Original Article

Binding of hepatic lipase to heparin: identification of specific heparin-binding residues in two distinct positive charge clusters

Correspondence to: Andre Bensadoun

	ABSTRACT

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The interaction of hepatic lipase (HL) with heparan sulfate is critical to the function of this enzyme. The primary amino acid sequence of HL was compared to that of lipoprotein lipase (LPL), a related enzyme that possesses several putative heparin-binding domains. Of the three putative heparin-binding clusters of LPL (J. Biol. Chem. 1994. 269: 4626;–4633; J. Lipid Res. 1998. 39: 1310;–1315), one was conserved in HL (Cluster 1; residues Lys 297;–Arg 300 in rat HL) and two were partially conserved (Cluster 2; residues Asp 307;–Phe 320, and Cluster 4; residues Lys 337, and Thr 432;–Arg 443). Mutants of HL were generated in which potential heparin-binding residues within Clusters 1 and 4 were changed to Asn. Two chimeras in which the LPL heparin-binding sequences of Clusters 2 and 4 were substituted for the analogous HL sequences were also constructed. These mutants were expressed in Chinese hamster ovary (CHO) cells and assayed for heparin-binding ability using heparin-Sepharose chromatography and a CHO cell-binding assay. The results suggest that residues within the homologous Cluster 1 region (Lys 297, Lys 298, and Arg 300), as well as some residues in the partially conserved Cluster 4 region (Lys 337, Lys 436, and Arg 443), are involved in the heparin binding of hepatic lipase. In the cell-binding assay, heparan sulfate-binding affinity equal to that of LPL was seen for the RHL chimera mutant that possessed the Cluster 4 sequence of LPL. Mutation of Cluster 1 residues of HL resulted in a major reduction in heparin binding ability as seen in both the cell-binding assay and the heparin-Sepharose elution profile.

These results suggest that Cluster 1, the N-terminal heparin-binding domain, is of primary significance in RHL. This is different for LPL: mutations in the C-terminal binding domain (Cluster 4) cause a more significant shift in the salt required for elution from heparin-Sepharose than mutations in the N-terminal domain (Cluster 1).—Sendak, R. A., D. E. Berryman, G. Gellman, K. Melford, and A. Bensadoun. Binding of hepatic lipase to heparin: identification of specific heparin-binding residues in two distinct positive charge clusters. J. Lipid Res. 2000. 41: 260;–268.

Supplementary key words: hepatic lipase, heparin binding domains, lipoprotein lipase, heparin sulfate proteoglycans, Chinese hamster ovary cells, heparin-Sepharose chromatography

	INTRODUCTION

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Hepatic lipase (HL) is synthesized primarily by hepatocytes \n (1) (2) (3) and is secreted into the sinusoidal space where it binds to both the capillary endothelium and hepatocytes (4). In concert with lipoprotein lipase (LPL), HL plays several major roles in plasma lipoprotein metabolism (5). Hepatic lipase has both triglyceride hydrolase and phospholipase activity. In vivo inhibition of HL activity by intravenous injections of anti-HL antibodies results in the accumulation of intermediate density lipoproteins and an increase in high density lipoprotein (HDL), cholesterol, and phospholipids (6) (7) (8). Similarly, patients with hepatic lipase deficiency exhibit increased plasma triglyceride, cholesterol, intermediate density lipoproteins, and HDL when compared to control subjects (9) (10). In a report on HL-deficient mice produced by homologous recombination, the main change compared to control mice was an increase in large HDL with no major change in apoB-containing lipoproteins (11). A recent report (12) on the phenotype of young (10;–14 weeks of age) homozygous (-/-) HL-deficient mice demonstrated a role of mouse HL in the formation of dense LDL as well as in the metabolism of HDL. It has been postulated that HL also modifies the cholesterol to phospholipid ratio of HDL thus facilitating transfer of cholesterol to liver cells (13). In addition to these roles in lipid metabolism that are dependent on the catalytic activity of the enzyme, HL has been shown to enhance the uptake of lipoproteins by lipoprotein receptors (5). This stimulation is not contingent on catalytic activity of the enzyme but is highly dependent on the presence of heparan sulfate proteoglycans on the cell surface (14) (15) (16) (17) (18).

