Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Henrik Laurell1,2, Juan Antonio Contreras1, Isabelle Castan3, Dominique Langin3 and Cecilia Holm1,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these {alpha} hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.

Keywords: {alpha}/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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Triglycerides stored in adipose tissue constitute by far the largest source of energy in mammals. Under conditions where energy expenditure exceeds dietary energy supply, such as during overnight fasting, free fatty acids (FFA) are released from adipose tissue in order to maintain energy homeostasis. The hydrolysis of stored triacylglycerols, the rate-limiting step in lipolysis, is catalysed by hormone-sensitive lipase (HSL). HSL has been cloned from rat (rHSL) (Holm et al., 1988Go), humans (hHSL) (Langin et al., 1993Go), mouse (Li et al., 1994Go) and pig (Harbitz et al., 1999Go). HSL shows significant sequence homology with a growing number of bacterial proteins, including lipase 2 from the psychrotroph bacterium Moraxella TA144 (Feller et al., 1991Go; Langin et al., 1993Go), a lipase from the Alaskan psychrotroph, Pseudomonas sp. B11-1 (Choo et al., 1998Go), and a recently reported esterase from an oil-degrading bacterium strain HD-1 (Mizuguchi et al., 1999Go). The identification of the homology between HSL and the Moraxella lipase prompted us to investigate the cold adaptation of HSL and compare it with two other mammalian lipases, carboxylester lipase and lipoprotein lipase. At 10°C HSL was found to retain 3–5-fold more of its 37°C catalytic activity than both of these lipases (Langin et al., 1993Go). This psychrotolerant property is probably of substantial survival value for obligatory hibernators, whose body temperature can decrease to less than 5°C during hibernation. These animals are dependent on a cold-adapted HSL for the mobilization of fatty acids from endogenously stored triacylglycerols in white adipose tissue (WAT) and brown adipose tissue (BAT). Fatty acids are the major fuel during the hibernation period and are also needed for the activation of uncoupling protein (UCP) in the process of non-shivering thermogenesis, which allows the animals to return quickly to a euthermic state.

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, {alpha}-amylase (Aghajari et al., 1998Go), a Ca2+–Zn2+ protease (Villeret et al., 1997Go), triose–phosphate isomerase (Alvarez et al., 1998Go), citrate synthase (Russell et al., 1998Go) and malate dehydrogenase (Kim et al., 1999Go), 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., 1998Go; Kim et al., 1999Go). 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, 1991Go). 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., 2000Go; Jaenicke, 2000Go).

Accumulating evidence has suggested that the catalytic domain of most lipases and esterases share a common folding, designated the {alpha}/ß-hydrolase fold (Ollis et al., 1992Go; Wei et al., 1999Go). 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., 1990Go), 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, 1993Go). 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., 1996Go, 1999Go). Based on secondary structure predictions, we have proposed a three-dimensional model of the catalytic domain of HSL (Contreras et al., 1996Go) (Figure 1Go). 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., 1999Go).



View larger version (80K):
[in this window]
[in a new window]
 
Fig. 1. The position of the HGGG loop in the proximity of the active site serine (S) is illustrated in this ribbon representation of the model for the catalytic domain of HSL (Contreras et al., 1996Go). The side chain of the His is shown, and the positions of the three contiguous glycine residues are indicated with black balls. Also, two glycine residues mutated in this work are indicated. These are G412, at the bottom of the central ß-strand, and G326, in the putative hinge region between the N-terminal domain (not modelled) and the catalytic domain. The two spheres (R and R') indicate the borders of the regulatory module (not modelled).

