From the Departments of Pediatrics and of
¶ Molecular Biology and Pharmacology, Washington University School
of Medicine and St. Louis Children's Hospital, St. Louis, Missouri
63110
Received for publication, November 2, 2000, and in revised form, December 29, 2000
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ABSTRACT |
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Pancreatic triglyceride lipase (PTL) requires
colipase for activity. Various constituents in meals and in bile,
particularly bile acids, inhibit PTL. Colipase restores activity to
lipase in the presence of inhibitory substances like bile acids.
Presumably, colipase functions by anchoring and orienting PTL at the
oil-water interface. The x-ray structure of the colipase·PTL
complex supports this model. In the x-ray structure, colipase
has a hydrophobic surface positioned to bind substrate and a
hydrophilic surface, lying opposite the hydrophobic surface, with two
putative lipase-binding domains,
Glu45/Asp89 and
Glu64/Arg65. To determine whether the
hydrophilic surface interacts with PTL in solution, we introduced
mutations into the putative PTL binding domains of human colipase. Each
mutant was expressed, purified, and assessed for activity against
various substrates. Most of the mutants showed impaired ability to
reactivate PTL, with mutations in the
Glu64/Arg65 binding site causing the greatest
effect. Analysis indicated that the mutations decreased the affinity of
the colipase mutants for PTL and prevented the formation of
PTL·colipase complexes. The impaired function of the mutants was most
apparent when assayed in micellar bile salt solutions. Most mutants
stimulated PTL activity normally in monomeric bile salt solutions. We
also tested the mutants for their ability to bind substrate and anchor
lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each
mutant colipase bound to tributyrin to the same extent as wild type
colipase. These results demonstrate that the hydrophilic surface of
colipase interacts with PTL in solution to form active colipase·PTL
complexes, that bile salt micelles influence that binding, and that the
proper interaction of colipase with PTL requires the
Glu64/Arg65 binding site.
Lipases participate in a variety of physiologic and industrial
processes (1, 2). They aid in the preparation of foods and
pharmaceuticals. They serve to generate second messengers, participate
in cytotoxicity reactions, and have become a model system for the study
of molecular interactions occurring at lipid-water interfaces. Most
importantly, they allow for the digestion and trafficking of dietary
triglycerides and phospholipids by converting apolar dietary lipids to
more polar products, fatty acids and monoacylglycerols. An
understanding of the digestive process has implications for the
treatment of human diseases, including malnutrition and obesity.
The digestion of dietary triglyceride begins in the stomach through the
process of emulsification and the actions of gastric lipase (3).
Digestion continues in the duodenum where pancreatic triglyceride
lipase (PTL)1 releases
50-70% of dietary fatty acids (3). Although PTL requires bile acids
to function efficiently, bile acid micelles inhibit PTL unless another
pancreatic protein, colipase, is present (2). Colipase restores PTL
activity in the presence of physiologic concentrations of bile acids.
Without colipase, PTL could not cleave fatty acids from dietary
triglycerides, and fat malabsorption with its consequences would result.
Previous models of colipase function uniformly proposed that colipase
acts as a bridge between PTL and the lipid substrate by binding to both
PTL and the lipid emulsion surface. The strongest support for this
model comes from the crystal structures of the porcine colipase·human
PTL complex solved under different conditions (4, 5). In the first
structure, PTL resides in an inactive conformation with the catalytic
site sterically blocked by a surface loop, called the lid. The other
structure, solved in the presence of mixed micelles, reveals the active
conformation of PTL in which the lid moves away from the catalytic site
and forms new interactions with colipase. Colipase has similar
conformations in both structures (6). Importantly, colipase has two
opposing surfaces that can potentially anchor PTL to the substrate. One
surface, which contains predominantly hydrophilic residues, forms
interactions with the C-terminal domain of PTL. The opposite surface,
which faces away from PTL, has four hydrophobic loops positioned to
interact with the substrate surface. In this conformation, colipase
brings PTL into the proper proximity and orientation for lipid hydrolysis.
