and one Glc-NAc sugar bound to Asn334 was identified.
Seven molecules of ethylene glycol were modeled in the density. The
R-factor calculated with 36,060 reflections between 10.0 and 1.80Å
resolution was 20.3% and the final R-free factor calculated with 5%
reflections was equal to 24.2% (Table I). The Ramachandran plat and
the electron density map (see Fig. 1) further demonstrate the quality
of the model. Coordinates have been deposited in the Protein Data Bank
with the accession number 1bu8.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
conformation found in other lipases and in the 
/ hydrolase fold family and is therefore located in a "generously allowed region" (28, 29). The glycosylated Asn334 is
conserved in all PLRP2 lipases except the coypu. This glycosylation site was not present in the structure of the gPLRP2/hPTL chimera because the C-terminal domain originated from hPTL. The PLRP1 glycosylation site at position 138 is not present in PTL or in PLRP2
lipases (30). Another potential glycosylation site in classical lipases
is located at position 166 but is only partially conserved (14) and is
not present in PLRP1 or PLRP2 lipases. Due to cryocooling, high
resolution, and low B-factors, seven molecules of cryoprotectant can be
observed in the electron density map. The ethylene glycol molecules are
stabilized mainly by hydrogen bonds and in part by hydrophobic
interactions. Hydrogen bonds are established with Arg side chains (four
cases) and with main and side chains of various semi-polar and polar
residues. Hydrophobic interactions involve aromatics (four cases) and
aliphatic residues (two cases).
/ hydrolase fold family of
proteins (29). The protein consists of two main domains, a globular
N-terminal domain and a -sandwich C-terminal domain. This structure
closely resembles the structure of hPTL and the other members of the
pancreatic lipase fold family (see Fig. 2A) (10). The core
of the N-terminal domain of the molecule consists of a tightly packed
-sheet surrounded by five helices. The rPLRP2 lid, which adopts a
closed conformation, has an electron density of excellent quality and
does not show any sign of disorder or particular flexibility (Fig.
1). Its B-factors display the same pattern as in other closed pancreatic lipases (data not shown). The lid
is located between the two sides of the bridged cysteine residues
237-261. Together with the 5 loop, it adopts the same conformation
as that observed in the closed structure of hPTL (10). The
active site is located at the bottom of a hydrophobic crevice that is
covered by the lid. The catalytic triad (Ser152,
His263, and Asp176) includes the nucleophile
belonging to the usual consensus sequence G-X-(nucleophile)-X-G (Fig.
2A).
View larger version (73K):
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Fig. 1.
Stereo view of the sigma weighted electron
density map of the closed lid contoured a 1 
.
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Fig. 2.
Structure of rPLRP2. A,
stereo view of the C trace of rPLRP2. The side chains of the N and C
terminus residues as well as those of the catalytic Ser152
and of the lid residue Trp252 are represented. The ethylene
glycol molecules (blue) and the GlcNAc residue linked to
Asn334 are shown. Green, C terminus domain;
brown, N-terminal catalytic domain; yellow,
catalytic domain following the lid; pink, lid. B,
stereo view of the C trace of rPLRP2 superimposed on those of the
other known closed pancreatic lipase structures.
5
loop), which switch between the closed and open conformations. The
degree of structural homology of rPLRP2 compared with the other lipases
found in the closed conformation (dog PLRP1, hPTL, and horse PTL) is
remarkable (Fig. 2B). As described previously, the
C-terminal domain orientations of the structures included in the
comparisons were found to differ due to rotations of a few degrees
occurring around hinge residue 337 (Fig. 2B) (11-14).
5 loop were taken from the open hPTL
structure and grafted onto the core of the PLRP2 enzyme, and the lid
residues were substituted according to the rPLRP2 sequence. No residues
were found to be substituted between hPTL and rPLRP2 in a 10 Å radius
around the nucleophilic Ser152 O
.
Consequently, the C11 phosphonate inhibitor positioned into the open
rPLRP2 model exhibits the same protein contacts as in the classical
lipase.
View larger version (14K):
[in a new window]
Fig. 3.
Interfacial activation of rPLRP2.
A, interfacial activation demonstrated in the laboratory of
R. Verger. The tripropionin solutions were made in 1% gum arabic as
described under "Materials and Methods." 25 µg of rPLRP2 was
added with a 5-fold molar excess of pure porcine colipase.
B, interfacial activation demonstrated in the laboratory of
M. Lowe. The tripropionin solutions were prepared in 2% gum arabic as
described under "Materials and Methods." 15 µg of rPLRP2 and a
5-fold molar excess of pure human colipase was added. C,
interfacial activation with p-nitrophenylbutyrate as the
substrate. The assay included 20 µg of rPLRP2 and a 5-fold molar
excess of pure human colipase. The vertical dashed line in
each figure shows the concentration when saturation of the tripropionin
or p-nitrophenylbutyrate solutions occurs.
Tyr, Ile401 Leu,
Tyr403 Asn, and Glu441 Asp (31). The
I401L substitution does not alter the interaction. The E441D
substitution may abolish the interaction with colipase Arg65, but very limited changes, such as side chain
torsion, could restore the ion pair. The stacking interaction of
Tyr403 with Arg65 is also lost but is readily
replaced by a hydrogen bond between Asn403 and
Arg65. The fourth substituted residue, Tyr360,
clashes severely with colipase Glu45. This unfavorable
interaction of the tyrosine hydroxyl group can be easily turned to a
favorable hydrogen bond through side chain torsion of these two surface
residues. To summarize, most interactions between colipase and PTL
would be conserved in rPLRP2, the two unfavorable interactions yielding
from substitutions can be relaxed easily, and no new unfavorable
interactions appear. This conclusion is consistent with kinetic data
showing that rPLRP2 does interact with colipase.
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Fig. 4.
Inhibition of rPLRP2 by tetrahydrolipstatin
and E600. The activity of rPLRP2 against tributyrin was
measured after incubating rPLRP2 with either E600 or
tetrahydrolipstatin. 
, 100-fold molar excess of tetrahydrolipstatin
in the presence of 4 mM sodium taurodeoxycholate in 10%
isopropanol. 
, 100-fold molar excess of E600 in 5% isopropanol
without bile salts. Another aliquot of E600 equivalent to a 100-fold
molar excess was again added after the 1-h time point was
sampled.
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Fig. 5.
Monolayer activity of PLRP2 lipases and PTL
against various substrates. Activities were determined in a
monolayer trough as described under "Materials and Methods." The
substrate legend is given in the figure. n-gPLRP2, guinea
pig PLRP2 isolated from pancreas; gPLRP2, recombinant guinea
pig PLRP2; hPTL, human PTL isolated form pancreas;
cPLRP2, recombinant coypu PLRP2; rPLRP2,
recombinant rat PLRP2.
Activities of lipases against various triglycerides in bulk phase
assays
5 loop between
the two enzymes, but they do not obviously explain the substrate
differences when compared with the open, active form of hPTL. It will
be necessary to solve the open structure of rPLRP2 before differences
in the active sites become apparent. Possibly, residues away from the
active site will affect substrate specificity as found in the serine proteases.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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