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Correspondence to:
Klaus A. Dugi, To whom correspondence should be addressed., klaus_dugi{at}med.uni-heidelberg.de (E-mail)
LPL, like other lipases, has the ability to hydrolyze water-insoluble lipid substrates, but the mechanism is incompletely understood. We previously demonstrated a 22-amino acid loop in the amino-terminal domain of LPL to be essential for interaction with lipid substrates (Dugi, K. A., H. L. Dichek, G. D. Talley, H. B. Brewer, Jr., and S. Santamarina-Fojo. 1992. J. Biol. Chem. 267: 25086;–25091) and mediation of substrate specificity (Dugi, K. A., H. L. Dichek, and S. Santamarina-Fojo. 1995. J. Biol. Chem. 270: 25396;–25401). The carboxy-terminal domain, LPL415;–438, contains two highly conserved hydrophobic stretches, and represents a candidate region for substrate interactions. Specific point mutations or deletion of the region between the hydrophobic stretches (LPL419;–430) caused up to 90% selective loss of hydrolyzing activity against water-insoluble triolein, but not against water-soluble tributyrin, implicating a crucial function for LPL419;–430 in the interaction with lipid substrates. In contrast, mutations introduced into the hydrophobic regions led to concomitant changes in tributyrin and triolein activities. The presence of an additional positive charge at position 416 yielded a gain of function mutant with 3-fold increased activity. This mutant was about three times more stable at 37°C than wild-type LPL, suggesting an important role for the hydrophobic regions in LPL dimer stability.
In summary, our data demonstrate that the carboxy-terminal region LPL415;–438 plays an important role in both the interaction of LPL with lipid substrates and the stability of the LPL homodimer. — Keiper, T., J. G. Schneider, and K. A. Dugi. Novel site in lipoprotein lipase (LPL415;–438) essential for substrate interaction and dimer stability. J. Lipid Res. 2001. 42: 1180;–1186.
Supplementary key words:
hepatic lipase, loop, structure-function, triglycerides
LPL plays a key role in human lipid metabolism by hydrolyzing triglycerides in chylomicrons and VLDL. It thereby generates free fatty acids, which are used either for storage or energy production (1). LPL is a 55-kDa glycoprotein that is primarily synthesized in adipocytes, muscle cells, and macrophages. The enzyme is active as a noncovalent homodimer (2) and is bound to the surface of endothelial cells via glycosaminoglycans, from which it can be released by heparin. Dissociation of the LPL dimer into monomers leads to an irreversible loss of catalytic function (3).
LPL, hepatic lipase (HL), pancreatic lipase (PL), and endothelial lipase (4) (5) belong to the mammalian triacylglycerol lipase gene family (6). As serine esterases, their catalytic triad consists of serine, aspartic acid, and histidine (7) (8). Because of the conservation of all disulfide bonds, a high degree of structural homology between these four lipases is expected (9).
The distinct property of all lipases, including LPL, is their ability to interact with water-insoluble emulsified substrates, but the exact mechanism is not fully understood. We have previously shown that a 22-amino acid loop covering the catalytic site of LPL is crucial for the lipid-binding properties of the enzyme because of two highly amphipathic
Treatment of an HL-LPL chimera with a monospecific antibody indicated that the carboxy-terminal domain of LPL may also be involved in lipid substrate interaction (12). Bovine LPL, truncated at Trp390, showed no binding to chylomicrons, and a greatly reduced enzymatic activity against water-insoluble substrates. The activity against water-soluble substrates such as tributyrin, however, was only minimally impaired (13). Even though these data imply an important role for the carboxy-terminal domain in lipid binding, the exact sites involved have not been elucidated. We investigated LPL415;–438, a region containing two stretches of three and six hydrophobic residues, respectively, which are brought into close proximity via a disulfide bond. This region is of interest, because hydrophobic residues could be involved in the interaction of LPL with liposoluble substrates, and the three-dimensional model of PL and LPL suggests an outward loop formation for this region (14) (15). We generated nine mutants in which the amino acid profile of the hydrophobic stretches or the residues between these stretches were altered to evaluate their potential role in LPL-substrate interaction.
