Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans.

Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic α2-macroglobulin receptor/low density lipoprotein receptor-related protein (α2MR/LRP). Whereas it is established that the C-terminal folding domain binds to α2MR/LRP, it remains uncertain whether it binds to heparin and to HSPG. To identify segments important for binding to α2MR/LRP and to clarify possible binding to heparin, we produced constructs of the human C-terminal folding domain, LpL-(313-448), and of the fragment LpL-(347-448) in Escherichia coli. In addition to binding to α2MR/LRP, LpL-(313-448) displayed binding to heparin with an affinity similar to that of the LpL monomer, whereas it bound poorly to lipoprotein particles. Moreover, LpL-(313-448) displayed heparin sensitive binding to normal, but not to HSPG deficient Chinese hamster ovary cells. LpL-(313-448) and LpL-(347-448) showed similar affinities for binding to both purified α2MR/LRP and to heparin. Deletion of LpL residues 380-384 abolished the binding to LRP, whereas binding to heparin was unperturbed. The binding to both heparin and α2MR/LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448). We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids within residues 404-430. Two segments of the domain are necessary for efficient binding to α2MR/LRP, one comprising residues 380-384 and another overlapping the segment important for binding to heparin.

nants can be taken up via the endocytic ␣ 2 -macroglobulin receptor/LDL receptor-related protein (␣ 2 MR/LRP) either mediated by apolipoprotein E, or by lipoprotein lipase (LpL) or hepatic lipase associated with the lipoproteins (1)(2)(3)(4). Recent results provide evidence that the uptake via ␣ 2 MR/LRP occurs in vivo, since remnant lipoproteins accumulate in mice that express reduced amounts of ␣ 2 MR/LRP and also lack functional LDL receptors (5). It has been suggested that the LpL-mediated pathway may be particularly important in the vascular wall, since ␣ 2 MR/LRP is abundant in macrophages and smooth muscle cells of atherosclerotic lesions and since LpL is secreted by macrophages in the lesions (6). In addition, it was recently shown that, whereas normal macrophages incubated with VLDL accumulate massive amounts of lipids, this does not occur in LpL-deficient macrophages (7).
LpL binds to cell surface heparan sulfate proteoglycans (HSPG) as reflected in its strong affinity for binding to heparin. This property is physiologically important for the docking on endothelial cells exposing the lipase to circulating lipoproteins. LpL also binds to HSPG of other cell types as recently shown directly by immunofluorescence studies (8), and studies in Chinese hamster ovary cells have shown that uptake and degradation of LpL can be mediated by HSPG (9). In addition, the docking of LpL on HSPG of cells rich in ␣ 2 MR/LRP is important for uptake of LpL and for LpL-mediated uptake of lipoproteins via this receptor (4,10,11).
LpL is a member of the mammalian lipase family also comprising the homologous hepatic and pancreatic lipases. The crystallographic structure of pancreatic lipase (12) shows that it consists of two folding domains, a larger N-terminal and a smaller C-terminal domain. Based upon the similarities in sequence it is thought that the domain organization is similar for LpL, and a three-dimensional model was recently proposed (13). LpL circulates both as a 96-kDa homodimer, which is the normally secreted and catalytically active form, and as a catalytically inactive monomer (14 -16). Although not known, it is likely that the LpL dimer is arranged head-to-tail in a way that allows enough space for conformational changes following substrate binding (13). The dimeric structure of LpL causes an increase in the affinity for heparin, presumably because sites in both monomers can participate in binding of one heparin molecule (13), and helps efficient ␣ 2 MR/LRP-mediated lipoprotein uptake, possibly because only the dimer can bind to a lipoprotein particle and to the receptor at the same time (10,17).