The binding of HL to heparan sulfate proteoglycans (HSPGs) is an important interaction, which may be necessary for many of the metabolic tasks that the lipase performs. To date, the regions of HL that are essential for the binding of the enzyme to heparin have not been identified. The heparin-binding regions of lipoprotein lipase (LPL), on the other hand, have been extensively studied (19) (20) (21) (22). LPL and HL are members of the triglyceride lipase superfamily that share extensive sequence homology, but differ significantly in substrate specificity, optimal hydrolysis conditions, cofactor requirements, and heparin-binding affinity. Several studies have been carried out that described the construction of chimeric lipases in which the N- and C-terminal domains of HL and LPL were exchanged (23) (24) (25). These studies revealed that HL and LPL each possess unique functional domains that can be localized to the N- or C-terminal domains of the proteins, further demonstrating the structural basis for the functional differences of these enzymes.

Putative heparin-binding regions of LPL were predicted in the primary amino acid sequence of the enzyme based on comparison with consensus heparin-binding sequences of other heparin-binding proteins (26) and in the tertiary structure based on molecular modeling (27). Four regions of positive charge were identified: these have been subjected to site-directed mutagenesis revealing that they may play a role in the heparin binding of the protein. Cluster 1 (avian sequence numbering): Arg 281, Lys 282, and Arg 284 (19) (20) (21) and Cluster 4: Lys 321, Arg 405, Arg 407, Arg 409, and Lys 416 (22) were shown to be involved in heparin binding while conflicting results were obtained for Cluster 2: residues 296;–302 (20) (21). Cluster 3 (residues 147;–151, human numbering) was not found to play a role in heparin binding (20). Alignment of the sequences of LPL and HL reveals that Cluster 1 is conserved between the two lipases while Clusters 2 and 4 are only partially conserved.

In this study, we have systematically analyzed the potential heparin-binding regions of rat HL (RHL) to identify those sequences involved in this important interaction. In addition, we have analyzed the putative LPL heparin-binding sequences that are poorly conserved in HL to characterize the basis for the difference in heparin-binding affinity between these two evolutionarily conserved proteins.

	MATERIALS AND METHODS

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Reagents and enzymes
Restriction enzymes and molecular biology reagents were purchased from New England Biolabs, MBI Fermentas, or Gibco BRL Life Technologies. Previously described procedures were used to purify native RHL (28) and chicken LPL (cLPL) (29) used for control experiments.

Construction of rat hepatic lipase heparin-binding mutants
Alignment of the RHL and cLPL primary sequences ((30), Table 1 and Table 2) allowed for comparison of the putative heparin-binding regions of LPL with the analogous sequences in RHL. We have identified discrepancies between the published cDNA sequence of RHL (31) and the results of our recent sequencing efforts. Both strands of the same clone isolated by Sensel et al. (31) were re-sequenced at the Cornell BioResource Center on PE-Biosystems 377 automated DNA sequencers. The published sequence reads: 5' ⁹⁹⁴ATC GGC CAC GTC¹⁰⁰⁵ 3', while our most recent sequence reads: 5' ⁹⁹⁴GAT CGG CCA CGC¹⁰⁰⁵ 3'. In addition, we found that the codon for Gly 11 is GGG (instead of GGA), amino acid 256 (AAC, Asn) is actually Lys (AAA), and amino acid 448 (ATG, Met) is actually Ile (ATA). The revised amino acid sequence, the nucleotide changes, as well as the three positive charge clusters studied are indicated on Table 1.