The crystal structure predicts that van der Waals forces and several
polar bonds stabilize the interaction between colipase and PTL. The
number of putative polar bonds varies between the inactive and active
conformation of PTL. In the active conformation of PTL two colipase
residues, Glu15 and Arg38, form polar bonds
with residues in the PTL lid. In both conformations of PTL polar
interactions occur between five residues of colipase, Arg44, Glu45, Glu64,
Arg65, and Asn89, and main-chain residues in
the C-terminal domain of PTL. Four residues form two potential binding
domains, one containing residues 45 and 89, which interact with
Lys400 of PTL, and the other containing residues 64 and 65, which interact with Gln369 of PTL. Arg44 is not
conserved and is replaced by a serine or methionine in most other
species. Additionally, Arg44 does not form a polar bond in
the porcine colipase·PTL complex. These points argue that
Arg44 may not play an important role in colipase·PTL
interactions in solution.
A few studies have examined the role of specific amino acids in the
formation of the colipase·PTL complex. One study examined the
interaction between Glu15 of colipase and the PTL lid by
site-specific mutagenesis of Glu15 (7). This study
concluded that the interactions between Glu15 and the PTL
lid stabilized the active confirmation of PTL. Two other studies
investigated the interaction between colipase and the C-terminal domain
of PTL. Jennens and Lowe (8) introduced substitution and deletion
mutations into the C-terminal domain of PTL. Single substitution
mutations of multiple PTL residues thought to interact with colipase
did not affect lipase activity. Deletion of the C-terminal domain
greatly decreased PTL activity, but the truncated PTL still required
colipase for activity in the presence of bile acids. These mutants
raised questions about the importance of the interactions between
colipase and the C-terminal domain of PTL. Another recent study
provided support for the predicted interaction between one colipase
residue, Glu45, and Lys400 of the PTL
C-terminal domain (9). Ayvazian et al. (9) demonstrated that
disruption of this interaction impaired lipase activity secondary to an
inability of the mutant colipase to bind PTL. None of these studies
addressed the relative importance of the other putative lipase binding
residues in the hydrophilic surface of colipase.
To determine if the hydrophilic surface of colipase interacts with
lipase in solution and to identify participating residues, we
introduced mutations into positions 44, 45, 64, 65, and 89 of human
colipase by site-specific mutagenesis. Each mutant colipase was
characterized for the ability to reactivate PTL in the presence of bile
salts and to anchor PTL at an oil-water interface. The results support
the orientation of colipase as defined by the crystal structures. That
is, the hydrophilic surface of colipase interacts with PTL in solution.
Furthermore, the results identify the 64/65 binding domain,
specifically position 64, as key to the normal function of colipase,
thereby adding additional details to the mechanism of PTL-mediated lipolysis.
Construction of Mutants--
Manipulations of DNA were done by
standard methods (10). Mutations were introduced into wild type human
colipase cDNA in the pPIC9 vector by polymerase chain reaction
using the Stratagene QuikChange site-directed mutagenesis kit per the
manufacturer's protocol. Oligonucleotide primers were designed to
introduce the desired mutations. Transformations into Epicurian
Coli XL-1 Blue Supercompetent cells were performed per the
QuikChange protocol. Minipreps were performed using either the Qiagen
Plasmid Mini kit or the Qiagen QIAprep Miniprep kit. Presence of the
desired mutation was confirmed by dideoxynucleotide sequence
analysis using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit.
Protein Methods--
Proteins were expressed and purified as
previously described (11). Protein concentrations were determined by
ultraviolet spectrophotometry at A280
using an extinction coefficient of 0.47. Equal quantities (3 µg) of
each mutant protein were run on SDS-PAGE gels and stained with GELCODE
Blue stain (Pierce, Rockford, IL) to confirm the homogeneity of the
protein product.
Stability in urea and TDC was determined for colipase and the colipase
mutants. Each colipase was incubated for 30 min at room temperature in
8 M urea or 4 mM TDC in 50 mM
Tris-HCl, pH 8.0, at a concentration of 0.1 mg/ml. Companion
incubations were done in buffer alone. After the incubation, 500 ng of
the colipase was assayed with 2.8 µg of PTL using tributyrin
emulsified with 0.5 mM TDC as described below. The
difference between PTL activity in the absence and presence of colipase
was defined as the colipase-stimulated PTL activity. Neither colipase
nor the colipase mutants lost any function after incubation in buffer
for 30 min at room temperature.
