1 Section for Molecular Signalling, Department of Cell and Molecular Biology, Lund University, PO Box 94, S-221 00 Lund, Sweden and 3 Unité INSERM 317, Institut Louis Bugnard, Hôpital Rangueil, F-31403 Toulouse Cedex 4, France
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Abstract |
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Keywords: /ß-hydrolases/cold adaptation/hormone-sensitive lipase/p-nitrophenyl butyrate/site-directed mutagenesis
Abbreviations: HSL, hormone-sensitive lipase hHSL, human HSL rHSL, rat HSL UCP, uncoupling protein WAT, white adipose tissue BAT, brown adipose tissue FFA, free fatty acids MOME, 1(3)-mono[3H]oleoyl-2-O-monooleylglycerol pNPB, p-nitrophenyl butyrate TBS, Tris-buffered saline BFA, Brefeldin A
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Introduction |
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The structural basis for cold adaptation of enzymes is not known. It has generally been assumed that cold adaptation or psychrophilicity is accompanied by increased flexibility, whereas thermophilic enzymes are characterized by a high degree of rigidity. Experimental evidence supporting the general validity of these hypotheses is, however, lacking. The crystal structure of only five psychrophilic enzymes have been solved to date, -amylase (Aghajari et al., 1998
), a Ca2+Zn2+ protease (Villeret et al., 1997
), triosephosphate isomerase (Alvarez et al., 1998
), citrate synthase (Russell et al., 1998
) and malate dehydrogenase (Kim et al., 1999
), all isolated from Antarctic bacteria. Although these enzymes have not yet all been analysed in detail with regard to comparisons with their mesophilic counterparts, it seems clear that increased overall flexibility is not a general characteristic of psychrophilic enzymes. Significantly lower average main-chain B-factors have been described for malate dehydrogenase and citrate synthase from cold-adapted microorganisms in comparison with their thermophilic enzyme counterparts (Russell et al., 1998
; Kim et al., 1999
). However, local increased flexibility in critical regions was observed in the structures for both psychrophilic enzymes, suggesting that increased flexibility and accessibility of, for instance, the active site region could be an important factor rendering enzymes more tolerant to low temperatures. Other structural features of enzyme cold adaptation have been suggested, such as increased relative flexibility between domains, longer loops, a decrease in the number of intermolecular ions pairs and an increase in the number of intramolecular ion pairs. The last feature has been proposed to protect psychrophilic enzymes from cold denaturation, a general phenomenon caused by the thermodynamically favourable hydration of non-polar protein groups (Dill and Shortle, 1991
). Concerning thermophilic enzymes, they are very similar to mesophilic enzymes with regard to sequence and structural features [for a review, see Vieille et al. (1996)] and recent amide hydrogen exchange experiments performed on Pyrococcus furiosus rubredoxin, which is the most thermostable protein characterized to date, provided no evidence for conformational rigidity (Hernandez et al., 2000
; Jaenicke, 2000
).
Accumulating evidence has suggested that the catalytic domain of most lipases and esterases share a common folding, designated the /ß-hydrolase fold (Ollis et al., 1992
; Wei et al., 1999
). The large divergence in primary structure between HSL and the first enzymes of this family to be crystallized, such as pancreatic lipase (Winkler et al., 1990
), however, contested a structural relationship. The cloning of a number of prokaryotic lipases and esterases revealed a group of enzymes related to HSL (Langin and Holm, 1993
). The existence of an HSL subfamily, closely related to the carboxylesterase B family and only distantly related to the family of classical mammalian lipases, was initially suggested by Hemilä et al. (1994). From a structural point of view, HSL appears to be the most complex member within the HSL family, with two structural domains and three distinct functional regions (Østerlund et al., 1996
, 1999
). Based on secondary structure predictions, we have proposed a three-dimensional model of the catalytic domain of HSL (Contreras et al., 1996
) (Figure 1
). This model was recently supported by the first determination of the crystal structure of a protein in the HSL family, Brefeldin A (BFA) esterase (Wei et al., 1999
).
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Materials and methods |
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All mutants were constructed by a two stepfour primer overlap/extension PCR method (Horton et al., 1990), using Vent DNA polymerase (New England Biolabs, Beverly, MA). The final product was digested with appropriate restriction enzymes to generate a cassette containing the mutation(s), which was used to replace the corresponding cDNA fragment in the HSL cDNA cloned in a eukaryotic expression vector. The expression vector pcDNA3 (Stratagene, La Jolla, CA) and the restriction enzymes KpnI and SfiI were used for all mutants except the human insertion mutant (hHSLins) and the rat deletion mutant (rHSLdel), for which SapI/DraIII and AflIII/BssHII, respectively, were used to introduce the mutated cassettes. The wild-type cDNA used in generation of the hHSLins and rHSLdel mutants was subcloned in pSVL (Amersham Pharmacia Biotech, Uppsala, Sweden). In each mutant, the cassette (spanning 943, 791 or 373 bp, respectively) was sequenced on both strands to verify the mutation(s) and to confirm the absence of undesired mutations. Sequencing was performed on an ABI 373A automatic sequencer with the use of AmpliTaq FS (Perkin-Elmer, Foster City, CA), in some cases complemented by manual dideoxynucleotide sequencing (US Biochemical, Cleveland, OH).
