From the Departments of Cell and Molecular Biology
and
Physical Chemistry 2, Lund University, S-221 00 Lund,
Sweden, Departments of § Chemistry and
§§ Molecular Physiology and Biological Physics,
University of Virginia, Charlottesville, Virginia 22906, ** Department
of Chemistry and Biochemistry, University of California, Los Angeles,
California 90024, and
Lipid Research Laboratory, West
Los Angeles Veterans Affairs Medical Center, Los Angeles, California
90073
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ABSTRACT |
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Structure-function relationship analyses of
hormone-sensitive lipase (HSL) have suggested that this metabolically
important enzyme consists of several functional and at least two
structural domains (Østerlund, T., Danielsson, B., Degerman, E.,
Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420;
Contreras, J. A., Karlsson, M., Østerlund, T., Laurell, H.,
Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in
more detail, we applied biophysical methods. Denaturation of HSL was
followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event.
Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one
corresponding exactly to the proposed N-terminal domain. Major cleavage
sites were found in the predicted hinge region between the two domains
and in the regulatory module of the C-terminal, catalytic domain.
Analyses of a hinge region cleavage mutant and calculations of the
hydropathic pattern of HSL further suggest that the hinge region and
regulatory module are exposed parts of HSL. Together, these data
support our previous hypothesis that HSL consists of two major
structural domains, encoded by exons 1-4 and 5-9, respectively, of
which the latter contains an exposed regulatory module outside the
catalytic The release of fatty acids from stored triglycerides in adipocytes
is accomplished by hormone-sensitive lipase
(HSL)1 and monoglyceride
lipase. Lipolysis is regulated by hormones and neurotransmitters, and
the major target of this regulation is HSL (1, 2). Catabolic hormones
and neurotransmitters (e.g. norepinephrine) activate
cAMP-dependent protein kinase, which, in turn,
phosphorylates and activates HSL. The major antilipolytic hormone is
insulin, which activates phosphodiesterase 3B that decreases cAMP
levels and thereby deactivates cAMP-dependent protein kinase (3). The regulation by reversible phosphorylation makes HSL
unique among lipases. Phosphorylation of HSL by
cAMP-dependent protein kinase occurs at three sites, the
serines 563, 659, and 660,2
both in vitro and in primary rat adipocytes (4). A fourth site of phosphorylation, Ser-565, may be a substrate of the
AMP-activated protein kinase, because this has been shown to be the
case in vitro (5). Also, preincubations of primary rat
adipocytes with the AMP-mimicking agent 5-aminoimidazole-4-carboxamide
1- HSL is known to associate with phospholipid vesicles (7) and to have an
overall amphipathic character (8). This suggests that HSL has one or
more sites at the surface, enabling interactions with detergent and
membranes such as the surface of intracellular lipid droplets. Specific
recognition of these lipid droplets, as opposed to other membrane
surfaces, would constitute another unique feature of HSL among lipases.
As for other lipases and esterases, it was expected that HSL, at least
in part, harbors the To establish the structural domain composition of HSL, we have
performed spectroscopic analyses during denaturation. Data from these
analyses support the idea that HSL has two major structural domains.
Limited proteolysis analyses (10) have been extended through
identification of generated peptides by mass spectrometry to identify
exposed regions of the intact protein and to identify more stable
fragments of the HSL molecule. Both the suggested hinge region and the
regulatory module are exposed to proteolysis, whereas the N-terminal
domain appears to be highly resistant to cleavage. Analysis by
proteolysis of a HSL mutant with an engineered Factor X site in the
predicted hinge confirms these results, and further support is provided
by analysis of the hydropathic pattern of HSL. Based on the generated
results, we present a more complete overall structural model for
HSL.