Colipase Assays--
Assays were performed at 37 °C using a
VIT 90 Radiometer pH-stat, in assay buffer containing 1 mM
Tris-HCl, pH 8, 2 mM CaCl2, 150 mM
NaCl, and taurodeoxycholate (TDC) concentrations ranging from 0 to 4 mM as described earlier (12). The rate of NaOH titration during the assay was set to maintain a constant pH of 8. Tributyrin (Fluka, Buchs Switzerland and Sigma Chemical Co., St. Louis MO) was
used as substrate. Assays were performed with 14.5 ml of assay buffer
and 0.5 ml of tributyrin except as noted below. Further analysis of the
mutants was performed by measuring lipase activity over a range of
colipase concentrations (0-1372 nM) with a constant concentration of lipase (1400 ng) and excess substrate. To determine the substrate dependence of PTL activity with the various colipase mutants, we measured PTL activity over a range of tributyrin
concentrations (0-43.4 mM), whereas holding the colipase
and lipase concentrations constant at a one to one molar ratio (2800 ng
of PTL, 560 ng of colipase).
Mutant Colipase Binding Assays--
Binding assays were
performed as previously described (13). Briefly, the reactions were
carried out in binding buffer containing 0.1 M Tris-HCl, pH
8, 150 mM NaCl, 2 mM CaCl2, and 4 mM TDC. 2800 ng of wild type lipase and 560 ng of mutant
colipase were added to 1 ml of binding buffer in a 1.5-ml
microcentrifuge tube that had been precoated with 1 mg/ml BSA in
binding buffer. The samples were vortexed for 1 min, then centrifuged
at 14,000 rpm for 5 min. 800 µl of the upper, aqueous phase was
carefully removed and placed in a microcentrifuge tube that had also
been precoated with 1 mg/ml BSA in binding buffer. The samples
were immediately placed on ice and stored at Radioimmunoassay for Colipase Quantitation--
The amount of
procolipase bound to the interface was determined by radioimmunoassay.
100-µl aliquots of the aqueous phase were tested for each mutant. The
wild type control curve samples (0-16 ng of wild type colipase), which
were linear over this range of colipase concentration, were brought up
to a total volume of 100 µl with binding buffer. All other dilutions
were performed with TBST (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% Tween 20) containing 1 mg/ml bovine serum
albumin. Wild type procolipase labeled with I125 was
diluted to a final concentration of ~120,000 cpm per 200 µl.
Polyclonal anti-colipase antibody was diluted 1:160. Each diluted
procolipase sample was placed in a 1.5-ml microcentrifuge tube that had
been precoated with 1 mg/ml BSA in TBST. 200 µl of the labeled
procolipase dilution was added to each tube, followed by 200 µl of
the anti-colipase antibody dilution. The contents of each tube were
mixed and allowed to sit at room temperature for 1 h. 30 µl of a
Protein A (Sigma Chemical Co., St Louis, MO) 50% slurry in TBST was
added to each tube, mixed, and incubated at room temperature for an
additional hour. The precipitate was collected by centrifugation and
then washed in 500 µl of TBST three times. The pellet was resuspended
in 100 µl of TBST and counted using a Packard Cobra II Auto Gamma counter.
Statistical Analysis--
Descriptive statistics were calculated
using Microsoft Excel. Regression analysis was performed with Sigma
Plot using a single rectangular hyperbola model.
R2 values Colipase Mutants--
The crystal structures of the pig
colipase-human PTL complex identified polar interactions between
residues Arg44, Glu45, Glu64,
Arg65, and Asn89 of porcine colipase and
residues in the C-terminal domain of PTL (6). Although the interactions
clearly form in crystals, the same bonds may not stabilize the
colipase·PTL complex in solution. To determine whether or not these
interactions contribute to the binding between human colipase and PTL
in solution, we created a variety of single- and double-substitution
mutants in human colipase (Fig. 1).