In this article we present a novel site in the carboxy-terminal domain of LPL (415;–438), with great impact on the interaction of the enzyme with emulsified, water-insoluble substrates and, in addition, on the stability of the LPL homodimer.
cDNA expression vector
Synthesis of mutant cDNA
The mutagenic primers used were as follows:
In vitro expression of cDNA in human embryonal kidney 293 cells
Determination of LPL activity
Determination of LPL mass
Prediction of protein secondary structure
To investigate the potential function of the carboxy-terminal region of amino acids 415;–438 in the interaction of LPL with lipophilic substrates, nine different LPL mutants were generated ( Table 1). Intracellular and extracellular LPL mass was measured to exclude the possibility that changed mutant enzymatic activity resulted from differences in the extent of protein secretion. For the mutants that had a low mass in the LPL ELISA (mutants D and E), triolein activity was determined instead of LPL mass. The rate of secretion could not be determined for mutants C and F, because they had undetectable levels of LPL mass and triolein activity. As presented in Table 2, all mutants with either measurable lipase mass or triolein activity were secreted to a similar extent as LPL wild type (
To characterize the mutants, tributyrin activity, triolein activity, and LPL mass were quantitated. The percentage of enzymatic activity compared with LPL wild type ± standard deviation was calculated from three independent transfection experiments for each mutant and is illustrated in Fig 1. In one transfection experiment, LPL mass was determined in duplicate, and the LPL mass data as well as specific tributyrin and triolein activities are presented in Table 3. In addition, the tributyrin activity, triolein activity, and triolein/tributyrin activity ratio of the mutants in this transfection are also listed in Table 3.
The LPL-related enzyme, HL, also contains a region analogous to LPL418;–438 at its C terminus. To gain insight into the importance of this region, and to examine whether the corresponding region of HL could assume the function of LPL418;–438 in lipid substrate interactions, a chimeric enzyme (mutant A) was generated, in which amino acids 418;–438 of LPL were replaced by the corresponding amino acids of HL. In this construct, the enzymatic activity against water-soluble tributyrin was fully intact compared with wild-type LPL, but the activity against water-insoluble triolein was greatly reduced, by more than 80% (Table 3 and Fig 1A), indicating impaired interaction of the mutant lipase with lipid substrates. These data were supported by chimeric protein B, in which only the sequence LPL418;–433 was replaced by the corresponding sequence of HL, while maintaining the hydrophobic stretch LPL434;–436 (Table 3 and Fig 1A). As in mutant A, the decrease of triolein activity was greater than 80%, demonstrating the great importance of region LPL419;–433 in the interaction of LPL with lipid substrates.
To examine the region further, an LPL418;–438 deletion mutant (mutant C) was generated, in which the original 19 amino acids were replaced with the short fragment IEEI, believed to form a loop and thereby maintaining a functional disulfide bond (10). In this construct, triolein activity was nearly completely abolished, while the tributyrin activity was maintained, albeit at only about 48% of LPL wild type (Fig 1A). This mutant was not detectable in the LPL mass assay. Because tributyrin activity could be consistently demonstrated, this was most likely due to the fact that one of the antibodies used in the LPL ELISA did not recognize this particular LPL mutant.
By analogy to the three-dimensional structure of PL ( Fig 2), the region between the hydrophobic stretches is predicted to form an outward loop and may therefore be important in substrate interaction. The conserved, positively charged lysine (LPL428) was changed to leucine in mutant D. In this construct, the activity against tributyrin was nearly fully preserved, whereas the activity against triolein was reduced by more than 70% (Fig 1A). This effect was even more pronounced in mutant E, in which the second conserved, positively charged lysine residue was also changed to leucine (LPL428L430L). Construct E had only 15% of LPL wild-type triolein activity, whereas the activity against the water-soluble tributyrin was close to the wild-type level (Fig 1A). Mutants D and E were detectable in the LPL mass assay, but their concentration was consistently only about 10% of LPL wild type and other mutants and therefore specific tributyrin and triolein activities were not calculated (Table 3). Because their esterase (tributyrin) activities were similar to that of LPL wild type, the lower LPL mass values are probably due to an impaired binding of the antibody used in the LPL ELISA to mutants D and E.