The N-terminal folding domain of human LpL, comprising residues 1-312, harbors the catalytic triad with its covering loop, which is important for interaction with lipid substrates (18), sites for binding to heparin (16), and the site for binding of the LpL cofactor apolipoprotein C-II (2). The C-terminal folding domain of the human lipase, comprising residues 313-448, is important for binding to ␣ 2 MR/LRP and also participates in binding to lipoprotein particles and to heparin. Thus, it is known that sequences within aa 378 -423 largely account for the affinity to ␣ 2 MR/LRP (11). The presence of a secondary lipid binding site in the C-terminal folding domain is strongly suggested by the finding that truncation of LpL at residue 390, which does not disrupt the dimeric structure, abolishes binding to particles such as chylomicrons (19) and rabbit ␤-VLDL (10). In addition, LpL-(313-448) has been reported to mediate binding of lipoproteins to ␣ 2 MR/LRP (20). Mutational studies have suggested that the secondary lipid binding site includes the tryptophan residues 390 and 393 (17,20). Previous results concerning heparin binding affinity of the C-terminal domain have been ambiguous. Studies including LpL-hepatic lipase chimeras strongly suggest that the C-terminal folding domain has a site for binding of heparin (21,22). This is in accordance with the result that heparin abolishes binding of a C-terminal LpL fragment (residues 378 -423) to ␣ 2 MR/LRP, which itself does not bind heparin. Earlier studies of mutated LpL species have shown that candidate segments comprising residues 390 -393 and 439 -448 appear not to contribute to the heparin affinity (16). Finally, the result that dimeric LpL-(1-390) and LpL-(1-448) are eluted at the same salt concentrations following heparin affinity chromatography (19) may be taken as an argument against a separate functional heparin binding site in the C-terminal folding domain. The purpose of the present work was to identify segments important for the receptor recognition of the C-terminal folding domain, to clarify whether the domain binds to lipoproteins, and to characterize possible binding to heparin and to HSPG. We report that the C-terminal folding domain binds poorly to lipoproteins, whereas the domain does bind to heparin and to cellular HSPG. Moreover, we provide evidence that two segments are important for binding to the receptor, one of them overlapping with the heparin binding domain.
Expression and Purification of Recombinant Proteins-The construct H 6 FX-LpL-(347-448), containing the hexahistidine-factor X substrate sequence MGSH 6 SIEGR and aa 347-448 of human LpL, was prepared as described by Nielsen et al. (24). This and other constructs containing human LpL sequences are referred to as LpL constructs. H 6 FX-LpL-(313-448) was generated in the same way, except that the primer 5Ј-CAGGATCCATCGAGGGTAGGGTCTTCCATTACCAAGTA-3Ј was used for the N-terminal. The mutated H 6 FX-LpL-(347-448) constructs were generated using full-length human LpL cDNAs containing different point mutations and deletions generated as described previously (16,17). All LpL fragment constructs were cDNA-sequenced to eliminate possible PCR errors. The recombinant C-terminal folding domain construct, H 6 FX-LpL-(313-448), and the recombinant wild-type and mutated LpL fragment constructs were expressed in Escherichia coli DH1 cells and purified on a Ni 2ϩ nitriloacetic acid column (11,24). The recombinant proteins were more than 95% pure, as judged from Coomassie Brilliant Blue-stained SDS-polyacrylamide gels, and were generally soluble in concentrations up to 20 -50 M in aqueous buffer. The H 6 FX-LpL-(313-448) construct, referred to as LpL-(313-448), was 125 Iiodinated to a specific activity of about 4 ϫ 10 6 Bq/mol using chloramine T as the oxidizing agent (11,24).
Heparin Preparations-Unless otherwise stated, heparin was from Leo Pharmaceuticals, Denmark. Tinzaparin, a low molecular mass heparin, and [ 3 H]tinzaparin with a specific activity of about 8 mCi/mg (27), were kind gifts from Dr. P. B. Østergaard, Novo Nordisk A/S, Denmark. Heparin-Sepharose (Cl6B) was from Pharmacia, Sweden.
Solid Phase Assays-␣ 2 MR/LRP was immobilized in Maxisorp, and bLpL and recombinant LpL fragments were immobilized in Polysorp microtiter wells (both from NUNC, Denmark) by incubation for 2 h at 20°C in 50 mM NaHCO 3 , pH 9.6, as described previously (10,11,24). Separate experiments showed that the bLpL was catalytically inactive after immobilization to the wells, 2 and the immobilized lipase is therefore thought to be in monomeric form. After washing, the wells were blocked in 5% bovine serum albumin when using 125 I-␣ 2 MR/LRP or 125 I-LpL-(313-448), and with 2% Tween 20 when using [ 3 H]tinzaparin. All incubations in microtiter wells were performed for 16 h at 4°C in 100 l of 140 mM NaCl, 10 mM Hepes, 2 mM CaCl 2 , 1% bovine serum albumin, pH 7.8. Following washing with 2 ϫ 200 l of incubation buffer, bound radioactivity was removed with 10% SDS and counted. Competition-inhibition curves were drawn as the best least square fit to triplicate determinations using the equation where b/f is the ratio of bound and free labeled ligand, R is the concentration of binding sites immobilized in the well, and F is the concentration of free competitor.