Initially, we substituted a small neutral amino acid, alanine, in
positions Glu45, Glu64, and Arg65,
which are conserved in human colipase, and in positions
Ser44 and Asp89, which are not conserved. We
next substituted amino acids with identical charge but different
side-chain length, with opposite charge in the side chain or with a
bulky side chain to determine which properties of the side chain
influence binding of colipase to PTL. Each of the mutant proteins was
expressed in a yeast system and purified by immunoaffinity and gel
filtration chromatography. Analysis of the purified proteins by
SDS-PAGE revealed a single broad band for each protein indicative of an
uncontaminated purified protein suitable for further characterization
(Fig. 2).
Activity of Colipase Mutants at Various TDC
Concentrations--
The effect of bile salts on PTL varies depending
on the concentration of the bile salts (2, 14). Below their critical micelle concentration (CMC) bile salts stimulate PTL activity. Above
their CMC bile salts inhibit PTL activity. Colipase increases PTL
activity in both cases, indicating that colipase and PTL interact below and above the CMC of a given bile salt. Although colipase and PTL
probably form a complex at all bile salt concentrations, the
interactions stabilizing the complex in the presence of bile salt
monomers could differ from those in the presence of bile salt micelles.
Thus, we tested the ability of the colipase mutants to stimulate PTL
activity at various concentrations of TDC, which has a CMC of about 1.1 mM under the assay conditions (Fig.
3). In the absence of colipase, the
activity of PTL increased at 0.5 mM TDC and, then, dropped
sharply as the TDC concentration increased (Fig. 3, A,
We extended these observations with studies on additional colipase
mutants. These mutants were created to alter the physical properties of
the side chains in specific ways. We changed the side-chain charge,
length, and bulk in Glu45, Asp89,
Glu64, and Arg65. Several of these mutants,
E45D, E45Q, and D89R, did not express well and could not be analyzed.
Of the remaining mutants, only the substitution of lysine for arginine
at position 65 was tolerated. PTL had full activity with the R65K
colipase mutant (Fig. 3B, Stability of the Mutants--
Decreased stability of the mutants
in 4 mM TDC could also explain the above results. To test
the stability of the mutants, we incubated them in 8 M urea
or 4 mM TDC for 30 min at room temperature. After the
incubation we determined the ability of colipase and each colipase
mutant to activate PTL. Under both conditions native colipase retained
full function, as did each of the colipase mutants. These results make
it unlikely that any of the mutations altered the stability of colipase
to a significant degree.
Concentration Dependence of the Alanine Mutants--
The assays at
various TDC concentrations suggested that bile salt micelles might
affect formation of productive complexes between colipase and PTL. To
better characterize any differences in the interaction between colipase
and PTL in the presence and absence of bile salt micelles, we measured
the ability of the colipase mutants to reactivate PTL over a range of
colipase concentrations in the presence of excess substrate and a
constant amount of PTL (Fig. 4). From
these data, we determined the concentration of the colipase mutants
that restored half-maximal activity to PTL (Kd) and
the concentration that restored maximal PTL activity
(Bmax) in 0.5 and 4 mM TDC. The
apparent Kd and Bmax were
determined by nonlinear regression with a rectangular hyperbola
function. Given the large excess of substrate in the assays, the
apparent Kd and Bmax most
likely reflect the properties of interactions between colipase and PTL
rather than between colipase and substrate. The experimental curves for the activity measured in 0.5 mM TDC are shown in Fig. 4
(A and B). All of the tested mutants had an
apparent Kd and Bmax equal to
that of wild type colipase with the exception of the R65E mutant, which
had an apparent Kd value ~4-fold weaker than wild
type (Table I). The preserved function of
the colipase mutations under these conditions indicated that the
mutations did not grossly disrupt the structure of the colipase
mutants.
At 4 mM TDC, we see differences in the curves (Fig. 4,
C-F). The curves shift to the right for most of the mutants
indicating a change in affinity of the colipase mutants for PTL. The
apparent Bmax for all mutants equals that of
native colipase, whereas the apparent Kd increased
for all of the mutants (Table II). Mutations of the Glu64/Arg65 binding site
affected the apparent Kd to a greater degree than
mutations of the Glu45/Asp89 binding site. The
greatest effect was seen with the R65E mutant, which had an apparent
Kd for lipase that was ~260-fold weaker than that
of native colipase, a further 60-fold reduction in apparent
Kd from that measured in 0.5 mM TDC. The decrease in apparent Kd of the colipase mutants
agrees with the hypothesis that the targeted residues participate in the formation of colipase·PTL complexes through direct interactions with PTL. The large effect of TDC micelles on the apparent
Kd suggests that micelles may influence the binding
of colipase and PTL either directly or indirectly.