Because of their potential for lipid interaction, the two hydrophobic stretches were also targeted for mutation. The introduction of a positive charge in the hydrophobic stretch LPL431;–436 (mutant F) led to a greater than 60% reduction of both tributyrin and triolein activities (Fig 1B). This mutant was also not detectable in the LPL ELISA. This fact, together with the near total loss of enzymatic activity, suggests that the introduction of a positive charge at position 435 leads to a significant change in overall LPL structure and subsequently LPL function. Two additional constructs were analyzed, in which one or two neutral amino acids replaced hydrophobic residues (mutants G and H, respectively). In both mutants, activities against tributyrin and triolein were not significantly different from wild-type LPL (Table 3 and Fig 1B).
To investigate the potential role of the shorter hydrophobic stretch, LPL415;–417, one mutant was generated in which two hydrophobic residues (415V416I) were replaced by hydrophilic residues (mutant K: 415Y416R). The insertion of the positively charged amino acid arginine, together with an uncharged hydrophilic residue, led to a nearly 3-fold increase in both tributyrin and triolein activities (Table 3 and Fig 1B). The LPL mass of this mutant was not higher than that of LPL wild type, demonstrating that the gain of function was not caused by a greater expression of the LPL mutant.
To elucidate the effect of the inserted positive charge on LPL stability, mutant K was subjected to heat inactivation. The results are illustrated in Fig 3. After 2 h of incubation at 37°C, the triolein activity of LPL wild type was quickly reduced to 14% of the starting material. In contrast, the triolein activity of construct K was reduced far more slowly. After 2 h of incubation, for instance, 55% of the triolein activity was preserved, roughly four times that of LPL wild type. The greater stability of mutant K was also true for its activity against water-soluble tributyrin (data not shown).
To gain insight into the changes in secondary structure of the generated mutants, the predicted secondary structures of LPL wild type and LPL mutants were analyzed by a new prediction method based on a neural network system. The exchange of an LPL sequence stretch with the corresponding amino acids of HL (mutants A and B) was predicted to result in a loss of loop formation of amino acid regions 421;–423 and 428;–431. The introduction of one or two leucine residues in constructs D and E was predicted to change the secondary structure of five amino acids (LPL 423;–427) into a helical region, suggesting that this one- or two-residue mutation led to a substantially altered secondary structure of the entire region. Concurrently, the solvent accessibility of these regions was predicted to be lower in mutants D and E compared with LPL wild type.
The characteristic feature of LPL, like other lipases, is its ability to hydrolyze water-insoluble lipid substrates. Although the carboxy-terminal domain of LPL is involved in the interaction with liposoluble substrates, the precise region has not been delineated. LPL415;–438 represents a candidate region by virtue of two hydrophobic stretches (LPL415;–417 and LPL431;–436) that are highly conserved between different species (cow, mouse, guinea pig, and chicken). In addition, the region LPL419;–430, lying between the hydrophobic sites, is predicted to form an outward loop in the three-dimensional model of the homologous PL (24), theoretically enabling it to bind substrate particles (Fig 2).
Nine different constructs were generated to investigate this C-terminal region and were analyzed for their ability to hydrolyze different triglyceride substrates. Preserved esterase (tributyrin) activity reflects intact dimer formation and overall structural integrity, because a shift in the monomer-dimer equilibrium over to the monomer results in irreversible loss of catalytic function demonstrated by the inability to hydrolyze the water-soluble substrate p-nitrophenyl butyrate (3). Preserved lipase (triolein) activity reflects the presence of sites in LPL needed for the interaction with water-insoluble substrates. Thus, a reduction of both tributyrin and triolein activities would indicate a disruption of the LPL dimer or overall structure, whereas a selective loss of triolein activity would suggest structural integrity, but a loss of sites important for the interaction of LPL with liposoluble substrates as has been shown for the lipase lid (10).