Recombinant HSL--
Recombinant rat HSL was expressed and
purified as described previously (10, 14). After this purification,
which includes the use of Q- and phenyl-Sepharose columns, HSL was
concentrated using small (1 - 3 ml) Q-Sepharose columns (14). HSL was
applied to these columns either directly in the phenyl-Sepharose
elution buffer or after dialysis against 20 mM Tris
acetate, pH 7.5, 1 mM dithioerythritol (DTE), 0.2%
C13E12 (a non-ionic alkyl polyoxyethylene ether-type detergent; see Ref. 14), and 10% glycerol. Washing was
performed with Buffer A (50 mM Tris acetate, pH 7.5, and 1 mM DTE) supplemented with different types and
concentrations of detergents and different concentrations of glycerol,
depending on the type of analysis after concentration (14). Elution was performed in one step with the particular Buffer A supplemented with
300 mM sodium acetate.
Circular Dichroism Spectroscopy--
HSL subjected to
denaturation by guanidine hydrochloride (GdnCl) or thermal denaturation
was monitored using circular dichroism spectroscopy (CD) on a J-720
spectropolarimeter (Jasco). HSL samples (50 µl; 2 mg/ml in Buffer A
with 0.2% C13E12 and 5% glycerol) were mixed
with 200 µl of Buffer P (100 mM potassium phosphate, pH
7.25, 0.9% sodium chloride, and 1 mM DTE) with different
concentrations of GdnCl, and the CD signal was measured at 222 nm in a
1-mm cuvette. Final concentrations of glycerol and detergent
(C13E12) were 1% and 0.04%, respectively.
After measurements by CD, the samples were recovered and stored at
For analyses during thermal denaturation, 200 µl of HSL (0.4 mg/ml)
in 5 mM potassium phosphate, pH 7.4, 1 mM DTE,
50% glycerol, and 0.2% C13E12 were mixed with
300 µl of Buffer P without GdnCl and monitored by CD at a gradually
increasing temperature ranging from 4 °C to 90 °C.
Fluorescence Spectroscopy--
Fluorescence measurements were
performed on a LS50B luminescence spectrometer (Perkin-Elmer). HSL
samples (200 µl) recovered from the CD measurements were further
diluted with either 600 µl of Buffer P or Buffer P supplemented with
0.04% C13E12 and 1% glycerol, containing the
same concentration of GdnCl as the original sample. All samples were
then excited at both 280 and 295 nm, and data were collected from 305 to 420 nm. 50 µl of glycerol or 10 µl of 20%
C13E12 were then added to the samples diluted in Buffer P, and 50 µl of glycerol were added to the samples diluted in detergent/glycerol-supplemented buffer P, and then the fluorescence was measured again.
Analytical Ultracentrifugation--
For sedimentation
equilibrium centrifugations of HSL, Brij-96 was used as detergent
instead of C13E12 because the partial specific
volume of Brij-96 is close to 1. The exchange of
C13E12 for Brij-96 was performed on the last
Q-Sepharose column used in the purification of recombinant HSL (see
above). After application of the enzyme from the phenyl-Sepharose step,
the column was washed with 50 column volumes of 50 mM Tris
acetate, pH 7.5, 0.1 mM DTE, 5% glycerol, and 0.006%
Brij-96 (Fluka). Elution was performed in the same buffer supplemented
with 0.3 M sodium acetate, and then the pooled material was
dialyzed against phosphate-buffered saline, 0.1 mM DTE, 5%
glycerol, 0.006% Brij-96, and 0.5 mM EDTA. Sedimentation
equilibrium centrifugations were performed at an initial HSL
concentration of 0.08 mg/ml in the absence and presence of 0.2 M GdnCl in a Beckman Optima XL-A analytical centrifuge at
7,000 rpm at 4 °C, using absorption optics and data analysis software provided by the manufacturer.
Proteolysis--
The HSL preparation (2 mg/ml) used for CD and
fluorescence analysis was also used for proteolytic digests.
Endoproteinase Lys-C (EndoL) was suspended to 3 units/ml in the
supplied buffer (Promega). Both initial and comprehensive proteolysis
of HSL were performed in the same reaction. Proteolysis was initiated
by mixing 50 µl of HSL with 1 µl of protease at room temperature.