Transfections
Purified plasmids (Qiagen, Hilden, Germany) were transfected in COS-1 cells using lipofectin (Gibco/BRL, Gaithersburg, MD), following the recommendations of the manufacturer. Amounts of 5 µg of plasmid DNA and 30 µl of lipofectin were used per 60 mm culture dish. After 18 h, the transfection mixture (3 ml) was removed from the cells and 6 ml of normal culture medium (Dulbecco Modified Eagle Medium with 10% foetal calf serum) were added. Cells were harvested 6772 h post-transfection and rinsed twice with PBS and the dried plates were kept at 80°C for at least 1 h. Each construct was transfected in duplicate in at least three different transfection experiments. Immediately upon thawing, 300 µl of ice-cold homogenization buffer (0.25 M sucrose, 1 mM EDTA, pH 7, supplemented with protease inhibitors: 20 µg/ml leupeptin, 2 µg/ml antipain and 1 µg/ml pepstatin) was added. The plates were scraped twice on ice using a rubber `policeman' and the 600 µl of cell mixture were homogenized by rapid passages (10 times) through a G-25 needle. Protein concentration was determined in triplicate according to Bradford (1976) and/or a modified method of Lowry (Schacterle and Pollack, 1973).
Western blot analysis
Proteins from crude COS cell homogenates (6080 µg) were separated by SDSPAGE (8% polyacrylamide in the separation gel) and electroblotted to nitrocellulose membranes (Hybond C extra, Amersham Pharmacia Biotech). Prestained molecular markers (Sigma, St. Louis, MO) were used as standards. Membranes were blocked in TBS (20 mM TrisHCl, 137 mM NaCl, pH 7.6) containing 5% dry skimmed milk and all washes were done in TBS-T (TBS supplemented with 0.25% Tween-20). Affinity-purified polyclonal rabbit anti-human HSL and 125I-labelled goat anti-rabbit IgG were used as primary and secondary antibodies, respectively. The radioactive signals were quantified in a PhosphoImager system (Fujix BAS 2000, Fuji, Tokyo, Japan).
Assays of lipase and esterase activities
Lipase activity was measured using the diacylglycerol analogue 1(3)-mono[3H]oleoyl-2-O-monooleylglycerol (MOME) as substrate at 37°C (Østerlund et al., 1996). Esterase activity was measured using p-nitrophenyl butyrate (pNPB) (Sigma) as substrate, which is water soluble under the conditions employed. pNPB was chosen as substrate in cold adaptation experiments in order to avoid the effect of the temperature on phase transitions of lipid substrates. In 10 ml glass tubes, assay buffer (0.9% NaCl, 0.1 M sodium phosphate, pH 7.25) was pre-incubated at the appropriate temperature. Enzyme (1550 µl of COS cell homogenate) and 10 µl of substrate (50 mM in acetonitrile) were added to each tube. The total reaction volume was 1 ml and the final substrate concentration was 0.5 mM. After incubation for 10 min, the reactions were terminated by addition of 3.25 ml of methanolchloroformheptane (10:9:7, v/v/v). After vortex mixing (10 s per tube) and centrifugation (20 min at 800 g) the tubes were heated at 42°C for 3 min. The absorbance (optical density) of 1 ml of the upper phase was measured in glass cuvettes at 400 nm. Equal amounts of total protein were used in all samples within the same assay and to ensure linearity the maximum activity did not exceed 2030 nmol pNPB/min [molar absorptivity (
) = 12 000 l/mol.cm]. An extract from cells transfected with the expression vector alone served as a negative control. The HSL-derived activity was 610-fold higher than the endogenous esterase activity at all temperatures. Experiments were run in triplicate. Results are expressed as percentage of wild-type activity or percentage of activity at 37°C (mean ± SEM) and were further analysed using Student's unpaired two-tailed t-test.