The full-length RHL cDNA was previously cloned into pcDNA3 to create pcDNA3/RHL (28). Several RHL mutants of Clusters 1, 2, and 4 were constructed ( Table 3). The polymerase chain reaction in combination with the overlap extension method was used to construct the substitution mutants HB-A, HB-B, HB-C, HB-E, and the chimeric mutants RHL2LPL and RHL4LPL (32). The mutagenic primers are listed in Table 4. The flanking oligonucleotides used had the sequences 5' ⁵⁷⁴AC AGGGCTGGACCCTGCAGGA⁵⁹⁴ 3' and 5' ⁵⁷⁴CTCATCTGCT CTTTTGCTTCT¹⁵⁰⁶ 3'. The final PCR product containing the mutation was subcloned into pcDNA3/RHL using EcoN I and Esp3IA in the case of RHL4LPL and RHL2LPL, and Bsg I in the case of HB-A, HB-B, HB-C, and HB-E. The region containing the mutated fragment was sequenced. HB-F and HB-G were constructed using the QuikChange^TM Site-Directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The entire cDNA of these mutants was sequenced to confirm the absence of any misincorporated bases by the polymerase.

Cell culture and protein expression
Chinese hamster ovary (CHO) cells (American Type Culture Collection, CCL 61) were cultured in Ham's F12 medium (Gibco BRL Life Technologies) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, and 10 mM HEPES, pH 7.2. Cells were maintained in a humidified incubator at 5% CO₂. The CHO cells were stably transfected with the RHL DNA constructs using Lipofectamine^TM according to a previously described procedure (22).

Lipase mass and activity measurements
Sandwich ELISAs with affinity-purified immunoglobins were used to measure the mass of cLPL (33) and RHL (34). In its most sensitive form, the standard curve for the RHL ELISA ranged from 0.02 ng to 1 ng with an OD₄₉₀ of 1.05 at 1 ng (r² = 0.999). The cLPL ELISA standard curve spanned from 0.01 to 1 ng with an OD₄₉₀ of 1.27 at 1 ng (r² = 0.999). Catalytic activity of RHL constructs was determined using a ³H-labeled triolein substrate with a specific activity of 500,000 cpm/µmol oleic acid (35).

Heparin-Sepharose chromatography of wild-type RHL, LPL, and RHL heparin-binding mutants
Heparin-Sepharose chromatography was carried out using a 1-ml HiTrap Heparin-Sepharose column (Pharmacia) at 4°C in an FPLC system (Pharmacia). The column was equilibrated in buffer A (0.1 M NaCl, 1 mM decyl-sucrose (Calbiochem), 10 mM phosphate, pH 7). Medium was incubated for 5 h in four 75-cm² flasks containing cells of a given construct at 80;–90% confluency. The medium, containing heparin (Sigma, 5 units/ml), was centrifuged at 3,000 g for 15 min and loaded onto the HiTrap column at a rate of 0.5 ml/min. The matrix was washed with buffer A (5 ml) and the lipase was eluted with a 40 ml linear gradient from buffer A to buffer B (2 M NaCl, 1 mM decyl-sucrose, 10 mM phosphate, pH 7). The flow rate was 0.5 ml/min and either 40 or 80 fractions were collected. The column was then washed with 5 ml of buffer B and equilibrated in buffer A. Three runs were completed for each mutant construct and the fractions were assayed for lipase by ELISA. A fourth chromatography was carried out where both enzyme mass and catalytic activity were measured. For this run, buffer A contained 2 mM decyl sucrose. The specific activity for each mutant for a given HL peak was calculated ( Table 5) by taking the average and standard deviations of specific activities of fractions with an activity equal or larger than 0.1 µmol/ml per h. Conductivity was determined using a conductivity meter (Radiometer, Copenhagen). Three 40-fraction runs and three 80-fraction runs were selected at random and assayed for conductivity. The average results were converted to NaCl concentration using a standard curve (r² = 0.998) and were used to plot the data sets. To determine peak salt concentration for each mutant, the fraction at which the peak lipase value was detected in the individual runs was assayed by conductivity. The average and standard deviation of these three values were calculated for each mutant.