Aliquots of 5 µl were taken for analysis by SDS-PAGE at three time
points up to 5 min. At 5 min, an additional 5 µl of protease were
added, and proteolysis continued for an additional 90 min. From the
more extensive digest, five more aliquots of 6 µl were taken for
SDS-PAGE analysis. All aliquots were added directly to tubes containing 2× SDS-PAGE loading buffer.
SDS-PAGE and Peptide Blotting--
The HSL digests were analyzed
on SDS-PAGE using the Tris/HCl-tricine buffer system (15). Samples from
initial digests were run on 8% gels, and samples from the more
extensive digests were run on 12% gels. After electrophoresis,
peptides were blotted onto polyvinylidene difluoride membranes
(Immobilon P; Millipore) and visualized with 0.1% Ponceau Red (Sigma).
Mass Spectrometric Analysis--
From the HSL peptide blots,
distinct bands were excised, destained in 200 µM sodium
hydroxide, and rinsed in water. The membrane pieces were then subjected
to digestion using sequencing-grade modified trypsin (Promega) at
37 °C overnight. The tryptic peptides were separated on a
C18 reverse phase high pressure liquid chromatography microcapillary column using a gradient from 0% to 80% acetonitrile in
0.1 M acetic acid and eluted into a triple quadrupole mass spectrometer (Finnigan) equipped with an electrospray ionization source. Several tryptic peptides from each EndoL-derived HSL fragment were subjected to collision-induced dissociation analysis, generating even smaller heterogenous and overlapping peptide fragments from which
the primary structures were deduced.
HSL Cleavage Mutant--
A cleavage mutant of rat HSL was
constructed by a polymerase chain reaction-based overlap extension
method (16), as described previously (12). The codons for amino acids
320-323 (SLAK) were mutated to encode a specific Factor X site (IEGR)
at which cleavage will take place after the arginine. A cassette
approach was used to replace a fragment of the full-length rat HSL
cDNA in pVL1393 (10) by the equivalent polymerase chain
reaction-generated fragment containing the mutations. Recombinant
baculovirus was generated using the BaculoGold transfection kit as
described by the manufacturer (PharMingen). Production in Sf9
cells and subsequent purification of the mutant HSL
(HSLFacX), was performed as described for the wild-type HSL
(see above). The purified mutant was subjected to proteolysis at
37 °C with Factor Xa (New England Biolabs) by mixing 10 µl of
HSLFacX (2 mg/ml in Buffer A with 0.2%
C13E12, 10% glycerol, and 300 mM
sodium acetate) with 39 µl of 20 mM Tris/HCl, pH 7.5, 100 mM sodium chloride, 2 mM calcium chloride, and
1 µl of protease (1 mg/ml). Aliquots of 6 µl were taken at
different time points and subjected to SDS-PAGE analysis on 8% gels as
described above. Peptides were visualized by staining with Coomassie
Brilliant Blue (Serva). As a control, the same procedure was performed
with wild-type HSL.
Denaturation Experiments--
To analyze the unfolding of HSL, CD
and fluorescence spectroscopy were used to follow changes in the
secondary and tertiary structure, respectively, brought about by
increasing GdnCl concentrations. For the fluorescence spectroscopy, two
different series of denaturation were created. One series had identical
detergent and glycerol concentrations as the CD measurements, and one
had lower detergent and glycerol concentrations (diluted in salt
buffer). Fig. 1 shows the fluorescence
scans of HSL in low GdnCl (20 mM) and high GdnCl (5.44 M) concentrations, thus comparing the folded and denatured states. The spectrum appears to be composed of three overlapping peaks,
of which two decrease significantly upon denaturation (peak 1 at 321 nm
and peak 2 at 333 nm). At these wavelengths, only tryptophan residues
have fluorescence emission. Five tryptophans are found in rat HSL, two
are found in the suggested N-terminal domain (Trp-238 and Trp-242), two
are found in the
It is clear from Fig. 2 that HSL is stabilized in the detergent and
glycerol series as compared with the salt-diluted series. This is
probably due to the interaction of these compounds with the protein,
shielding it from GdnCl and shifting the denaturation toward higher
GdnCl concentrations. The effects of detergent and glycerol are most
markedly seen during denaturation of the more stable domain. Replotting
of the series diluted in salt buffer and the series diluted in
detergent/glycerol-supplemented salt buffer in the range from 1 to 6 M GdnCl (data not shown) allowed estimations of the changes
in free energy of transition from the native (N) to the unfolded (U)
state in the absence of denaturant (
Additions of glycerol to either series had only marginal effects (data
not shown), whereas the addition of detergent (0.23% final
concentration) to the salt-diluted series markedly increased relative
fluorescence in the range from 1 to 5 M GdnCl. Whether this
addition induces some refolding or the detergent interacts directly
with tryptophans to increase fluorescence is not clear. Because
glycerol on its own had very little effect, it is likely that the
stabilizing effect in the detergent/glycerol/salt buffer-diluted series
is mainly due to the detergent.