 
One of the well-conserved motifs in the HSL family is a stack of glycine residues, preceded by a histidine residue (HGGG motif). This motif is not unique to the HSL family, since it is also found, with minor variations, in most of the enzymes from the carboxylesterase B family, which includes acetylcholine esterase and several lipases (Cygler et al., 1993Go). The HGGG motif forms a loop that is in close proximity to the active site (Figure 1Go) and contributes to the formation of the oxyanion hole (Sussman et al., 1991Go). Therefore, it is likely to participate directly in the catalytic process by stabilizing the oxyanion intermediate of the reaction. The presence of multiple glycine residues could confer an increased flexibility to this loop, which may be one of the factors contributing to facilitate catalysis at low temperatures (Feller et al., 1992Go). Although the presence of the HGGG motif is not limited to the enzymes of the family showing cold adaptation, preliminary experiments comparing HSL, carboxyl ester lipase and lipoprotein lipase indicated that the first two enzymes performed better at low temperatures than the last (Langin et al., 1993Go), in which the corresponding loop is formed by the sequence HGWT. In this study, we used a site-directed mutagenesis approach to investigate the importance of the last two glycine residues in this conserved motif for the catalytic capacity of HSL, and also the putative contribution of the HGGG loop to the psychrotolerant characteristics of HSL. Moreover, the cold-adapted character of the rat and human enzymes was compared using p-nitrophenyl butyrate as substrate. The role of an additional 12 amino acids in the regulatory module of the rat protein, which constitute the major difference in the primary structure between the two proteins, was also investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Site-directed mutagenesis

All mutants were constructed by a two step–four primer overlap/extension PCR method (Horton et al., 1990Go), 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 67–72 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, 1973Go).

Western blot analysis

Proteins from crude COS cell homogenates (60–80 µg) were separated by SDS–PAGE (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 Tris–HCl, 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., 1996Go). 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 (15–50 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 methanol–chloroform–heptane (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 20–30 nmol pNPB/min [molar absorptivity ({varepsilon}) = 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 6–10-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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Site-directed mutagenesis of the HGGG loop in human HSL

To test the importance of the multiple glycine residues in the HGGG loop (Figure 1Go) 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 2Go. 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., 1993Go). The other two double mutants generated, HGAA and HGSS, were designed to cause less drastic structural rearrangements than the previous one (Bordo and Argos, 1991Go). 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 3Go). Despite this, all three mutants exhibited no lipase activity and only minimal esterase activity (Figure 2Go). Similar results were obtained for a deletion mutant, in which one of the glycines was deleted (HG-G) (Figures 2 and 3GoGo).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2. Hydrolysis of the diacylglycerol analogue MOME (A) and p-nitrophenyl butyrate (pNPB) (B) in homogenates of COS cells transfected with wild-type (HGGG) or mutated hHSL cDNAs. Results are expressed as percentage of wild-type hHSL activity (defined as 100%) and represent the mean values (±SEM) from the indicated number of experiments in triplicate from 2–6 independent transfection experiments. Results were normalized for total protein and for background activity, i.e. the activity in homogenates of cells transfected with the empty expression vector.

 


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3. Western blot analysis of COS cells transfected with wild-type (HGGG) hHSL, hHSL mutants or with the empty pcDNA3 expression vector. Homogenized cell extracts (60 µg of total protein) were subjected to SDS–PAGE and Western blot analysis, using affinity-purified polyclonal rabbit anti-human HSL as primary antibody and 125I-labelled goat anti-rabbit IgG as secondary antibody. The signals were quantified through digital imaging of 125I. The mobility of one of the prestained molecular markers, fructose-6-phosphate kinase, is indicated.

 
Two single mutants (HGGA and HGAG) retained partial hydrolytic activity compared with wild-type HSL. The HGGA mutant was significantly more active than the HGAG mutant with regard to both lipase activity (60.0 ± 10.7 compared with 9.2 ± 2.3% of wild-type activity, p < 0.005) and esterase activity (34.2 ± 1.9 versus 6.7 ± 0.9% of wild-type activity, p < 0.0001). The data suggest that the HGGA mutant has a preference for larger substrates, since it retained more activity towards MOME than towards pNPB (60.0 ± 11 versus 34.2 ± 1.9% of wild-type activity, p < 0.005). However, a corresponding difference was not observed for the HGAG mutant (p < 0.5).

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 3Go). 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 2Go).