Substrate Dependence of the Colipase Mutants--
To assure that
mutations of the hydrophilic surface of colipase did not also affect
the interaction of the colipase·PTL complex to substrate, we measured
the activity of the mutants over a range of substrate concentrations
(Fig. 5). Enzymatic lipolysis does not
lend itself to standard steady-state analysis due to the interfacial mechanism, constantly changing substrate, and the requirement of a
cofactor (14). As a result, true Michaelis-Menten parameters cannot be
easily measured. Still, parameters determined under constant,
predetermined conditions provide a measure of the relative function
among the colipase mutants. The assays contained 0.5 or 4.0 mM TDC and equimolar amounts of PTL and the colipase
mutants. In 0.5 mM TDC, wild type colipase and all of the
colipase mutants increased PTL activity to the same level. In 4.0 mM TDC, two mutants, E45A and R65K, had curves identical to
that of native colipase (Fig. 5, A, Anchoring of PTL to Substrate by the Colipase Mutants--
To
address the possibility that the mutant proteins anchor PTL to
substrate, but in an inactive confirmation, we directly measured the
ability of each colipase mutant to bind substrate and to anchor lipase
on the substrate surface. We measured binding with a centrifugation
assay that rapidly separates the organic lipid phase from the aqueous
phase. Each assay contained an equimolar ratio of colipase and PTL in
an emulsion of tributyrin and 4.0 mM TDC. After separation
of the phases the amount of colipase and PTL remaining in the aqueous
phase was determined as described under "Materials and Methods." In
this assay, all of the colipase mutants partitioned with the substrate
phase to a degree similar to native colipase, 80% compared with 67%
to 93% for the various mutants, which indicates that binding of the
colipase mutants to tributyrin was minimally impaired (Fig.
6, black bars). The amount of
lipase anchored to the substrate surface varied from 85% for E45A to
8% for E45W, as compared with 90% for wild type colipase (Fig. 6,
light gray bars). The amount of lipase anchored to the
substrate was proportional to the activity of each mutant (Fig. 6,
dark gray bars). Even though the colipase mutants bind to
substrate normally, they cannot anchor PTL efficiently, which is
predicted by the hypothesis that the targeted residues in colipase mediate complex formation between colipase and PTL.
In the crystal structure of porcine colipase bound to PTL, polar
interactions form between colipase residues Glu45,
Asn89 (Asp89 in human colipase),
Glu64, and Arg65 and residues in the C-terminal
domain of PTL (6). We mutated each putative lipase-binding residue of
colipase to determine whether these amino acids participate in complex
formation when the proteins are in solution. Characterization of the
colipase mutants showed that substitutions in each targeted site
decreased the ability of colipase to bind PTL in the presence of bile
salt micelles. Several conclusions can be made from these results. First, residues in the hydrophilic surface of colipase mediate the
formation of colipase·PTL complexes in solution as predicted by the
crystal structure. Second, bile salt micelles influence the assembly of
productive colipase·PTL complexes. Third, both the
Glu45/Asp89 and
Glu64/Arg65 binding domains contribute to
complex formation, but the Glu64/Arg65 binding
site predominates. Although the Glu45/Asp89
sites tolerated alanine substitutions, any changes, with the exception
of a conserved mutation at position 65, in the
Glu64/Arg65 binding site greatly diminished the
activity of these colipase mutants when assayed above the CMC of
TDC.
The increased sensitivity of the Glu64/Arg65
site compared with the Glu45/Asp89 site was not
expected, based on the crystal structure of the PTL·colipase complex
(6). The carboxylate side chain of Glu64 forms a hydrogen
bond with the main-chain nitrogen of PTL residue Gln369 and
serves as a contact point for a water molecule bridge involving Glu45 of colipase and Ser366 of PTL.