The selective loss of lipase activity seen in mutants A;–E infers that the entire region of LPL418;–438 is essential for lipid substrate interaction. The three mutants C;–E, involving only amino acid changes in LPL419;–430, exhibited a selective reduction of lipase activity compared with esterase activity, indicating an important role of this region in the interaction of LPL with its liposoluble substrates. Because the mutations introduced in constructs D and E led to an increase in hydrophobic residues, which theoretically should improve lipid binding, the observed effect is most likely due to an altered secondary and/or tertiary structure of this region, making the hydrophobic residues of LPL419;–430 and/or neighboring regions less accessible for lipid substrates. The effect on secondary or tertiary structure is most likely mediated by the loss of positive charge. This hypothesis is supported by secondary structure prediction using neural networks, in which the alteration of 428K or 428K430K changed the predicted secondary structure of a stretch of five amino acids in this region into a helical conformation, and to a reduction of solvent accessibility. This alteration in the secondary structure and the minimization of a 19-amino acid region to 4 residues in mutant C may be responsible for the greater decrease in lipase activity compared with esterase activity. The binding of water-soluble substrates such as tributyrin would also be impaired, but these small molecules could gain easier access to the catalytic pocket of the head-to-tail-oriented, adjacent monomer.
We (10) and others (25) (26) have previously suggested that the initial interaction of LPL with its lipid substrates may be via the carboxy-terminal domain, ultimately leading to a conformational change in the lid region, which in turn would enable access of the substrate to the catalytic pocket. In addition, it has been shown that the LPL homodimer is likely to be oriented in a head-to-tail fashion (27). Therefore, it is possible that the introduced changes in LPL419;–430 lead to an impairment of the interaction of LPL with lipid substrates by interfering with the necessary conformational change in the altered monomer, and subsequently to reduced lipid-binding ability. An alternative explanation would be that the alterations in LPL419;–430 impede the opening of the lipase lid in the neighboring monomer, leading to a reduced ability of the lid region to interact with lipid substrates.
Surprisingly, in the mutants of the hydrophobic stretches LPL415;–417 (mutant K) and LPL431;–436 (mutants F;–H), a selective loss of lipase activity, which would have suggested a role for this hydrophobic region in lipid binding, was not observed. Esterase and lipase activities were either unchanged, both reduced, or both increased, suggesting that the hydrophobicity in this region does not play a major role in lipid substrate binding. Interestingly, mutant K, in which the positively charged arginine and the uncharged hydrophilic tyrosine replace two uncharged lipophilic amino acids, showed a nearly 3-fold increase in both esterase and lipase activities, thus representing a gain of function mutant.
These data imply that the modifications in the hydrophobicity profile and/or charge of constructs F and K strongly influence enzyme stability. We hypothesized that the modifications influence the stability of the LPL protein. This hypothesis was supported by the mutant's 4-fold greater preservation of lipase activity during heat inactivation. It has been shown in vitro that the LPL dimer quickly dissociates into catalytically inactive monomers (3). The preservation of triolein activity in mutant K therefore most likely reflects a more stable LPL homodimer. The reduction of lipase and esterase activities in mutant F was too large to include it in the LPL stability assay, but it is probable that the mutation led to a reduced formation and/or stability of the LPL dimer, as has been shown for mutations at positions 176, 188, 195, and 244 in the amino-terminal domain (28) and at position 410 in the carboxy-terminal domain (29).