Fig. 4 shows the normalized CD
measurements of HSL with increasing GdnCl. The unfolding seems quite
complex, but two major reductions in the secondary structure content
can be seen at 0.5-1.5 and 2.5-4 M GdnCl, respectively.
The fluorescence data from the detergent/glycerol-diluted series have
been replotted (Fig. 4) because these are measured under the same
conditions of detergent and glycerol as the CD measurements. The
reduction in fluorescence does not seem to be accompanied by a loss of
secondary structure content from 0 to 0.4 M GdnCl. In
contrast, as GdnCl concentration increases to 1.5 M, there
is a substantial loss in the CD signal but only a small reduction in
fluorescence. From 2.5 to 4 M GdnCl, there is a clear
reduction in both the secondary and tertiary structure, corresponding
to the complete unfolding of a second domain. It is possible to
envisage that at low GdnCl concentrations, one domain unfolds without
the disruption of secondary structure elements (particularly
HSL was also analyzed by CD during thermal denaturation. Fig.
5 shows the result of one such
experiment, in which it can be seen that the reduction in the secondary
structure content takes place in two steps. Absorption measurements
(data not shown) indicate that some protein aggregation takes place
during thermal denaturation. However, comparing CD scans from different
temperatures (data not shown) and comparing the results in Fig. 5 with
the results of a thermal denaturation in which complete aggregation
occurred (data not shown) provide evidence that protein aggregation
does not contribute to the loss in the CD signal. Together, the
spectroscopy data strongly support the notion that HSL is composed of
two major structural domains.
Proteolytic Analysis--
To identify those putative proteolytic
sites in HSL that are accessible to enzymatic cleavage, we turned to
limited proteolysis. From our previous study using limited proteolysis,
it was clear that lipase activity (hydrolysis of
phospholipid-stabilized lipid substrate emulsions) demanded an almost
intact protein, whereas esterase activity (hydrolysis of soluble
substrates) could be measured after extensive proteolysis (10). Among
the regions in the suggested domain structure (10, 11) predicted to be particularly accessible to proteolysis are the regulatory module (approximately residues 460-680), because this probably contains few
secondary structure elements (11) and serves as a substrate for kinases
and phosphatases, and the putative hinge region between the two
structural domains (approximately residue 315-335). HSL was digested
with EndoL, and the peptides generated were identified by mass
spectrometry. Both initial proteolysis with a low concentration of
EndoL and extensive digestion with a much higher concentration of
protease were performed. Fig. 6 shows the
generated HSL peptides on a nylon membrane. Several products are
observed in the initial digest (Fig. 6A), of which one is
more abundant, whereas three stable fragments can be seen even after 90 min of extensive proteolysis (Fig. 6B). The major band
appearing after initial proteolysis (p64) corresponds to
residues 1-583. Therefore, a major site of cleavage is probably
located at Lys-583 in the anticipated regulatory module. The three more
stable bands (p36, p25, and p20) cover three
different parts of HSL. The most abundant is p36, which encompasses
residues 1-323 and thus corresponds exactly to the suggested
N-terminal domain (10). Another major band is p25, which corresponds to
the C-terminal 229 amino acids (540-768). Because cleavage at Lys-539
(generation of p25) and Lys-583 (generation of p64) cannot generate
both p25 and p64, there must be more than one pathway of cleavage. In
the generation of p25, cleavage has taken place at another site in the
regulatory module as compared with the cleavage that generates p64. In
fact, in these two pathways, cleavage takes place at opposite sides of
two of the phosphorylation sites (Ser-563 and Ser-565). The nature of
the p20 band has not been definitely established because it appears
that at least two peptides are present. However, there is strong
evidence that one peptide is residues 359-540. This fits well with a
previously identified stable tryptic peptide in bovine HSL
corresponding to residues 333-499 (18). The p20 (residues 359-540)
peptide, together with p36 and p25, covers almost the entire HSL
protein. There is also evidence that another peptide in the p20 band
could be residues 1-161. This would indicate that p36 is not
completely resistant to proteolysis, although it takes several hours of
continued proteolysis to observe a reduction in the intensity of this
band (data not shown).