Culture and Incubation of Cells-COS-1 cells (American Type Culture Collection CRL 1650) were incubated in monolayers (about 3 ϫ 10 5 cells/ml) as described previously (24). After incubation for 4 h at 37°C, degradation of 125 I-␤-VLDL was assessed by measuring radioactivity in the medium soluble in 12% trichloroacetic acid. After washing, the cells were lysed in 1 M NaOH and assayed for cell-associated radioactivity. Normal human skin fibroblasts were obtained as described previously (8) and cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing glutamine (2 mM), penicillin, and streptomycin. Wild-type and mutant (CHO-745) Chinese hamster ovary cells lacking heparan sulfate and chrondroitin sulfate glycosaminoglycans (28) were kindly provided by Dr. J. D. Esko, Department of Biochemistry, University of Alabama at Birmingham. The CHO cells were grown in Ham's F-12 culture medium supplemented with 10% fetal calf serum, glutamine, and antibiotics. Cells were plated on glass coverslips for immunofluorescence experiments and used between the 3rd and 4th day of seeding.
Immunofluorescence Experiments-The binding of bLpL (2.5 g/ml) or LpL(313-448) (25 g/ml) to fibroblasts and CHO cells was performed by incubating prechilled cells for 30 min at 4°C in Dulbecco' modified Eagle's medium containing 20 mM Hepes (pH 7.4) and 1% defatted bovine serum albumin. The cells were then fixed for 15 min at room temperature in 3% paraformaldehyde, 2% sucrose, 0.1 mM phosphate buffer, pH 7.4. After rinsing twice in phosphate-buffered saline (PBS, 10 mM phosphate, 150 mM NaCl, pH 7.4) containing 20 mM glycine, the fixed cells were blocked with PBS-glycine containing 1% bovine serum albumin. As a primary antibody, we used the monoclonal antibody 5D2 (Oncogene) at dilution 1:100. This antibody is reported to react within residues 396 -405 of the C-terminal folding domain (11,29), and separate analysis showed that it did not react with the peptide comprising aa 402-423 used in inhibition experiments. A fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (F(abЈ) 2 fragment, DAKO, Denmark) at dilution 1:50 was used to visualize the primary antibody. Both antibodies were diluted in PBS-glycine, 1% bovine serum albumin. The primary antibody was applied for 45 min at 37°C and, following washing in PBS-glycine for 15 min, the secondary antibody was applied for 45 min. The coverslips were then labeled with the nuclear stain Hoechst 33342, mounted upside-down on a glass slide with a drop of Immunofluore (ICN Biomedicals) and viewed on a Zeiss Axioskop equipped for epifluorescence. For confocal microscopy studies we used a Leica TCS 4D confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope and a 63ϫ (numerical aperture 1.4, oil) Leitz Plan-Apo objective. Fluorescein isothiocyanate was excited at 488-nm lines from a 75-mW krypton-argon laser. Each image was the average of four serial scans at the confocal microscope normal scan rate (30).
Image sizes were 512 ϫ 512 and were photographed on a color high resolution video printer. The results are representative for at least five separate experiments.

RESULTS
Segments Important for Binding to ␣ 2 MR/LRP-We first examined whether the the C-terminal folding domain of the human lipase, LpL-(313-448), has an affinity toward ␣ 2 MR/ LRP similar to that of the fragment, LpL-(347-448). Fig. 1 shows binding of 125 I-labeled folding domain to immobilized purified ␣ 2 MR/LRP. The binding was completely inhibited by unlabeled LpL-(313-448) or LpL-(347-448) at high concentrations. The potency of the fragment toward inhibition of the binding of 125 I-LpL-(313-448) was similar to or slightly lower than that of the folding domain. Other experiments (not shown) demonstrated complete inhibition of the binding of tracer in the presence of heparin (10 units/ml).
