INTRODUCTION

Lipoprotein lipase (EC) (LPL)¹ is a key enzyme in lipid homeostasis in vertebrates, providing intravascular release of fatty acid from circulating triacylglycerol (1, 2). A genetic deficiency of LPL results in chylomicronemia and Type I hyperlipoproteinemia (1). LPL is produced by heart, adipose tissue, muscle, as well as in small amounts by many other tissues (3). It is a glycoprotein anchored on the luminal surface of capillary endothelium through its interaction with cell surface glycosaminoglycans (4). The normal targets for LPL action are the triacylglycerol-rich lipoproteins, especially chylomicrons and very low density lipoproteins (VLDL). Triacylglycerols and monoacylglycerols are preferred substrates, whereas phosphatidylcholine is hydrolyzed at a slower rate. The activity of LPL is markedly stimulated in the presence of a plasma activator, apolipoprotein C-II.

Recently, another role for LPL has been suggested by studies in a number of laboratories (5, 6, 7, 8, 9, 10, 11, 12, 13). It has been demonstrated that LPL stimulates the cellular binding and uptake of various lipoproteins including chylomicrons, rabbit -VLDL, and low density lipoproteins (LDL). The enhancement of the catabolism of these lipoprotein particles by LPL has been reported to be mediated by the LDL receptor-related protein (LRP) (5, 6, 7, 10, 11), the LDL receptor (9), as well as by non-receptor-dependent pathways (8, 9, 12). The C-terminal domain of LPL is thought to be important for this action of LPL in some of these studies (13). LPL is thought to bind to the lipoprotein particles, concentrating them on the cell surface in the vicinity of the lipoprotein receptors to which they subsequently transfer the lipoproteins for uptake and internalization. A proportion of the LPL-lipoprotein complex may also be directly internalized without the participation of a specific lipoprotein receptor (8, 9, 12).

In its native state, LPL has a defined secondary and tertiary structure (14, 15). Based on extensive mutagenesis experiments, the structure of LPL is projected to be very similar to that of pancreatic lipase whose crystal structure was published several years ago (16). The protein consists of two domains, an N-terminal domain comprising three-quarters of the sequence, and a distinct C-terminal domain containing the last ~100 amino acids. The N-terminal domain shows similarity to the fungal lipases; it is an / polypeptide with a central core of a mixed -sheet containing the catalytic triad of Ser/His/Asp, and a surface loop which restricts access of the substrate to the active site and must be repositioned for catalysis to take place. The C-terminal domain is unique to pancreatic lipase, LPL, and hepatic lipase, and is missing in the fungal lipases. It is almost entirely made up of -sheets. Observations on a large number of site-specific LPL mutants have provided information on the structure-function relationship of enzyme catalysis involving LPL based on the two-domain model (e.g. Refs. 17, 18, 19, 20, 21, 22). However, the structural basis of LPL in its noncatalytic role to act as a bridge for circulating lipoproteins and their receptors is poorly understood. There is evidence that enzyme activity may not be required for this function: (i) enzymatically active bacterial lipase fails to enhance binding of chylomicrons to HepG2 cells (5), and (ii) guanidinium chloride-treated LPL, (iii) tetrahydrolipstatin (an irreversible inhibitor of LPL catalysis)-inactivated LPL both retain the capacity to mediate the binding of -VLDL to LRP (10), and (iv) C-terminal domain fragments which lack enzyme activity may display similar function (13). However, studies have not been conducted that make use of LPL mutants with minimal structural perturbations, e.g. mutants involving only one or two amino acid residues producing limited but specific changes in LPL function or conformation. Such an approach should provide insight into the structural requirement for LPL in its bridge function. We note that although the bridge function does not require enzyme activity, lipolysis may play a role in some circumstances (23).