HSL Cleavage Mutant--
Encouraged by the finding that
proteolysis takes place in the predicted regions according to the
domain structure model (see above), we constructed a mutant of HSL with
a specific cleavage site for Factor X (HSLFacX) in the
proposed hinge region. The mutant HSL was expressed in insect cells to
the same level as the wild-type protein (data not shown). After
purification of HSLFacX, its specific activity was
estimated to be the same as that of wild-type HSL (10). Cleavage by
Factor X takes place at an introduced Arg-323 in the putative hinge
region between the two suggested structural domains. This arginine
replaces Lys-323 that was shown to be a cleavage site for EndoL (Fig.
6). Fig. 7 shows the cleavage of
HSLFacX with Factor X compared with the cleavage of
wild-type HSL. It is clear that initial cleavage primarily takes place
at the introduced site in HSLFacX, indicating that in the
intact protein, this site is accessible to the protease. After cleavage
of the two domains, the C-terminal domain is subjected to further
nonspecific cleavage, whereas the N-terminal domain is not. Under the
same conditions, there is also significant nonspecific cleavage of the
wild-type protein. From the pattern of cleavage, it is suggested that
there are three major nonspecific cleavage sites for Factor X in
wild-type HSL, probably all in the regulatory module. This suggestion
is based on two observations. First, the three larger bands range from
50 to 70 kDa in size, comparable to the major band generated initially
by EndoL (Fig. 6), and secondly, only the C-terminal domain of
HSLFacX undergoes any further significant cleavage.
Intriguingly, some of the smaller bands generated by cleavage of this
domain correspond exactly in size to bands in the wild-type HSL digest.
Attempts to crystallize HSL have been unsuccessful thus far, most
likely due to the demand for detergent in HSL preparations, heterogeneity of the protein (it has four phosphorylation sites), and
instability of the protein (see above). To generate the separate domains for further crystallization attempts, procedures for the purification of the two domains after cleavage of HSLFacX
will be worked out.
In this study, further evidence for our previously proposed model
for the domain structure of HSL is provided (10, 11). The denaturation
analyses support that HSL has at least two (and probably only two)
structural domains. The fluorescence data clearly suggest the unfolding
of two individually folded domains. Although HSL appears to be a
homodimer in solution, it is unlikely that the dissociation of dimers
contributes significantly to the loss in fluorescence at low GdnCl
concentrations. CD measurements during denaturation by GdnCl and heat
indicate that the unfolding of secondary structure elements occurs in
two major steps. It cannot be ruled out that some protein aggregation
occurs in the thermal denaturation. However, it does not seem to have
any significant influence on the CD signal.
From both spectroscopic data and proteolytic analyses, it appears that
one domain is more fragile than the other. The fragile domain is
unfolded by low GdnCl concentrations and probably represents the
C-terminal domain, because all HSL activities are lost concomitant with
its unfolding (10). This domain is also highly sensitive to proteolytic
digestion, especially in the regulatory module, as shown by proteolytic
cleavage by both EndoL and Factor X. The second unfolding, which occurs
at high GdnCl concentrations, probably represents the N-terminal
domain. Because detergent and perhaps glycerol had a more protective
effect on this domain than on the other domain, it is speculated that
some sites of detergent and membrane interactions are located here.