The different effect of an additional positive charge at position 416 (mutant K) and position 435 (mutant F) indicates that the charge distribution rather than the alterations of secondary structure due to the mutations in the analyzed carboxy-terminal region of LPL plays a crucial role in the formation and/or stability of the LPL homodimer. The importance of LPL415;–438 in maintaining the LPL homodimer is supported by the three-dimensional structure of the homologous PL (Fig 2), which illustrates that this loop region of one monomer lies in close proximity to the other monomer. Our data are in agreement with a naturally occurring mutation in LPL, in which the loss of a negative charge at position 410, which approximates the mutation in construct K, led to a greater than 70% loss of lipase activity, attributed to a shift of the monomer-dimer equilibrium to the monomer (29). Taken together, these data demonstrate that the hydrophobic regions LPL415;–417 and LPL431;–436, which are brought into close proximity by the formation of the disulfide bond between Cys418 and Cys438, do not play an important role in the interaction of LPL with its liposoluble substrates but rather in the formation and/or stability of the LPL homodimer.
Conflicting data exist about the importance of the disulfide bridge Cys418;–Cys438 for lipase activity. While in some studies a change of Cys418 and Cys438 to serine led to little (30) or no (31) reduction in lipase activity, a truncation within this region at position 436 led to a 78% reduction in lipase activity (32). A mutation at position 421 (Glu421Lys) was described in a patient who died of pregnancy-induced hypertriglyceridemic pancreatitis, and was shown to lead to a 30% reduction in lipase activity in vitro (33). Our data confirm that the region between the two cysteines is centrally involved in the interaction with lipid substrates and dimer stability, and suggest that the charge distribution in this region may be more important than preservation of the disulfide bond.
In summary, we have identified a novel region in LPL, LPL415;–438, which plays a crucial role in the interaction of LPL with lipid substrates, and suggest that the initial interaction of LPL with lipoproteins may be via its carboxy-terminal domain. In addition, we have shown that LPL415;–438 is also essential for the stability of the LPL homodimer and therefore provide important new insights into the structure-function relationship of LPL.
Abbreviations: CMV, cytomegalovirus; HL, hepatic lipase; PL, pancreatic lipase.
This work was supported by grant Du 207/2-1 from the Deutsche Forschungsgemeinschaft. The authors thank Dr. Silvia Santamarina-Fojo (Molecular Disease Branch, National Institutes of Health, Bethesda, MD) for providing the expression vector pCMV and labeled tributyrin. The authors also thank Dirk Ramacher for expert technical assistance.
Manuscript received February 22, 2001; and in revised form April 6, 2001
Copyright © 2001 by Lipid Research, Inc.
Original Article
Novel site in lipoprotein lipase (LPL415;–438) essential for substrate interaction and dimer stability
Tanja Keipera,
Jochen G. Schneidera, and
Klaus A. Dugia
a Department of Internal Medicine I, Endocrinology and Metabolism, Heidelberg University, 69115 Heidelberg, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structures (10). We subsequently showed that this amino-terminal structure not only mediates lipid binding but also substrate specificity (11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The parent plasmid (pCMV) used for site-directed mutagenesis and transfection was a pUC18-derived vector containing the cytomegalovirus (CMV) immediate-early promoter and the polyade-nylation site of simian virus 40 as described previously (16) and was a generous gift from S. Santamarina-Fojo (Molecular Disease Branch, National Institutes of Health, Bethesda, MD). A 1,473-bp fragment of wild-type human LPL cDNA (17) was cloned into the XbaI and HpaI restriction sites of pCMV (pCMV-wtLPL). The DNA sequence of each fragment, which spanned the signal peptide through to the termination codon, was confirmed by DNA sequence analysis, using the dideoxynucleotide chain termination method (18) and T7 DNA polymerase (T7 Sequenase version 2.0; Amersham Pharmacia Biotech, Uppsala, Sweden).