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Results |
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To test the importance of the multiple glycine residues in the HGGG loop (Figure 1) on the catalytic activity of HSL and whether these residues contribute to the ability of HSL to retain activity at low temperatures, we substituted one or two of the glycine residues in human HSL and expressed the mutants in COS-1 cells. Activity measurements for these mutants are presented in Figure 2
. Three double mutants were generated. In one of them, G352 and G353 (HGGG) were replaced by the corresponding residues in lipoprotein lipase (HGWT), which shows a typical mesophilic behaviour (Langin et al., 1993
). The other two double mutants generated, HGAA and HGSS, were designed to cause less drastic structural rearrangements than the previous one (Bordo and Argos, 1991
). The level of expression of these three mutants in the COS cell system was at least 50% of that of wild-type HSL (HGWT not shown, HGAA and HGSS shown in Figure 3
). Despite this, all three mutants exhibited no lipase activity and only minimal esterase activity (Figure 2
). Similar results were obtained for a deletion mutant, in which one of the glycines was deleted (HG-G) (Figures 2 and 3
).
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In the Western blot analyses of the mutants a band with slightly higher mobility than that of the intact HSL protein was consistently observed (Figure 3). This band presumably corresponds to a proteolytic fragment of the HSL protein. Since the proportion between the band corresponding to the intact HSL protein (88 kDa) and the slightly lower band was the same for wild-type HSL and the various mutants, as quantified by PhosphoImager analysis, the existence of the lower band did not significantly affect the evaluation of the expression levels. The lower expression levels (60%) of the HGAA, the HGSS and the HG-G mutants compared with the other constructs did not affect the conclusions significantly, since all these mutants were inactive (Figure 2
).
Moreover, two other glycine mutants were generated, involving glycine residues conserved in the known HSL sequences and located far from the HGGG loop. One of these, G412 in hHSL, is conserved in the carboxylesterase B family, whereas the other, G326 in hHSL, is a non-conserved glycine residue, located in the putative hinge region between the N-terminal domain and the catalytic domain (Contreras et al., 1996; Østerlund et al., 1996
, 1999
). Replacing G326 with alanine had no significant effect on either lipase or esterase activity. In contrast, mutating G412 into alanine caused a substantial reduction in enzymatic activity for both substrates (Figure 2
). These results are coherent with the present model where G412 is predicted to be located in a sharp turn between an
-helix and the central ß-strand preceding the active site serine (Figure 1
), and presumably adopts
/
angles that are outside the allowed values for residues other than glycine. Thus, replacing G412 by alanine probably causes a distortion of the structure that affects the positioning of the active site serine.
Table I summarizes the analysis of the hHSL mutants tested with regard to cold adaptation. The results are expressed as a percentage of the activity shown at 37°C for each construct. The reduced activity of the mutants affecting the HGGG loop hampered their analysis at temperatures below 17°C. No significant differences were found between the mutants and wild-type HSL at temperatures from 17 to 37°C (Table I
). Like wild-type hHSL, both mutants showed a temperature optimum of 32°C, and retained almost full activity at 27°C and close to 80% at 22°C. Only at the lowest temperature tested (17°C) was a slightly lower relative activity observed for the HGAG mutant, although not statistically significant. These data suggest that the HGGG loop of HSL is not a major structural element responsible for the high catalytic capacity of HSL at low temperatures.
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When the relative activities of human and rat HSL at different temperatures were compared, we observed that rHSL was significantly more active than hHSL at temperatures between 10 and 37°C (p < 0.0001 at 25°C, p < 0.0005 at 16°C and p < 0.01 at 10°C). Figure 4A shows a temperature-dependence plot of the pNPB-hydrolysing activity of rat and human HSL expressed in COS cells, normalized against their respective activity at 37°C. When normalized in this manner, rHSL consistently showed higher activity at 32°C than at 37°C, whereas hHSL showed similar or slightly reduced activity at 32°C than at 37°C. The only major difference between the primary structure of rHSL and hHSL, apart from the extreme C-terminal region, is found in a hypervariable region of the regulatory module, immediately preceding two of the four known phosphorylation sites (Anthonsen et al., 1998
). In this region, rHSL and mouse HSL accommodate 12 and six additional amino acids, respectively, compared with hHSL and porcine HSL. In order to investigate whether the length of this hypervariable region could influence the temperatureactivity profile, two mutant HSL constructs were made in which the 12 amino acid residues were deleted from rHSL and inserted into hHSL. Both mutants behaved very similarly to their respective wild-type enzymes (Figure 4B
), indicating that the different behaviour of rHSL in the 1037°C interval is not due to the presence of the longer, alternatively extra, loop in the regulatory module.