Other sites might be found in the /
-hydrolase fold core.
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INTRODUCTION
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-D-ribofuranoside inhibit isoprenaline-induced
lipolysis (6), thus lending support for a role of AMP-activated protein
kinase in the regulation of HSL.
/
-hydrolase fold and has a catalytic triad
(Ser, Asp/Glu, His) (9). To map these and other features in HSL, we
have initiated analyses into its structural and functional domains by
alignments to other lipases and esterases and by limited proteolysis,
denaturation, site-directed mutagenesis, and molecular modeling.
Analysis by limited proteolysis and denaturation suggested that HSL has
at least two structural domains (10). Those regions in the C-terminal
part of the molecule that align to other lipases and esterases are
thought to adopt the
/
-hydrolase fold and to harbor the catalytic
triad (10, 11). In fact, it was possible to build a model for the
catalytic
/
-hydrolase fold core (11). The proposed residues of
the catalytic triad have been probed by site-directed mutagenesis and
found to be essential for activity (12, 13), strongly supporting the
structural model for the catalytic core. In the primary structure, this
core is interrupted by approximately 200 residues, including the four
phosphorylation sites, which are thought to form a regulatory module
(10, 11). This part is a substrate of kinases and phosphatases and has
been predicted to contain only a few secondary structure elements (11). Thus, it is suspected to be an exposed and rather flexible module. The
N-terminal part of the protein (approximately 320 residues) is believed
to constitute a separate structural domain and shows no significant
sequence similarity to other proteins (10).
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80 °C until they were analyzed by fluorescence spectroscopy (see below).
RESULTS
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/
-hydrolase fold core (Trp-368 and Trp-402), and
one is found in the regulatory module (Trp-525). An isofluorescent
point was identified at 362 nm. The fluorescence at peak 1 and peak
2 was normalized for all samples using the value at 362 nm to
compensate for the differences in HSL concentrations between samples.
The results of these calculations are shown in Fig.
2. Only results from excitation at 280 nm
measured at peak 1 are shown, because very similar results were
obtained at peak 2 and when samples were excited at 295 nm. It is clear that loss of fluorescence takes place in two steps, at a Cm
of approximately 0.15 and 3.5 M GdnCl, respectively,
indicating the unfolding of two independent domains. Gel filtration
studies using small amounts of rat HSL purified from adipose tissue
have suggested that HSL may exist as a homodimer in solution (1). The
loss in fluorescence at a low GdnCl concentration may therefore arise due to the dissociation of dimers. We have initiated analyses of the
subunit composition of HSL by ultracentrifugation and gel filtration.
The results obtained strongly indicate that HSL is a homodimer (Fig.
3).3
Ultracentrifugation experiments of HSL in the presence of GdnCl were
also performed. The results of sedimentation equilibrium of HSL at an
initial concentration of 0.08 mg/ml are presented in Fig. 3. The
equilibrium gradients, both in the absence and presence of 0.2 M GdnCl, are best fit by a single species of about 170,000 Da, and the goodness of fit is indicated by the random distribution of
the residuals. Therefore, at the concentration used for fluorescence
measurements, there is no significant dissociation of dimers at 0.2 M GdnCl. Thus, it is likely that most of the loss in
fluorescence at low GdnCl concentrations is due to changes in the
tertiary structure.
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Fig. 1.
Fluorescence scans of HSL excited at 280 nm. HSL in 20 mM (solid line) or 5.44 M GdnCl (dashed line) was measured from 305 to
420 nm. Peak 1 (1) is at 321 nm, peak 2 (2) is at
333 nm, and peak 3 (3) is at 351 nm, whereas an
isofluorescent point (iso) is seen at 362 nm.
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Fig. 2.
Fluorescence measurement of HSL during
denaturation by GdnCl. Fluorescence of HSL at peak 1 (Fig. 1) was
measured in salt buffer ( ) or in salt buffer with detergent and
glycerol (
). Detergent was added to some of the salt buffer samples,
after which they were measured again (
). The right panel
shows the results expanded over 0-1 M GdnCl (dotted
rectangle) due to the many data points in this area. The data
points for each series are from one experiment. Very similar results
were obtained at peak 2 (Fig. 1) and when excitation was performed at
295 nm instead of 280 nm (see "Experimental Procedures" and
"Results").