Arg65 contributes another hydrogen bond with the side chain
of Gln369. In contrast, Glu45 forms the only
salt bridge with the C-terminal domain of PTL (Lys400),
forms a hydrogen bond with Asn366, and contributes to the
water bridge noted above. Asp89 has a hydrogen bond with
Lys400 of PTL and contributes to a water bridge with
Gly66 of colipase and Leu444 of PTL.
Additionally, site-specific mutagenesis of Glu45 and
Lys400 of PTL suggested that the ion pairing between these
two residues was essential for lipolysis (9). Our results question this conclusion. The preserved function of the E45A colipase mutant suggests
that the salt bridge between Glu45 of colipase and
Lys400 of PTL is not essential or, alternatively, may not
form in solution.
Regardless of which binding site was mutated, bile salts altered the
behavior of most single substitution mutants. The magnitude of the
effect depended on the bile salt concentration. At 0.5 mM
TDC, when monomers of bile salts spread at the oil-water interface, the
affinity of the colipase mutants for PTL was unchanged except for R65E.
At higher TDC concentrations, above the CMC, the affinity of the
colipase mutants decreased appreciably for all but the E45A and S44A
mutants. At least two models can be offered to explain this
observation. The first model proposes that colipase and PTL must form a
complex with bile salt micelles in aqueous solution before binding to
the interface. There is support for this model in the literature.
Multiple studies have demonstrated that bile salt micelles bind to
colipase and to PTL (15-18). A recent study with small angle neutron
scattering found a detergent micelle associated with the PTL·colipase
complex indicating that a complex can form between PTL, colipase, and
micelles (19).
The second model states that colipase and PTL have poor affinity for
each other in aqueous solution. In the absence of substrate PTL
preferentially binds to bile salt micelles. When a substrate is added,
colipase binds to the substrate first. After formation of a complex
with substrate, the bound colipase has a higher affinity for PTL than
that of the bile salt micelles. PTL then binds to colipase on the
substrate surface. Several studies support this model. The concept of a
sequential mechanism where colipase binds to the substrate first
followed by PTL has been previously proposed and supported by various
experiments (2). Other studies using microcalorimetry, two-phase
partitioning, and affinity chromatography with immobilized colipase
demonstrated that bile salt micelles bind PTL and weaken the binding of
colipase to PTL, which may favor the binding of colipase to the
substrate surface rather than to complexes of PTL and bile salt
micelles (20-22). In another study, monomeric bile salt solutions
allowed the absorption of PTL to an interface, but a micellar bile salt
solution completely inhibited PTL binding to an interface (23). Two
kinetic studies suggested that bile salt micelles or multiple bile
salts bind to PTL and inhibit absorption of PTL to the substrate-water
interface even in the presence of colipase (24, 25).
Both models could account for our data. Mutations in the hydrophilic
surface of colipase could destabilize the complex of PTL, bile salts
and colipase predicted to form by in the first model. This disruption
could occur because the colipase mutants have decreased affinity for
the PTL·bile salt complex or because the mutants bind to PTL in an
incorrect conformation producing an inactive complex. The fact that the
colipase mutants have lowered affinity for PTL and can bind to the
substrate but not anchor PTL even as an inactive complex argues against
the last possibility. The weakened binding affinity of the colipase
mutants for PTL is also consistent with the second model. In this
instance the decreased affinity of the colipase mutants for PTL allows
the bile salt micelles to compete for PTL binding, and thereby inhibit the formation of an active colipase·PTL complex.
Although the role of bile salt micelles in the lipolytic mechanism
remains unclear, many molecular details of lipolysis have been
described in the past decade. X-ray crystallography and site-specific mutagenesis identified the catalytic triad of PTL. The crystal structures of the colipase·PTL complex and the characterization of
naturally occurring and designed lipases with mutations in the lid have
demonstrated a role for the lid in substrate selectivity, in substrate
binding, and interactions with colipase. Monolayer experiments, kinetic
analysis, and site-specific mutagenesis have broadened the role of
colipase in the lipolytic mechanism. It stabilizes the lid in the open,
active conformation and may direct PTL to substrate patches on the
surface of dietary emulsions, which contain high concentrations of
phospholipids and cholesterol esters. This work extends these
observations to better define the crucial interactions between colipase
and PTL. An increased understanding of the molecular details of this
important physiologic process provides the information needed to
rationally modulate PTL activity for therapeutic purposes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. The amount of
lipase remaining in the aqueous phase was determined by activity assay
using 200 µl of the aqueous phase and 270 ng of wild type
procolipase. The amount of lipase bound to the substrate surface was
calculated as the difference between the amount of lipase initially
added and the amount recovered in the aqueous phase. We developed a radioimmunoassay to determine the amount of colipase remaining in the
aqueous phase (see below). The amount of procolipase bound to the
substrate was calculated as the difference between the total amount
initially added, and the amount remaining in solution.