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., 1996Go; Østerlund et al., 1996Go, 1999Go). 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 2Go). These results are coherent with the present model where G412 is predicted to be located in a sharp turn between an {alpha}-helix and the central ß-strand preceding the active site serine (Figure 1Go), and presumably adopts {Psi}/{Phi} 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 IGo 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 IGo). 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.


View this table:
[in this window]
[in a new window]
 
Table I. Relative activities of wild-type hHSL (HGGG) and single mutants (HGGA/HGAG) at different temperatures compared with activity at 37°Ca
 
Comparison of the relative activities of human and rat HSL at different temperatures

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 4AGo 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., 1998Go). 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 temperature–activity 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 4BGo), indicating that the different behaviour of rHSL in the 10–37°C interval is not due to the presence of the longer, alternatively extra, loop in the regulatory module.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. (A) Influence of temperature on hydrolysis of p-nitrophenyl butyrate (pNPB) by HSL. Homogenates of COS cells, transfected with expression vectors containing rat (squares) and human (circles) HSL cDNA, were incubated for 10 min at 3, 10, 16, 25 and 37°C in the presence of 0.5 mM pNPB as described under Materials and methods. Enzyme activities are shown as percentage of activity at 37°C. **, p < 0.005; *, p < 0.05; NS, p > 0.05 rHSL vs hHSL (unpaired t-test). Data represent four independent experiments in triplicate from two different transfections (mean ± SEM). (B) Similar experiment as in (A) but with homogenates of COS cells transfected with wild-type rHSL (open squares), rHSL deletion mutant lacking the 12 amino acids in the regulatory module that are not present in hHSL (rHSLdel) (filled squares), wild-type hHSL (open circles) and hHSL with an insertion of the 12 amino acids lacking in hHSL (hHSLins) (filled circles). The figure shows the mean of two experiments performed in triplicates on homogenates from two independent transfections at incubation temperatures of 17, 22, 27, 32 and 37°C.