In the reverse experiment, 125 I-labeled ␣ 2 MR/LRP was bound to immobilized bovine LpL, and the inhibitory potencies of LpL-(313-448) and LpL-(347-448) were again found to be similar and inhibitable by heparin (not shown). We decided to use the LpL-(347-448) fragment as the basis for mutational analysis since the yields of the recombinant products were satisfactory. The inhibitory potencies toward binding of 125 I-␣ 2 MR/LRP to immobilized bLpL were measured as illustrated in Fig. 2. The results summarized in Table I show that deletion of aa 380 -384 caused a 10 -20-fold reduction in the inhibitory potency. Deletion of aa 390 -393, or exchange with the equivalent aa in human hepatic lipase, had no effect. Deletion of a large segment comprising aa 404 -430 also caused a marked reduction in the inhibitory potency, whereas the deletion of aa 404 -414 caused only an approximately 4-fold reduction. The displayed point mutations had little or no effects. Previous results have shown that LpL-(378 -448) and LpL-(378 -423) have the same affinity to ␣ 2 MR/LRP (11). When taken together, the results therefore indicate that two segments rich in basic residues are important for the interaction with receptor: aa 380 -384 (LKWKS) and aa 404 -423 (IRVKAGETQK-KVIFCSREKV).
Binding to ␤-VLDL-It has been reported that LpL-(313-448) (20), in contrast to the fragment LpL-(378 -448) (11) can mediate the the binding of lipoproteins to ␣ 2 MR/LRP. We therefore compared the abilities of the folding domain, LpL-(313-448), and the fragment, LpL-(347-448), to bind 125 I-labeled rabbit ␤-VLDL. Fig. 3A shows that LpL-(347-448) does not bind ␤-VLDL. The binding of ␤-VLDL to LpL-(313-448) was confirmed, although this binding is poor compared with the binding to bLpL. Fig. 3B shows that LpL-(313-448) can mediate binding of ␤-VLDL to ␣ 2 MR/LRP, although poorly, and that the concentrations of LpL-(313-448) required for the mediation of lipoprotein binding are more than 100-fold higher than those needed for bLpL. Finally, Fig. 4 shows that, whereas 100 nM bLpL clearly mediates degradation of 125 I-␤-VLDL in COS-1 cells, which express abundant ␣ 2 MR/LRP (24), 1 M of the C-terminal folding domain is incapable of mediating degradation of the labeled lipoprotein. We therefore conclude that the C-terminal folding domain cannot on its own mediate efficient uptake of lipoproteins.  Binding to Heparin-We next turned to the question whether the C-terminal folding domain has a site for heparin binding. Fig. 5 shows that LpL-(313-448) was retained on heparin-Sepharose and eluted at 0.8 M NaCl. The position of elution was similar to that of the bLpL monomer, whereas dimeric bLpL eluted at 1.3 M NaCl. This result suggests that a segment in the C-terminal folding domain may account for a large part of the heparin affinity of the LpL monomer. We used binding of tinzaparin, a low molecular mass heparin (average M r ϳ4,500) for further analysis of the putative heparin binding segment. Fig. 6 shows an experiment in which bLpL, LpL-(313-448), or LpL-(347-448) immobilized in microtiter wells were incubated with [ 3 H]tinzaparin and increasing concentrations of unlabeled tinzaparin. The results show that tinzaparin bound with approximately equal affinities to LpL-(313-448) and LpL-(347-448), and that the affinity was only moderately lower than that for binding to bLpL that was catalytically inactive after immobilization 2 and therefore thought to be in monomeric form. Similar experiments with immobilized ␣ 2 MR/ LRP showed no binding of heparin (data not shown) confirming that heparin inhibits binding of the C-terminal folding domain due to interaction with the ligand and not with the receptor. Table II summarizes the results concerning binding of tinzaparin to LpL and wild-type and mutated LpL fragments. It appears that the deletion of aa 380 -384, which essentially abolished the binding to ␣ 2 MR/LRP (Table I), had no effect on the binding of tinzaparin. Deletion of the large segment comprising aa 404 -430 completely abolished the binding of labeled tinzaparin, whereas deletion of the aa 404 -414 segment had only a minor effect.
The results show that heparin can bind directly to the Cterminal folding domain and that residues within the segment comprising aa 404 -430 are essential. Since heparin inhibits the binding of LpL-(378 -423) to ␣ 2 MR/LRP (11), it seemed possible that aa 424 -430 are not essential. This prompted us to prepare the peptide comprising aa 402-423 and analyze for possible binding to heparin-Sepharose. The peptide was retained on the heparin column used for chromatography of LpL-(313-448) and eluted at about 0.3 M NaCl, whereas the irrelevant basic peptide urokinase-(148 -159) (AGQKTLRPRFK) was not retained (not shown).