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Discussion |
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Extended surface loops have been suggested to render enzymes more cold adapted (Davail et al., 1994). In the case of HSL, the difference in the relative catalytic capacity at low temperatures observed between rHSL and hHSL is not due to the 12 additional amino acids immediately preceding two of the four phosphorylation sites in rHSL (Anthonsen et al., 1998
). The deletion in rHSL and the insertion of the same sequence in hHSL had no effect on the activitytemperature profiles (Figure 4B
). The overall amino acid distribution of both enzymes is very similar, as given by an 82% sequence identity. One noticeable difference is the reduced number of arginine residues and the lowered Arg/(Arg + Lys) molar ratio in the rat enzyme. Arginine residues often have a stabilizing effect on enzyme structure because each residue is able to form up to five hydrogen bonds (Borders et al., 1994
). The conversion of Arg residues into Lys residues is one strategy that psychrophilic enzymes have adapted to reduce structural rigidity without affecting the net charge (Arpigny et al., 1997
; Feller and Gerday, 1997
). In the case of HSL, a net total of four (five minus one) Arg-to-Lys substitutions are found in the C-terminal catalytic domain of the rHSL protein. This factor may contribute to the more psychrotolerant character of rHSL.
HSL from the two species analysed in the present study retained 13.2 ± 2.2% (hHSL) and 18.6 ± 2.9% (rHSL) of their respective 37°C activity at 3°C. In fact, the activity profile between 3 and 37°C for rHSL towards pNPB is identical with that described for the lipase of Psychrobacter immobilis B10, an Antarctic bacterium (Arpigny et al., 1997), and displays relatively higher activities at low temperatures than was recently described for an esterase from an Alaskan Pseudomonas sp. (Choo et al., 1998
), reflecting that HSL indeed behaves as a cold adapted enzyme.
The HSL subgroup of /ß-hydrolases consists of a growing number of enzymes from primarily prokaryotic organisms. Interestingly, HSL and a liver arylacetamide deacetylase (Probst et al., 1994
) are so far the only mammalian enzymes in this family. The other members originate from organisms that are exposed to a wide variety of living conditions (Feller et al., 1991
; Choo et al., 1998
; Manco et al., 1998
). The thermophilic member of the HSL family is an esterase (EST2) of the thermo-acidophilic eubacterium Bacillus acidocaldarius. This was originally reported as an open-reading frame (ORF3) sharing sequence homology with HSL (Hemilä et al., 1994
), but was recently purified and characterized biochemically (Manco et al., 1998
). A three-dimensional model of EST2 supports a protein folding similar to that of HSL (Manco et al., 1999
). Even though EST2 exhibits its optimum activity at 70°C, 5% of this activity is retained at 5°C, which is unusual for thermophilic enzymes (Jaenicke, 1990
; Arpigny et al., 1997
). It appears as though the general scaffolding that the proteins in the HSL subfamily have acquired is well adapted for an enzyme such as HSL, which needs to function at both low and mesophilic temperatures. However, the thermal stability of HSL is limited. The enzyme loses all activity against pNPB after 30 min of incubation at 50°C, whereas 70% remains after 30 min at 42°C compared with enzyme incubated on ice (H.Laurell, unpublished observation).
The cold adaptation of HSL most likely plays a critical role in hibernating animals for the supply of fatty acids to energy metabolism and BAT non-shivering thermogenesis. Free fatty acids, released mainly through the action of HSL, are believed to activate UCP1, a mitochondrial protein which mediates the uncoupling between O2 consumption and ATP synthesis and thereby promotes heat production. In contrast to a number of other genes expressed in BAT, such as lipoprotein lipase and UCP1, there is no cold-induced activation of HSL at the transcriptional level (Holm et al., 1987). It can be hypothesized that, during cold exposure, sympathetic nervous system activation, followed by phosphorylation and activation of a constitutively highly expressed, cold-adapted HSL, constitute the first adaptive step. Prolonged duration of the cold stress leads to severe depletion of the endogenous triglyceride reserves and activation of the second line of defence, cAMP-mediated transcriptional activation of a number of genes, including LPL (Carneheim et al., 1988
). This leads to increased LPL-mediated uptake of lipids from the circulation and increased delivery of fatty acids from white adipose tissue.
In conclusion, we have shown that the HGGG loop is a critical structural element for the catalytic capacity of HSL, although the cold adaptation of HSL cannot be attributed to the presence of a glycine stack in this element as previously hypothesized. At 3°C, HSL from humans and rat retain 16% of their activity at 37°C. However, at temperatures between 10 and 32°C, the rat enzyme is relatively and significantly more active than hHSL. This feature was not due not to the presence of an extra loop in the regulatory module in the rat enzyme. Identification of the specific structural features involved in the cold adaptation of HSL will most likely require the crystal structure of the enzyme and also structural information for other members of the HSL subfamily of carboxylesterases.
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Notes |
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4 To whom correspondence should be addressed. E-mail: cecilia.holm{at}medkem.lu.se
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Acknowledgments |
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Received March 20, 2000; revised August 2, 2000; accepted August 10, 2000.