 
The possibility of an increased thermal inactivation of rHSL compared with hHSL, that could explain the reduced activity at 37°C compared with 32°C, was ruled out in two types of experiments. First, enzymes were preincubated at 37°C for 0, 5, 10 and 15 min, followed by the standard 10 min of incubation with pNPB. In a second experiment, the enzymes were preincubated at 32, 37 and 42°C for 10 min and then transferred to 37°C, where the esterase activity was measured immediately with pNPB for 10 min. Both experiments showed a minimal loss of activity due to preincubation at 37°C (<10%), but no significant differences were observed between rat and human HSL. Hence the higher activity of rHSL at 32°C observed in the experiments illustrated in Figure 4Go may reflect a lower optimum temperature for this species compared with the hHSL. To test this, three additional experiments were performed in which rat and human HSL activities were monitored at 27, 32, 37 and 42°C, to determine the optimum temperature for each of these HSL species. Figure 5Go shows one representative example of these experiments. As expected, a bell-shaped curve was obtained for both rHSL and hHSL, with a difference of ~5°C in the estimated optimum temperature for rHSL (~32°C) and hHSL (~37°C).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Determination of the optimum temperature for the esterase activity of rat and human HSL. COS-cell homogenates containing rat (squares) or human (circles) HSL were incubated for 10 min at 27, 32, 37 and 42°C in the presence of 0.5 mM pNPB. Hydrolytic activity is shown as percentage of the maximum activity for each HSL species. The curve shown is from one representative experiment out of three performed, with homogenates from two independent transfections. Each point is the mean of duplicate determinations.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of the present study was to investigate the functional importance of some well-conserved glycine residues in the HSL family and to study their potential role in the psychrotolerant properties of HSL. An HG-dipeptide motif (van Oort et al., 1989Go) is, apart from the GXSXG motif around the catalytic site serine, one of the most conserved motifs in lipases and esterases. This dipeptide is located in a loop that flanks the active site and plays an important role in the catalytic machinery by stabilizing the oxyanion intermediate of the hydrolysis reaction (Cygler et al., 1993Go; Derewenda, 1993Go). In HSL and related enzymes, a highly conserved HGGG motif is found in this loop. The three-dimensional model of HSL (Contreras et al., 1996Go) and the recently crystallized BFA esterase (Wei et al., 1999Go) suggest that the last of the three glycine residues (G353 in hHSL) is the hydrogen donor to the oxyanion intermediate of the reaction. Glycine-rich loops are likely to contribute to a local increased flexibility and, therefore, a stack of glycine residues could be a contributing factor for maintaining a catalytic capacity at low temperatures (Feller and Gerday, 1997Go). To probe whether the HGGG motif contributes to the notably psychrotolerant character of HSL, a series of double and single mutants affecting this loop were generated. Feller et al. (1991) mutated the histidine in the HG-dipeptide of Moraxella TA144 lipase 2 with a concomitant loss of function. Here, we show that even mild mutations of the last two glycine residues in the HGGG motif (single mutations from glycine to alanine) result in a significant loss of activity, and double mutations in this loop completely inactivate the enzyme. Interestingly, the integrity of the loop did not seem to be essential for the stability of the structure, since all mutants (including the HGWT and the single glycine deletion HG-G) were found to be expressed to comparable levels as wild-type HSL in COS cells. This is in contrast to what has been observed for lipoprotein lipase, where a mutation of the tryptophan in the loop (W55) into an alanine resulted in a very poorly expressed protein (Lookene et al., 1997Go). Hence the effect of mutating residues in the HGGG loop on the activity of HSL is likely to be due to a direct interference with the catalytic process. An intriguing observation was that the HGGA mutant retained significantly higher activity towards the large diglyceride analogue MOME than towards the relatively small pNPB ester. This may have a kinetic basis, since large lipidic substrates probably have a slower turnover rate, and therefore stay in a favourable position in the active site long enough to be hydrolysed by a sub-optimum catalytic machinery. With regard to cold adaptation, only two of the single mutants (HGGA and HGAG) retained sufficient enzymatic activity to allow a comparison with wild-type HSL. We analysed these mutants in the interval 17–37°C and found that the temperature-dependence curve displayed by the two mutants was almost identical with that obtained for the wild-type enzyme. Altogether, these data corroborate the critical role of the HGGG loop in the overall catalytic performance of HSL, but argue against a major role of this loop in the psychrophilicity of HSL.

Extended surface loops have been suggested to render enzymes more cold adapted (Davail et al., 1994Go). 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., 1998Go). The deletion in rHSL and the insertion of the same sequence in hHSL had no effect on the activity–temperature profiles (Figure 4BGo). 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., 1994Go). 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., 1997Go; Feller and Gerday, 1997Go). 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., 1997Go), and displays relatively higher activities at low temperatures than was recently described for an esterase from an Alaskan Pseudomonas sp. (Choo et al., 1998Go), reflecting that HSL indeed behaves as a cold adapted enzyme.

The HSL subgroup of {alpha}/ß-hydrolases consists of a growing number of enzymes from primarily prokaryotic organisms. Interestingly, HSL and a liver arylacetamide deacetylase (Probst et al., 1994Go) 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., 1991Go; Choo et al., 1998Go; Manco et al., 1998Go). 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., 1994Go), but was recently purified and characterized biochemically (Manco et al., 1998Go). A three-dimensional model of EST2 supports a protein folding similar to that of HSL (Manco et al., 1999Go). 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, 1990Go; Arpigny et al., 1997Go). 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., 1987Go). 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., 1988Go). 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.