Native purified RHL was run on the column under the same conditions to verify that the recombinant RHL produced in CHO cells possessed the same affinity for heparin-Sepharose. Native purified chicken LPL was also run on the column for comparison with the RHL/LPL chimeric mutants.

A control Sepharose column was constructed. One gram of CNBr-activated Sepharose (Pharmacia) was suspended in 1 mM HCl, washed with 300 ml of HCl, then with 200 ml of 0.1 M sodium carbonate, pH 8.3. The matrix was then incubated with 20 ml of 1 M ethanolamine, pH 8.0, for 2 h at room temperature. The Sepharose was then washed three times consecutively with 0.5 M NaCl, 0.1 M acetate, pH 4, then 0.5 M NaCl, 0.1 M Tris-HCl, pH 8.0. Purified native RHL was run on the Sepharose column under the same conditions used for heparin-Sepharose chromatography.

Cell binding assay
A cell-binding assay was used to quantify changes in the equilibrium binding affinity of the lipase constructs for cell surface HSPGs (16). CHO cells transfected with wild-type cLPL (19), RHL, RHL2LPL, RHL4LPL, HB-A, HB-B, HB-C, HB-E, HBF, and HB-G were cultured on 60-mm dishes. Fresh medium (3 ml) was added to eight dishes per construct when the transfected cells reached ~90% confluency. After a 4-h incubation, the dishes were placed on ice at 4°C for 2 h. The medium was removed and saved; two dishes were pooled per sample yielding 4 samples/construct. The dishes were then washed with 3 ml of ice-cold PBS, and the bound lipase was eluted with medium containing 100 units heparin/ml. The "bound" and "free" lipase samples were assayed for mass. The cells were harvested and assayed for DNA content using a previously described procedure (36).

	RESULTS

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Construction of RHL heparin-binding mutants
Several mutants were made to characterize the regions in hepatic lipase that are responsible for the binding of the enzyme to heparin and heparan sulfate. The mutations were of two types: substitution mutants in which putative heparin-binding residues of RHL were mutated to Asn, and chimera mutants which involved the substitution of previously identified heparin-binding sequences of LPL for the corresponding regions of RHL. The substitution of a polar amino acid for a basic residue is a conservative one and therefore would most likely preserve the relative solvent exposure of the residue, minimizing the chance of a disruption in structure of the enzyme. The mutants were assayed for heparin-binding ability using heparin-Sepharose chromatography and a cell surface HSPG binding assay, and for enzymatic activity to verify that the overall structure was not disrupted by the mutations.

Heparin-Sepharose chromatography of lipase constructs
Heparin-Sepharose chromatography has been used in several studies to demonstrate a loss in heparin affinity of site-directed mutants of LPL (19) (20) (21) (22). The salt concentration required for elution of the protein from the column matrix provides a measure of the affinity of the lipase for heparin. Dimeric LPL elutes at ~1.2;–1.3 M NaCl; elution of heparin-binding mutants at lower salt concentration indicates a loss in affinity for heparin relative to the wild-type protein (19) (22). The high-affinity binding of proteins to heparin is due to specific interactions between heparin and precisely arranged residues within a binding region on the protein (37). As a control we constructed a Sepharose column with matrix derivatized with ethanolamine. RHL eluted from such a column at 0.35 M NaCl.