0.94 were obtained for all
regression lines used to determine Kd,
Km, Bmax, or
Vmax. Data are presented as the mean ± S.D. of the fit.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Crystal structure of colipase. The
-carbon backbone and disulfide bonds of colipase are presented as
tubes. The side chains of the colipase residues that the
crystal structure predicts will form polar interactions with PTL are
shown. They form two potential binding sites,
Glu64/Arg65 and
Glu45/Asp89. The side chain of
Ser44 is not shown but sits next to the side chain of
Glu45. The coordinates were obtained from the Protein Data
Bank, entry 1LPB. The Protein Structure and Macromolecular Graphics
Core of the Digestive Disease Research Center made the figure.
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Fig. 2.
Analysis of the purity of mutant colipase
proteins. 3 µg of each mutant protein were run on a denaturing
SDS-PAGE gel and stained with GELCODE Blue. M, molecular
weight markers; 1, native colipase; 2, S44A;
3, E45A; 4, E64A; 5, R65A;
6, D89A; 7, E45A/D89A; 8, E64A/R65A;
9, E45W; 10, E64D; 11, E64Q;
12, R65K; 13, R65E.
;
B,
). The addition of native colipase increased PTL
activity at all TDC concentrations (Fig. 3,
). The S44A mutant restored full PTL activity at all TDC concentrations tested, suggesting that Ser44 does not contribute to the binding of human
colipase to human PTL (Fig. 3A,
). The other alanine
mutants separated into two groups by putative binding site (Fig.
3A). Below the CMC, single alanine substitutions into the
Glu45/Asp89 and
Glu64/Arg65 binding sites had little effect on
PTL activity compared with native colipase. Both double-substitution
mutants had decreased function but still increased PTL activity above
the no colipase levels. By 4 mM TDC, the E64A and R65A
mutants restored less than 5% of the PTL activity compared with native
colipase, and the E64A/R65A double mutant restored no activity to PTL.
The function of the single-substitution mutants in the other binding
site remained relatively preserved even at 4 mM TDC. The
E45A restored full activity to PTL, and the D89A restored 45% of
activity. Mutating both Glu45 and Asp89
decreased the function of the double mutant to about 5% of native colipase. These findings indicate that both sites on colipase contribute to the binding between colipase and PTL on colipase. Of the
two sites, Glu64/Arg65 appears to form
essential interactions with PTL.
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Fig. 3.
Influence of bile salt concentration on
mutant colipase function. The function of the colipase mutants was
measured over a range of TDC concentrations with 1.5 µg of PTL and
0.3 µg of colipase. PTL activity was measured in a Radiometer pH-stat
as described under "Materials and Methods." The activity is
expressed as micromoles of fatty acid released per minute.
A, alanine mutants: , native colipase;
, S44A;
,
E45A;
, D89A;
, R65A;
, E64A;
, E45A/D89A;
,
E64A/R65A;
, no colipase. B, nonalanine mutants:
,
native colipase;
, R65K;
, E64D;
, E64Q;
, E45W;
, R65E;
, no colipase.
). The other mutants had
relatively preserved function below the CMC of TDC compared with their
greatly decreased function above the CMC. At position 45, the
substitution of a bulky side group abolished the ability to restore PTL
activity above the CMC of TDC (Fig. 3B,
). Likewise, the
substitution of a negatively charged amino acid, glutamic acid, for a
positively charged amino acid, arginine, at position 65 eliminated
function above the CMC (Fig. 3B,
). Position 64 was
sensitive to both chain length and charge with both the E64D and E64Q
mutant colipases showing decreased function (Fig. 3B,
and
). The results confirm the importance of the targeted sites in
the interaction between colipase and PTL.
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Fig. 4.