In this article, we have studied the role of LPL structure and action in mediating VLDL binding and catabolism in COS cells. By taking advantage of these fibroblast-like cells that can be readily induced to produce wild-type and mutant LPLs by transfection (17, 18, 19, 24), we have analyzed the structural requirement for the effect of LPL in LDL receptor-mediated uptake and catabolism of normal human VLDL. Our results indicate that in these cells, the LDL receptor can bind to and catabolize VLDL in the presence of LPL. By examining LPLs with limited site-specific mutations, we found that certain parts of the LPL molecule are much more important than others in mediating VLDL binding and catabolism (a function referred to as bridge function in this paper). Furthermore, the structural constraints for this nonenzymatic function of LPL are quite distinct from those required for enzyme catalysis.

MATERIALS AND METHODS

Wild-type and mutant LPL expression vectors were constructed as described previously (3, 17, 18). A cDNA containing 1786 bases extending from nucleotides 320 to 1466 (counting the first base of the translation initiation codon as nucleotide +1) was subcloned into M13 mp19 and used as a template for site-specific mutagenesis. It contains 320 nucleotides in the 5-untranslated region, the entire coding region, and 38 nucleotides in the 3-untranslated region and is bounded by an artificial EcoRI site in the 5 end and a natural EcoRI site in the 3 end.

Oligonucleotides synthesized on an Applied Biosystems Inc. 380A DNA synthesizer were 5-phosphorylated and annealed with single-stranded template. Mutagenesis was carried out as described by Taylor et al. (25) using an oligonucleotide-directed in vitro mutagenesis kit (Amersham Corp). Mutant and wild-type LPL cDNAs were used to transfect Escherichia coli TG1 cells, and positive clones were identified by direct sequencing. Replicative form DNAs were isolated, digested with EcoRI, and inserted into the EcoRI site of p91023(B) (26). After transformation of E. coli DH 5 cells, positive clones were isolated, and orientation of inserts was determined by restriction mapping. Many of the mutant LPL constructs have been used in previous studies on the structure-function relationships of LPL from our laboratory and have been described in detail before (17, 18, 27).

COS M-6 cells were inoculated on T75 flasks (1.5 × 10⁶ cells/flask) and allowed to grow for 2 days, reaching 90-95% confluence when they were used for transfection. Transfections were performed with 20 µg of plasmid DNA/flask by the technique of Selden et al. (28), using DEAE-dextran/dimethyl sulfoxide treatment for 30 min, followed by a 3-h incubation with chloroquine. In each experiment, a parallel flask was subjected to the transfection protocol without plasmid DNA. Transfected cells were incubated overnight in DMEM and 10% fetal calf serum. They were then washed, trypsinized, and transferred to 12-well plates. Cells from two T-75 flasks were split into three 12-well plates and grown in DMEM in 10% fetal calf serum for 65 h before being used for binding studies. Some cells and media were also used for LPL activity assay and ELISA for LPL immunoreactive mass (ng/µl).

Cell extracts and media were assayed for LPL activity. Cells were washed in phosphate-buffered saline, scraped into 1 ml of 50 mM NH₃/NH₄Cl (pH 8.1) containing heparin, and sonicated. Media and cell extracts were then flash-frozen in a dry ice/ethanol bath and stored at 70 °C. Just before determination of LPL activity or protein mass, samples were thawed in ice water, made 0.2% in sodium deoxycholate, and rotated at room temperature for 5 min. Cell extracts and media were assayed for LPL enzyme activity as described previously (3). LPL activity is expressed in milliunits (1 milliunit = 1 nmol of fatty acid released per min).

hLPL mass was determined by a two-antibody sandwich ELISA described previously (27, 29). A monoclonal antibody raised against purified bovine milk LPL (monoclonal antibody 40 (30)) was used as first antibody coated on plates, and polyclonal antibody generated from chicken against hLPL purified from stably transfected CHO cells was used as a second antibody to bind antigen (31).