The mutant LPL cDNAs were synthesized by overlap extension PCR (19), using pCMV-wtLPL as template. The PCR was performed in an automated DNA thermal cycler (Perkin-Elmer, Norwalk, CT) with native DNA polymerase from Pyrococcus furiosus (Stratagene, La Jolla, CA) and 30 cycles with 1 min of denaturation at 95°C, 1 min of annealing at 50°C, and a 2-min extension at 72°C in 1x native Pfu buffer (Stratagene), 250 µM each of dATP, dCTP, dGTP, and dTTP (Boehringer, Mannheim, Germany), and a 0.5 µM concentration of each primer. The mutagenic primers used are listed below. The mutant cDNAs were subcloned into the pCMV expression vector and amplified using TOP10 competent cells (InVitrogen, Groningen, The Netherlands). Clones carrying the mutant cDNA were grown overnight at 37°C in LB2Y broth (tryptone from Difco, Detroit, MI; yeast from GIBCO-Life Technologies, Paisley, UK) and correct insert size was verified by screening PCR. Large-scale plasmid preparation of desired clones was performed with a Qiagen (Hilden, Germany) Plasmid Maxi kit. All constructs were examined by sequence analysis of the complete cDNA insert. Oligonucleotide primers for overlap extension PCR were ordered and synthesized by MWG-Biotech (Ebersberg, Germany). Oligonucleotide primers for sequencing were synthesized by the phosphoramidite method on a DNA synthesizer (model 30B; Applied Biosystems, Foster City, CA).
Plasmids, prepared and purified with the Qiagen Plasmid Maxi kit, were transfected into human embryonal kidney 293 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Transfections were performed with LipoTAXI transfection agent (Stratagene) by adding 7 µg of plasmid DNA to each 100-mm plate of subconfluent human embryonal kidney 293 cells. After transfection, cells were incubated at 37°C, 5% CO2 for 5 h, and then 6 ml of DMEM (BioWhittaker, Walkersville, MD), supplemented with 10% FBS (BioWhittaker) and heparan sulfate (2 units/ml; Braun, Melsungen, Germany) were added. Medium for activity determination was harvested 12 h later. Intracellular protein was recovered as described by Chait, Iverius, and Brunzell (20). Aliquots of medium and intracellular extracts were kept at - 80°C until lipase assays were performed. Each plasmid was transfected in triplicate. Wild-type LPL was used as positive control, and the pCMV vector without insert was used as negative control.
Esterase activity was quantitated in duplicate with [14C]tributyrin (21), and triglyceride lipase activity was determined in duplicate with [14C]triolein (22). To test LPL stability, transfection medium was incubated at 37°C and analyzed for triglyceride lipase activity at 0, 30, 60, 120, and 240 min.
Intracellular and extracellular protein concentration was measured with the LPL-IFMA-ELISA kit (Immundiagnostik, Bensheim, Germany).
Secondary structures of LPL wild-type and LPL mutants were analyzed with a web-based neural network system (PredictProtein) (23).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
85%).
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Table 1.
Amino acid alignment of wild-type LPL, wild-type HL, and LPL mutants
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Table 2.
Rate of secretion of wild-type and mutant LPL constructs
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Figure 1.
Esterase activity (open columns) and lipase activity (solid columns) of LPL wild type and LPL mutants. Activities are expressed as a percentage of LPL wild type. The error bars represent the standard deviation of three independent transfection experiments.
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Table 3.
Enzymatic activities and LPL mass
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Figure 2.
Three-dimensional structure of the horse PL dimer, presented in a head-to-tail fashion. The carboxy-terminal loop corresponding to LPL418;–438 is highlighted for one monomer in red. Helices are illustrated in green, ß sheets in yellow. The red ball represents the catalytic pocket. Because this is a two-dimensional illustration of a three-dimensional image, monomer 2 is not an identical inversion of monomer 1. The loop is highlighted only in monomer 1, because in monomer 2 this loop is facing in the opposite direction.
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Figure 3.
LPL stability assay. The time points represent the triolein activity before and at various time points during incubation at 37°C. Triolein activity is expressed as a percentage of starting material activity before incubation. Dashed line, LPL wild type; solid line, LPL mutant K.
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FOOTNOTES 
ACKNOWLEDGMENTS
REFERENCES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
Thr) leads to abnormal heparin binding and loss of enzymic activity. Proc. Natl. Acad. Sci. USA. 87:3474-3478Val) leads to enzyme inactivation and familial chylomicronemia. J. Lipid Res. 35:1552-1560[Abstract].
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