The C-terminal Folding Domain Binds to Cellular HSPG-The binding to normal human fibroblasts and to wild-type as well as heparan and condroitin sulfate glycosaminoglycan-deficient CHO cells was investigated in order to evaluate the possible physiological significance of the heparin binding. Fig.  7, row 1, shows that LpL-(313-448) was able to bind to fibroblasts. As for dimeric bLpL, the binding of the C-terminal folding domain was mainly surface-associated. In addition, scattered bright fluorescence spots were observed when using LpL-(313-448), indicating some nonspecific binding. Heparin (50 units/ml) inhibited the binding to the fibroblasts (not shown). Wild-type CHO cells (row 2) also bound LpL-(313-448) as well as bLpL, and the binding was inhibited by heparin (row 3). Moreover, binding was inhibited by the peptide comprising aa 402-423 (row 4), but not by the irrelevant peptide (not shown), Finally, the binding of both bLpL and the folding domain was greatly reduced when using the CHO-745 cells deficient in HSPG (row 5). It should be noted that the apparent nonspecific binding persisted in the presence of heparin and in incubations of the CHO-745 cells. When taken together, the results indicate that the C-terminal folding domain binds to proteoglycans and might participate in the docking of LpL to cell surfaces. DISCUSSION The present results help defining the segments involved in the functions of the C-terminal folding domain of LpL. Fig. 8 shows a model of the folding domain (13) based on the known three-dimensional structure of pancreatic lipase (12) and highlights the segments important for binding to receptor and to heparin. We suggest that residues within the segment comprising aa 380 -384 are important for recognition of ␣ 2 MR/LRP and that residues within aa 404 -430 are important for binding to receptor as well as to heparin. This interpretation presumes that the functional changes of the C-terminal folding domain are the direct result of the deletion mutations. However, it cannot be excluded that some functional changes may be due to conformational changes of the domain induced by the mutations. This applies particularly to the large deletion of aa 404 -430.
Binding to ␣ 2 MR/LRP-The most notable result is perhaps the dramatic decrease in affinity following deletion of aa 380 -384 (LKWKS). The findings that the affinity for binding of heparin remains unperturbed, and that other deletions, e.g. of aa 390 -393, have little or no effect on the receptor binding affinity, argue that residues in this region interact with the receptor. Since basic residues of other ligands have been shown to be important for binding to the receptor (31), we propose that Lys-381, Lys-383, and possibly Lys-379 participate in the binding. Surprisingly, deletion of aa 404 -414, including Lys-407 which is thought to be critical for binding to ␣ 2 MR/LRP (20), caused only a moderate reduction in the affinity. On the other hand, deletion of the large segment comprising aa 404 -430 caused a marked decrease in the affinity for the receptor. Since previous results have shown the same receptor binding affinity of the fragments comprising aa 378 -448 and 378 -423 (11), it seems likely that residues within the segment aa 404 -423 participate in the binding. These may include Arg-405, Lys-407, Lys-413, Lys-414, and particularly, as judged from the minor effect of deletion of aa 404 -414, Arg-420 and Lys-422. Since neither deletion of aa 380 -384 nor of aa 404 -430 completely abolished the affinity to the receptor, we propose that two segments located on either side of the C-terminal folding domain interact with ␣ 2 MR/LRP to provide high affinity binding.  The exact location of sites on the receptor for binding of LpL is not known. However, we have previously shown that wildtype LpL-(347-448) binds to the same sites on the receptor as the C-terminal fragment of RAP (24) which primarily binds in the region of ␣ 2 MR/LRP containing a cluster of eight LDL receptor class A repeats (32,33). Recently, the three-dimensional structure of class A repeats has shown a clustering of many of the side chains of acidic residues, including the conserved Ser-Asp-Glu triad (34), thus emphasizing the importance of exposed basic residues in ligands. It is also of note that LpL, like RAP, binds to two other members of the LDL receptor family, megalin (35) and the VLDL receptor (36), with about the same affinity as to ␣ 2 MR/LRP, and it may be inferred that the same segments of LpL participate in binding to class A repeats of these receptors.