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Fig. 3.
Sedimentation equilibrium of HSL in the
absence and presence of GdnCl. HSL at an initial concentration of
0.08 mg/ml was analyzed by sedimentation equilibrium centrifugation as
described under "Experimental Procedures" in the absence
(A) and presence (B) of 0.2 M GdnCl.
Nonlinear square curve fitting of the equilibrium gradients, absorbance
(at 232 nm in A and at 234 nm in B)
versus radius, gave molecular weights of 164,000 for
A and 176,000 for B. The residuals (the
differences between the data and curve fit) are random, indicating that
a single species is a reasonable fit.
GNU(H2O)) and midpoint concentrations of
GdnCl (Cm) for the unfolding (17) of this
domain. The salt-diluted and detergent/glycerol/salt-diluted series had
an estimated
GNU(H2O) of 17 and 21 kJ/mol
and a Cm of 3.1 and 3.8 M GdnCl,
respectively. This confirms that there is a marked effect of detergent
and glycerol on the unfolding of this particular domain.
-helices). Then, as GdnCl increases from 0.5 to 1.5 M,
the secondary structure elements of this domain unfold with a
Cm of approximately 0.9 M GdnCl and
only minor additional exposure of fluorescent side chains. Above 2.5 M GdnCl, both the exposure of fluorescent side chains and
the disruption of secondary structure elements in a second domain
occur. Of course, the general picture can be reconciled with the
presence of even more structural domains in HSL, but hardly with only
one domain. It is noteworthy that the loss in fluorescence below 0.5 M GdnCl coincides with the loss of HSL activities measured
at these concentrations of GdnCl (10). This suggests that it is
primarily the C-terminal domain, harboring the catalytic core/machinery
(11), that is affected at these low GdnCl concentrations.
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Fig. 4.
GdnCl denaturation of HSL measured by CD and
fluorescence spectroscopy. Changes in relative CD signal at 222 nm
( ) and fluorescence at peak 1 (
) were measured as a function of
GdnCl concentration under the same conditions with respect to detergent
and glycerol. The fluorescence data are replotted from Fig. 2. The CD
data points are from one experiment repeated three times.
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Fig. 5.
Thermal denaturation of HSL measured by
CD. Denaturation of HSL by increasing temperature was measured at
222 nm and expressed as a normalized CD signal. Single data points are
from one experiment repeated four times.
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Fig. 6.
Proteolytic digestion of HSL by EndoL.
HSL was subjected to limited proteolysis by EndoL at (A) low
and (B) high concentrations of protease. HSL peptides were
analyzed by SDS-PAGE on 8% (A) and 12% (B)
gels, respectively. After electrophoresis, peptides were identified by
staining with Ponceau Red. Lanes 1 and 5 are
molecular mass markers (in kDa). Lanes 2-4 are proteolytic
digests for 0.5, 2, and 5 min, respectively. Lanes 6-10 are
digests for 2, 10, 30, 60, and 90 min, respectively, after a further
addition of EndoL (see "Experimental Procedures"). The different
peptides that have been identified are indicated to the
right of B, and the residues that they represent
in HSL, based on mass spectrometric analyses, are indicated in
parentheses. Tentative assignments are indicated by
question marks.
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Fig. 7.
Proteolytic digestion of
HSLFacX and wild-type HSL by Factor X. Proteolysis of HSLFacX (lanes 2-6) and HSL
(lanes 9-11) was performed for 2 min (lane 2), 5 min (lanes 3 and 9), 15 min (lane 4),
30 min (lanes 5 and 10), and 60 min (lanes
6 and 11). Intact (FL) HSLFacX
was run in lane 1. Lanes 7 and 8 contain molecular mass markers (in kDa). Full-length HSL
(FL), the C-terminal domain (C-dom), and the
N-terminal domain (N-dom) are indicated. Other proteolytic
fragments are indicated by arrows.