Colipase dependence of PTL activity at 0.5 and 4.0 mM TDC. Activity of 1.5 µg of PTL against
tributyrin was measured at increasing concentrations of native or
mutant colipases at both 0.5 and 4.0 mM TDC. The activity
with no colipase was subtracted from the experimental values obtained
in 0.5 mM TDC. The results are expressed as micromoles of
fatty acid released per minute. The regression lines are
plotted. A, C, and E, alanine mutants:
, native colipase;
, E45A;
, D89A;
, R65A;
, E64A.
B, D, and F, nonalanine mutants:
,
native colipase;
, R65K;
, E64D;
, E64Q;
, R65E.
E and F were replotted from the data in
C and D, respectively, to emphasize the lower
colipase concentration ranges.
Apparent Bmax and Kd of colipase mutants at 0.5 mM TDC
Apparent Bmax and Kd of colipase mutants at 4 mM TDC
; B,
).
With the remaining mutants, PTL had decreased maximal velocities. These
impressions were confirmed by calculation of the apparent
Km and maximal Vmax of the
colipase·PTL complex for tributyrin (Table
III). The mutants and wild type colipase had similar values for Km and
Vmax in 0.5 mM TDC. The complex
produced with all tested mutants in 4.0 mM TDC had an apparent Km equal to that of the complex with wild
type colipase. E45A and R65K·PTL complexes had normal apparent
maximal velocities. Complexes with the remaining mutants had decreased apparent Vmax. PTL had low activity with E64A,
and the data did not provide a good fit. Consequently, the apparent
constants were not reported, but the shape of the curve suggested that
the E64A mutation primarily affected the maximal velocity. The
decreased apparent Vmax indicated that some of
the mutants form fewer productive complexes with PTL in 4.0 mM TDC than does native colipase, a finding consistent with
decreased affinity of the colipase mutants for PTL. Once a complex
forms between the various mutant colipases and PTL, the complex has a
normal affinity for the substrate. That is, the mutations in the
hydrophilic surface of colipase do not affect the binding of the
complex to the substrate interface once the complex has formed.
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Fig. 5.
Mutant colipase dependence on substrate
concentration. The activity PTL against tributyrin with native and
mutant colipases was measured in 0.5 and 4.0 mM TDC. Each
assay contained 2.8 µg of PTL and 0.56 µg of colipase or mutant
colipase. PTL activity was determined using a Radiometer pH-stat as
described under "Materials and Methods." The regression
lines are plotted. A and B show the results
with 0.5 mM TDC. C and D show the
results with 4.0 mM TDC. A and C,
alanine mutants: , native colipase;
, E45A;
, D89A;
, R65A;
, E64A. B and D, nonalanine mutants:
,
native colipase;
, R65K;
, E64D;
, E64Q;
, R65E.
Apparent Vmax and Km of the mutant colipase · lipase complex
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Fig. 6.
Binding of colipase mutants to substrate and
colipase-mediated anchoring of PTL to substrate. Colipase binding
to tributyrin and its ability to anchor PTL to tributyrin was
determined as described under "Materials and Methods." The binding
and anchoring data are expressed as the percent bound of the total
colipase or PTL added. The ability of each mutant to activate PTL in
the presence of 4.0 mM TDC is graphed as a percentage of
PTL activity with native colipase. The activity was determined with a
1:2 molar ratio of colipase to PTL. A, alanine mutants;
B, nonalanine mutants. Black bars, colipase
binding; light gray bars, anchoring of PTL; dark gray
bars, PTL activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD33060 and DK52574.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.
§ Present address: Division of Pediatric Gastroenterology and Nutrition, Columbus Children's Hospital, 700 Children's Dr., Columbus, OH 43205.
To whom correspondence should be addressed: Washington
University School of Medicine, 660 South Euclid Ave., Campus Box 8208, St. Louis, MO. Tel.: 314-286-2857; Fax: 314-286-2894; E-mail: Lowe@kids.wustl.edu.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M009986200
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ABBREVIATIONS |
---|
The abbreviations used are: PTL, pancreatic triglyceride lipase; CMC, critical micelle concentration; TBST, Tris-buffered saline with Tween 20; TDC, taurodeoxycholate; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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