Site-directed mutagenesis of cluster 1 of cLPL (Arg 281;–Arg 284) was previously shown to disrupt heparin binding of the enzyme. This region is conserved in RHL, therefore, substitution mutants of RHL were made in which the basic residues in the region Lys 297;–Arg 300 were singly substituted (HB-A, HB-B, HB-C), or entirely replaced (HB-E) with Asn. These mutations lowered the affinity of RHL for heparin-Sepharose ( Fig 1a, Table 5) implying that Cluster 1 is involved in the binding of RHL to heparin. The single substitution mutants HB-A and HB-B showed a modest decrease in the salt required for elution, while HB-C eluted at a much lower salt concentration (0.48 M NaCl) than wild-type RHL (0.70 M NaCl). The mutant in which all residues were substituted for Asn (HB-E), eluted at 0.44 M salt, which is much lower than the value for wild-type RHL (0.70 M NaCl).

View larger version (45K): [in this window] [in a new window]   Figure 1. Heparin-Sepharose chromatography of wild-type RHL and the site-directed substitution mutants. Heparin-Sepharose chromatography was carried out as described in Materials and Methods. Both RHL enzyme mass (•) and catalytic activity () were measured on fractions; (A) wild-type (wt RHL) and mutant RHL in Cluster 1; (B) wild-type (wt RHL) and mutant RHL in Cluster 4.

A chimeric mutant was constructed in which Cluster 2 of LPL was substituted for the corresponding region in RHL. Several attempts to transfect RHL2LPL into CHO cells failed to produce stable transformants secreting catalytically active enzyme. In addition, the transfected cells secreted very low amounts of enzyme protein. CHO cells transfected with wild-type RHL secreted 16.5 ng/h per 75- cm² flask, whereas the corresponding value for RHL2LPL was 0.6. Accordingly we cannot draw any conclusions concerning the function of Cluster 2 in heparin binding.

Mutations in the Cluster 4 region of LPL were shown to affect heparin-binding (22). This region is partially conserved in RHL. The conserved residues (Lys 337, Lys 436, and Arg 443) were mutated to determine whether they are involved in the heparin binding of RHL. A substitution mutant in which Lys 337 (corresponding to Lys 321 in LPL) was mutated to Asn (HB-F), and one in which Lys 436 and Arg 443 (corresponding to Lys 409 and Lys 416 in LPL) were both changed to Asn (HB-G), were constructed. These mutants both eluted at a lower salt concentration from heparin-Sepharose than wild-type RHL (Fig 1b, Table 5) indicating that these conserved residues may be important in the binding of RHL to heparin. Part of Cluster 4 is not conserved in RHL, namely Thr 432;–Trp 434. This region (residues 431;–434) was mutated to that of LPL in the chimera mutant RHL4LPL (Table 3). This mutant showed an increased affinity for heparin-Sepharose, relative to wild-type RHL, as reflected by a higher salt concentration required for elution from the affinity matrix ( Fig 2, Table 5).

View larger version (22K): [in this window] [in a new window]   Figure 2. Heparin-Sepharose chromatography of wild-type RHL, wild-type LPL and the Cluster 4 chimeric mutant RHL4LPL. Heparin-Sepharose chromatography was carried out as described in Materials and Methods. Both RHL enzyme mass (•) and catalytic activity () were determined on wild-type RHL (wt RHL) and RHL4LPL. A chromatogram of cLPL is included for comparison.

Equilibrium binding of lipase constructs to cell-surface heparan sulfate
Heparin-Sepharose chromatography provides a measure of the relative affinity of different site-directed mutants for heparin, but yields no direct information regarding how the equilibrium binding constant of the enzyme has been affected by these mutations. An equilibrium binding experiment described by Berryman and Bensadoun (19) gives a direct measure of this effect. Briefly, after a medium change transfected CHO cells are permitted to secrete lipase for 4 h, then the cells are incubated at 4°C during which time the lipase partitions between the bound (cell surface) and free (medium) states. It has been shown previously that HSPGs are primarily responsible for the binding of LPL to CHO cells (38) and this is most likely the case for hepatic lipase as well. The enzyme concentrations are low (5 to 3 times less than K_d) so this simple relationship holds:

B = n K F

where B is the bound enzyme, n is the number of binding sites on the CHO cell surface, K is the affinity constant, and F is the free enzyme concentration. Because n, the number of binding sites for RHL on the cell surface, is not expected to change from construct to construct, the ratio of bound to free enzyme is proportional to the affinity constant.