DISCUSSION
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ABSTRACT
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/
-hydrolase fold core, although
most hydrophobic residues of this part are not exposed (11). To
directly compare the pattern of hydrophobicity in the primary structure
of HSL with the proposed domain structure, the mean hydropathic index
was calculated for every 60 residues, according to Kyte and Doolittle
(19). Fig. 8 shows the calculated
hydropathy indices with indications of the localization of suggested
domains in the primary structure. Hydrophilic regions correlate well
with the anticipated hinge region and regulatory module.
Arrows indicate the major cleavage sites as identified in
Fig. 6. The most hydrophobic regions are those of the
/
-hydrolase
fold, whereas the N-terminal domain has an amphipathic character.
Because the regulatory module is probably located at the surface of the
C-terminal domain, and most of the hydrophobic residues of the
/
-hydrolase fold are located in the core of the fold (11), there
are probably only a few exposed hydrophobic patches in this part. The
membrane/detergent binding sites are presumably located at these
patches as well as at hydrophobic patches in the N-terminal domain.
Overall, the hydropathy calculations support the suggested domain
structure, particularly by demonstrating that the exposed regions are
markedly hydrophilic.
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Fig. 8.
Calculated hydropathy pattern of HSL.
The mean hydropathy index was calculated in sections of 60 amino acids
using the Kyte and Doolittle (19) algorithm in the GeneWorks program
(IntelliGenetics Inc.) with a span of 11 residues, as recommended. The
mean hydropathy indices are directly compared with the proposed domain
structure as outlined in the schematic primary structure
(bar). The N-terminal domain is dark gray, the
hinge region is black, the regulatory part is light
gray, and the /
-hydrolase fold core is white.
Arrows indicate the major proteolytic cleavage sites
determined in Fig. 6.
The exact location of the hinge region in the primary structure has
been the subject of some speculation (10, 11). The cleavage at Lys-323
and the hydrophilic character of this region support the concept that
the N- and C-terminal domains are separated at the hinge region
(residues 315-335) as suggested from alignments (10). Cleavage of
HSLFacX by Factor X shows that this region is immediately
accessible to the protease. Our current view and working hypothesis of
the domain structure is illustrated in Fig. 9 with indications of cleavage sites and
action of GdnCl at different concentrations. The secondary structure
elements of the /
-hydrolase fold are outlined (11).
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In conclusion, the present study provides additional support for the
proposal that HSL is composed of two major structural domains encoded
by exons 1-4 and 5-9, respectively. The latter is the catalytic
domain formed by an /
-hydrolase fold core (11), which is
interrupted in the primary structure by the insertion of a regulatory
module. Both the regulatory module and the suggested hinge are exposed
hydrophilic parts. Additional investigations of HSLFacX
before and after cleavage and the expression of individual domains are
underway. Purified domains and fragments will be valuable tools for
structural and functional analyses as well as the determination of the
three-dimensional structure of HSL.
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ACKNOWLEDGEMENTS |
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We thank Birgitta Danielsson for excellent technical assistance regarding the expression and purification of wild-type HSL and Drs. Howard Wong and Henry Choy for contributing unpublished data on the subunit structure of HSL.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council (Grant 11284 to C. H.), A. Påhlsson's Foundation, the Crafoord Foundation, the Novo Nordisk Foundation, the Swedish Diabetes Association, and the Veterans Affairs Merit Review.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: Dept. of Medical Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-1333
¶¶ To whom correspondence should be addressed: Section for Molecular Signalling, Dept. of Cell and Molecular Biology, Lund University, S-221 00 Lund, Sweden. Tel.: 46-46-222-85-81; Fax: 46-46-222-40-22; E-mail: cecilia.holm{at}medkem.lu.se.
2 The residue numbering is for rat HSL.
3 P. Poon, T. Østerlund, M. C. Schotz, C. Holm, and H. Wong, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HSL, hormone-sensitive lipase; DTE, dithioerythritol; GdnCl, guanidine hydrochloride; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
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REFERENCES |
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