    Notes
 
2 Present address: Unité INSERM 397, Institut Louis Bugnard, Hôpital Rangueil, F-31403 Toulouse Cedex 4, France Back

4 To whom correspondence should be addressed. E-mail: cecilia.holm{at}medkem.lu.se Back


    Acknowledgments
 
We thank Ann-Helen Thorén for technical assistance with cell culture, Cecilia Falkenberg in the laboratory of Bo Åkerström for providing the labelled goat anti-rabbit IgG and Soojay Banerjee for valuable discussions. This work was supported by grants to C.H. from the Swedish Medical Research Council (No. 11284), the Swedish Diabetes Association and the Albert Påhlsson (Malmö, Sweden), Thelma Zoega (Helsingborg, Sweden) and Novo Nordisk (Copenhagen, Denmark) foundations and by a grant to D.L. from Produits Roche-France.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aghajari,N., Feller,G., Gerday,C. and Haser,R. (1998) Protein Sci., 7, 564–572.[Abstract/Free Full Text]

Alvarez,M., Zeelen,J.P., Mainfroid,V., Rentier-Delrue,F., Martial,J.A., Wyns,L., Wierenga,R.K. and Maes,D. (1998) J. Biol. Chem., 273, 2199–2206.[Abstract/Free Full Text]

Anthonsen,M.W., Rönnstrand,L., Wernstedt,C., Degerman,E. and Holm,C. (1998) J. Biol. Chem., 273, 215–222.[Abstract/Free Full Text]

Arpigny,J.L., Lamotte,J. and Gerday,C. (1997) J. Mol. Catal. B, 3, 29–35.[ISI]

Borders,C.L., Jr., Broadwater,J.A., Bekeny,P.A., Salmon,J.E., Lee,A.S., Eldridge,A.M. and Pett,V.B. (1994) Protein Sci., 3, 541–548.[Abstract/Free Full Text]

Bordo,D. and Argos,P. (1991) J. Mol. Biol., 217, 721–729.[ISI][Medline]

Bradford,M.M. (1976) Anal. Biochem., 72, 248–254.[ISI][Medline]

Carneheim,C., Nedergaard,J. and Cannon,B. (1988) Am. J. Physiol., 254, E155–E161.[Abstract/Free Full Text]

Choo,D.W., Kurihara,T., Suzuki,T., Soda,K. and Esaki,N. (1998) Appl. Environ. Microbiol., 64, 486–491.[Abstract/Free Full Text]

Contreras,J.A., Karlsson,M., Østerlund,T., Laurell,H., Svensson,A. and Holm,C. (1996) J. Biol. Chem., 271, 31426–31430.[Abstract/Free Full Text]

Cygler,M., Schrag,J.D., Sussman,J.L., Harel,M., Silman,I., Gentry,M.K. and Doctor,B.P. (1993) Protein Sci., 2, 366–382.[Abstract/Free Full Text]

Davail,S., Feller,G., Narinx,E. and Gerday,C. (1994) J. Biol. Chem., 269, 17448–17453.[Abstract/Free Full Text]

Derewenda,Z.S. (1993) In Schumaker,V.N. (ed.), Advances in Protein Chemistry. Academic Press, New York, pp. 1–52.

Dill,K.A. and Shortle,D. (1991) Annu. Rev. Biochem., 60, 795–825.[ISI][Medline]

Feller,G. and Gerday,C. (1997) Cell. Mol. Life Sci., 53, 830–841.[ISI][Medline]

Feller,G., Thiry,M. and Gerday,C. (1991) DNA Cell. Biol., 10, 381–388.[ISI][Medline]

Feller,G., Lonhienne,T., Deroanne,C., Libioulle,C., Van Beeumen,J. and Gerday,C. (1992) J. Biol. Chem., 267, 5217–5221.[Abstract/Free Full Text]

Harbitz,I., Langset,M., Ege,A.G., Hoyheim,B. and Davies,W. (1999) Anim. Genet., 30, 10–15.[ISI][Medline]

Hemilä,H., Koivula,T.T. and Palva,I. (1994) Biochim. Biophys. Acta, 1210, 249–253.[ISI][Medline]

Hernandez,G., Jenney,F.E., Jr., Adams,M.W. and LeMaster,D.M. (2000) Proc. Natl Acad. Sci. USA, 97, 3166–3170.[Abstract/Free Full Text]

Holm,C., Fredrikson,G., Cannon,B. and Belfrage,P. (1987) Biosci. Rep., 7, 897–904.[ISI][Medline]

Holm,C., et al. (1988) Science, 241, 1503–1506.[ISI][Medline]