We measured the bound to free ratios for all RHL mutants ( Table 6). Mutation of all basic residues in Cluster 1 in HB-E reduced the affinity of HL for the CHO cell surface dramatically. Single point mutations of basic residues in cluster 1 had major effect on affinity. Mutation of arg 300 (HB-C) caused the greatest decrease in binding affinity when compared to mutations of Lys 297(HB-A) and Lys 298(HB-B). The chimera mutant RHL4LPL demonstrated an increase in equilibrium binding as compared to wild-type RHL. In fact, this mutant showed a cell-binding affinity that was similar to that of wild-type LPL.

Enzyme activity of RHL constructs
The enzymatic activity of the constructs was assayed as an indicator of any global changes in the tertiary structure of the mutants (Table 5). All of the mutants possessed activity with the exception of RHL2LPL. RHL2LPL was secreted at very low levels, and enzyme activity could not be measured reliably. In some of the activity profiles (Fig 1), a peak is seen eluting at about 1.1 M NaCl. This is most likely due to endogenous LPL that CHO cells are known to secrete (39).

	DISCUSSION

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Several site-directed mutants of hepatic lipase were constructed to identify regions of the protein involved in heparin binding. These mutant constructs were expressed in CHO cells and evaluated for heparin-binding ability using heparin-Sepharose chromatography and a cell-based binding assay. Site-directed mutagenesis was used to confer increased or decreased heparin binding of various HL mutants. A limitation of this method is that there exists a possibility that the mutation causes a loss of function by altering the structure of the protein. Indicators such as preservation of enzymatic activity suggest that the overall structure has not been perturbed, but one cannot state with certainty that this is the case. Despite this limitation, in the absence of information such as X-ray data, site-directed mutagenesis is a useful tool for identifying potential structural features of proteins. The results of these experiments define many of the putative residues that could be involved in heparin binding of the protein, and allow for a comparison with the structurally related enzyme LPL.

The RHL mutant constructs, with the exception of RHL2LPL, exhibited enzymatic activities that were similar to that of wild-type RHL (Table 5). This suggests that for mutants of Cluster 1 and 4, the overall structure of the lipase was not affected by the mutations. Berryman et al. (28) saw a similar preservation of activity subsequent to epitope-tagging of the lipase. The stability of RHL is in contrast to that of LPL, which was shown to be much more sensitive to mutation, as reflected by large reductions in enzymatic activity, in similar studies (19) (20) (21) (22). The greater stability of RHL, as compared to LPL, indicates that it is a superior candidate for future structure/function studies.

Cluster 1 of LPL (Arg 281;–Arg 284, avian sequence numbering) was previously proposed to be involved in heparin binding (19) (20) (21). This region is conserved in RHL (Table 2). Mutation of the basic residues of this cluster resulted in a reduced heparin binding ability of RHL. This was reflected by a shift to lower salt concentration required for elution from a heparin-Sepharose column; 0.44 M NaCl for the triple mutant HB-E versus 0.70 M NaCl for wild-type RHL. RHL eluted from the control column at 0.35 M NaCl. HB-E eluted at a higher salt concentration (0.44 M NaCl) suggesting that other residues in addition to Cluster 1 might be involved in the heparin-RHL interaction. The effect of this triple mutation on the equilibrium affinity constant of RHL for cell surface HSPGs was shown in a cell-based binding assay (Table 6). This experiment indicated a major decrease in affinity of the lipase for heparan sulfate; the [bound]/[free] values for HB-E and RHL were 0.14 ± 0.03 and 6.20 ± 1.39 (ng/dish)/(ng/ml)/100 mg DNA, respectively. Both of these measures of heparin affinity suggested that Cluster 1 is a significant heparin-binding region of RHL.