Binding to Lipoproteins-Previous studies showed that the receptor binding fragment LpL-(378 -448) does not bind to ␤-VLDL and therefore inhibits the LpL-mediated uptake of lipoproteins via ␣ 2 MR/LRP. On the other hand, the C-terminal folding domain was reported to bind to lipoproteins and to mediate their uptake into cells (37). We find that although LpL-(313-448) does bind to ␤-VLDL, the concentrations needed are several magnitudes higher than for LpL. This also applies to the mediation of ␤-VLDL binding to ␣ 2 MR/LRP. We therefore conclude that the secondary lipid binding site in the C-terminal folding domain is insufficient on its own, even though it is important for binding of full-length LpL to lipoprotein particles (10,19). Although not proven, it is likely that Trp-393 and Trp-394 participate in binding to lipoproteins, since substitution of both residues with alanine abolishes binding of LpL-(313-448) to lipoproteins (20), and since deletion of aa 390 -393 reduces the ability of dimeric LpL to promote lipoprotein uptake by about 40% (17). The position of the putative secondary lipid binding site adjacent to residues 380 -384, which are necessary for binding to receptor, may explain why the dimeric structure of LpL appears important for binding to both lipoproteins and to ␣ 2 MR/LRP and thereby for efficient mediation of lipoprotein binding and uptake (10,11).
Binding to Heparin and to HSPG-The present results show for the first time that the C-terminal folding domain binds to heparin. Previous studies have examined two candidate regions in this domain by using mutational analysis of dimeric LpL. One region is the cluster of positive charges in the extreme C-terminal region (13,38). Ma et al. (16) found that replacement of the 10 C-terminal aa with the equivalent residues in pancreatic lipase did not disrupt the affinity for heparin, in agreement with our previous result that heparin at low concentration inhibited binding of both LpL-(378 -448) and LpL-(378 -423) to ␣ 2 MR/LRP (11). The other region is the Prechilled cells were incubated for 30 min at 4°C with bLpL (2.5 g/ml) or LpL-(313-448) (25 g/ml). Heparin, when present (row 3), was 50 units/ml. In some experiments (row 4), cells were preincubated for 30 min at 4°C with 50 M peptide comprising aa 402-423, followed by continued incubation for 30 min with bLpL or LpL-(313-448). After binding, the cells were fixed in paraformaldehyde, and bound bLpL or LpL-(313-448) was detected using the 5D2 antibody followed by a fluorescein isothiocyanate-conjugated anti-mouse IgG. Immunofluorescence staining was analyzed and photographed by a confocal microscope. Laser conditions were identical for all micrographs. Note the presence of nonspecific fluorescence spots when using LpL-(313-448), possibly due to the low solubility of the recombinant domain. Bar ϭ 25 m.  (13). The start of the C-terminal folding domain (residue 313) is at the upper left corner, and the Cterminal 10 residues (aa 439 -448) are deleted. Highlighted segments comprise residues 380 -384 important for binding to ␣ 2 MR/LRP and residues 402-423 important for binding to receptor as well as to heparin. The tryptophan residues thought to participate in binding to lipoproteins are also indicated. noncharged sequence aa 390 -393 (WSDW) homologous to the heparin binding type-1 repeat of thrombospondin, and, as in our experiments with the C-terminal folding domain, deletion or exchange with the equivalent sequence in hepatic lipase had little influence on the heparin affinity of LpL (16). A third candidate region is the cluster of basic residues originating from a noncontiguous peptide stretch and comprising Lys-319 -Lys-403-Arg-405-Lys-407-Lys-413-Lys-414 (13). In addition, contacts may be made to glutamine residues (e.g. aa 402 and 412) as shown for basic fibroblast growth factor (39). It appears that Lys-319 is not important since LpL-(313-448) and LpL-(347-448) showed similar affinities. Surprisingly, the heparin affinity was only moderately affected following deletion of aa 404 -414, whereas deletion of aa 404 -430 abolished the binding to heparin. This may suggest that Arg-420-and Lys-422 are important as supported by the heparin affinity, although weak, of the peptide LpL-(402-423). In addition, the ability of this peptide to inhibit binding of bLpL and LpL-(313-448) to wildtype CHO cells is presumably due to a blocking of the sites on HSPG.
The finding that the C-terminal folding domain binds to heparin with about the same affinity as the LpL monomer suggests that it is an important determinant for binding to HSPG and therefore presumably for catabolism of the monomer. The considerably higher affinity of the dimer is thought to be due to interaction of one heparin molecule (e.g. dodecamer or longer) with the two LpL monomers at the same time. The heparin molecule may span the three positively charged clusters in the N-terminal domains and even occupy positively charged residues of the C-terminal domains (13). It remains unknown whether the site for binding of heparin in the Cterminal domain may be shielded in the dimer and therefore contribute only to a limited extent to the heparin affinity of dimeric LpL. In conclusion, we have shown that the C-terminal folding domain has affinity for heparin and for HSPG, and we have identified segments important for the interactions with ␣ 2 MR/LRP and with heparin.