Horton,R.M., Cai,Z.L., Ho,S.N. and Pease,L.R. (1990) Biotechniques, 8, 528–535.[ISI][Medline]

Jaenicke,R. (1990) Philos. Trans. R. Soc. London, Ser B, 326, 535–551.[ISI][Medline]

Jaenicke,R. (2000) Proc. Natl Acad. Sci. USA, 97, 2962–2964.[Free Full Text]

Kim,S.Y., Hwang,K.Y., Kim,S.H., Sung,H.C., Han,Y.S. and Cho,Y. (1999) J. Biol. Chem., 274, 11761–11767.[Abstract/Free Full Text]

Langin,D. and Holm,C. (1993) Trends Biochem. Sci., 18, 466–467.[ISI][Medline]

Langin,D., Laurell,H., Holst,L.S., Belfrage,P. and Holm,C. (1993) Proc. Natl Acad. Sci. USA, 90, 4897-4901.[Abstract]

Li,Z., Sumida,M., Birchbauer,A., Schotz,M.C. and Reue,K. (1994) Genomics, 24, 259–265.[ISI][Medline]

Lookene,A., Groot,N.B., Kastelein,J.J., Olivecrona,G. and Bruin,T. (1997) J. Biol. Chem., 272, 766–772.[Abstract/Free Full Text]

Manco,G., Adinolfi,E., Pisani,F.M., Ottolina,G., Carrea,G. and Rossi,M. (1998) Biochem. J., 332, 203–212.[ISI][Medline]

Manco,G., Febbraio,F., Adinolfi,E. and Rossi,M. (1999) Protein Sci., 8, 1789–1796.[Abstract]

Mizuguchi,S., Amada,K., Haruki,M., Imanaka,T., Morikawa,M. and Kanaya,S. (1999) J. Biochem. (Tokyo), 126, 731–737.[Abstract]

Ollis,D.L., et al. (1992) Protein Eng., 5, 197–211.[Abstract]

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.[ISI][Medline]

Østerlund,T., Beussman,D.J., Julenius,K., Poon,P.H., Linse,S., Shabanowitz,J., Hunt,D.F., Schotz,M.C., Derewenda,Z.S. and Holm,C. (1999) J. Biol. Chem., 274, 15382–15388.[Abstract/Free Full Text]

Probst,M.R., Beer,M., Beer,D., Jeno,P., Meyer,U.A. and Gasser,R. (1994) J. Biol. Chem., 269, 21650–21656.[Abstract/Free Full Text]

Russell,R.J., Gerike,U., Danson,M.J., Hough,D.W. and Taylor,G.L. (1998) Structure, 6, 351–361.[ISI][Medline]

Schacterle,G.R. and Pollack,R.L. (1973) Anal. Biochem., 51, 654-655.[ISI][Medline]

Sussman,J.L., Harel,M., Frolow,F., Oefner,C., Goldman,A., Toker,L. and Silman,I. (1991) Science, 253, 872–879.[ISI][Medline]

van Oort,M.G., Deveer,A.M., Dijkman,R., Tjeenk,M.L., Verheij,H.M., de Haas,G.H., Wenzig,E. and Götz,F. (1989) Biochemistry, 28, 9278–9285.[ISI][Medline]

Vieille,C., Burdette,D.S. and Zeikus,J.G. (1996) Biotechnol. Annu. Rev., 2, 1–83.[Medline]

Villeret,V., Chessa,J.P., Gerday,C. and Van Beeumen,J. (1997) Protein Sci., 6, 2462–2464.[Abstract/Free Full Text]

Wei,Y., Contreras,J.A., Sheffield,P., Østerlund,T., Derewenda,U., Kneusel,R.E., Matern,U., Holm,C. and Derewenda,Z.S. (1999) Nature Struct. Biol., 6, 340–345.[ISI][Medline]

Winkler,F.K., D'Arcy,A. and Hunziker,W. (1990) Nature, 343, 771–774.[ISI][Medline]

Received March 20, 2000; revised August 2, 2000; accepted August 10, 2000.