Cluster 2 of LPL has previously been subjected to site-directed mutagenesis yielding conflicting results as to which specific residues are involved in heparin binding, but suggesting that, overall, this region has importance in the heparin-LPL interaction (20) (21). We constructed a chimeric mutant, RHL2LPL, in which the sequence of LPL within the vicinity of Cluster 2 has been substituted for the corresponding region in RHL. CHO cells transfected with this construct secreted very low levels of enzyme and therefore no conclusion can be drawn on the significance of Cluster 2 in heparin binding.

Cluster 4 of LPL has been shown to be involved in the heparin binding of LPL (22). This cluster is partially conserved in RHL (Table 2). Mutation of the conserved residues in this cluster (Lys 337, Lys 436, Arg 443) in the mutants HB-F and HB-G resulted in a loss in heparin-binding ability based on heparin-Sepharose elution data (Table 5, Fig 1b) and cell binding data. This suggests that these residues are involved in the heparin binding of RHL and that they may account for the residual heparin binding seen in the mutant HB-E (Fig 1a). The non-conserved residues in this region (residues 431;–434) were mutated to the corresponding LPL residues in the RHL4LPL chimera (Table 3). This mutant exhibited enhanced binding to heparin-Sepharose (Table 5, Fig 2) and cell surface HSPGs (Table 6), as compared to wild-type RHL. Production of an intact Cluster 4 region in RHL4LPL resulted in cell surface binding similar to that of LPL.

These data have provided a broad picture of putative regions in HL that may be required for binding to heparin. This interaction requires residues in Cluster 1 (Lys 297;–Arg 300) and Cluster 4 (Lys 337, Lys 436, and Arg 443). HL, which possesses a lower affinity for heparin than does LPL, does not have a fully intact Cluster 4 heparin-binding region. The biological significance of the different heparin-binding affinities of LPL and HL is not clear. Fromm et al. (37) demonstrated that heparin, with its closely spaced charge density, interacts with high affinity with peptides containing closely spaced basic residues, while heparan sulfate, with its more widely spaced charge clusters, binds most tightly to peptides with widely spaced basic residues. This indicates that the positioning of basic residues within the tertiary structure of a protein dictates whether that protein will interact with a given heparan sulfate chain. The differences in affinity for heparin seen between LPL and HL may indicate that the HSPG binding sites of these proteins in vivo differ. Evidence in support of this theory includes a rat liver perfusion study, which demonstrated that, in normal rat liver, additional perfused HL was not capable of binding to sites within the liver, while additional perfused LPL was capable of binding (40). This suggests that at least some of the binding sites of HL and LPL within the liver are not interchangeable. Additional experiments to determine the specificity of HL and LPL for particular HSPGs would be of interest to further characterize the binding of these enzymes to HSPGs in vivo.

In summary, we have produced site-directed mutants of HL and assessed these constructs for their ability to bind heparin and heparan sulfate. Our results suggest that all of the basic residues in Cluster 1 (Lys 297;–Arg 300) and some of the residues in Cluster 4 (Lys 337, Lys 436, and Arg 443) are involved in the heparin binding of HL. The lack of a completely intact Cluster 4 region, as compared to LPL, is responsible for the lower heparin-binding affinity seen for HL.

	ACKNOWLEDGMENTS

These studies were supported in part by NIH Training Grant GMO7273-23 (to R.A.S.) and NIH Grant HL14990.

Manuscript received August 31, 1998; and in revised form June 10, 1999; and in revised form October 29, 1999

Abbreviations: HL, hepatic lipase; LPL, lipoprotein lipase; CHO, Chinese hamster ovary; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycan; RHL, rat hepatic lipase; cLPL, chicken lipoprotein lipase; ELISA, enzyme-linked immunosorbent assay

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