VLDL and LDL for study were isolated by ultracentrifugation from the plasma of normolipidemic subjects following an overnight fast. Similar binding affinities were observed for apoE-3/E3 and apoE-3/2 VLDL for both wild-type and mutant LPL experiments; they were used interchangeably for the experiments. VLDL was isolated at a density of less than 1.006 g/ml. LDL was isolated from the infranatant at d = 1.02-1.063 g/ml. Gel electrophoresis and Coomassie Blue staining of the LDL fraction revealed no detectable apoE. -VLDL was obtained from the plasma of adult female New Zealand White rabbits fed a diet of normal chow pellets coated with 1% cholesterol (32). Rabbits were maintained on the diet for 4-6 weeks after which blood was collected in 1% EDTA by cardiac puncture. Cellular components were sedimented by low-speed centrifugation and Trasylol (Mobay Chemical Co.) was added immediately to inhibit proteolysis. -VLDL was obtained by ultracentrifugation for 18 h at plasma density (55,000 rpm, 4 °C). The whitish supernatant was removed and washed with buffered saline. The density of the solution was adjusted to 1.210 g/ml, and the lipoproteins were again ultracentrifuged at 4 °C for 18 h at 55,000 rpm to remove residual albumin. By SDS-polyacrylamide gel electrophoresis, the -VLDL was found to be enriched in apoE.

¹²⁵I-VLDL and ¹²⁵I--VLDL were prepared by the iodine monochloride procedure (33). The final specific activity was 300-600 cpm/ng.

Medium was removed from COS M-6 cells and all dishes were washed one time with serum-free DMEM. All experiments were performed in triplicate. One-half of the dishes were incubated for 20 min with DMEM containing 0.2% fatty acid-free bovine serum albumin and 200 units/ml of heparin. The remaining dishes were incubated with DMEM + 0.2% bovine serum albumin only. After 20 min, 1-ml aliquots of medium were removed and saved from each dish and all dishes were washed three times with serum-free DMEM and one time with DMEM + 0.2% bovine serum albumin. Experiments were then initiated by the addition of radioactive lipoproteins to the washed cells. After 5 h, binding, uptake, and degradation of radioactive lipoproteins at 37 °C was measured as described by Goldstein et al. (34). Up-regulation of the LDL receptor was accomplished by incubating cells for 48 h with media containing 10% lipoprotein-deficient serum and for 24 h with media containing 1 µg/ml lovastatin (Merck & Co.).

RESULTS

We first examined the capability of COS cells to bind to, take up, and degrade VLDL and the effect of human LPL expression by these cells on these processes. COS cells were transfected with a normal hLPL expression vector. The production of enzymatically active and immunoreactive LPL was monitored in all experiments using an activity assay and an ELISA described previously (18, 24). As shown in Fig. 1, transfection of COS cells with a hLPL cDNA expression vector significantly enhanced uptake and degradation of normal VLDL (Fig. 1, D-F) as compared to mock-transfected cells (Fig. 1, A-C). There were some variations in the exact amount of VLDL bound and catabolized between experiments but in all cases they were significantly enhanced above mock-transfected controls. Pretreatment of transfected cells with heparin to remove surface-bound LPL prior to initiating the binding experiments completely abolished the LPL-mediated effects.

In order to establish whether apoE or apoB-100 was the ligand for the LPL-mediated uptake of VLDL, we performed the following experiments. First, we examined whether monoclonal antibody ID7, which is known to associate with the receptor-binding domain of apoE and block its binding of the LDL receptor (35), would inhibit the LPL-mediated uptake of normal VLDL. We found that ID7 blocked the binding of ¹²⁵I-VLDL to the LPL-producing COS cells in a concentration dependent manner. Moreover, this monoclonal antibody also blocked the uptake and degradation of ¹²⁵I-VLDL by these cells (Fig. 2). The sensitivity of the binding, uptake, and degradation to the addition of ID7 suggests that apoE mediates the VLDL-lipoprotein receptor interaction, and, furthermore, that the normal receptor-binding domain of apoE may be crucial to all steps of the process. We therefore compared the uptake by hLPL-producing COS cells of normal VLDL and E2/E2 VLDL from a classical type III hyperlipidemic individual (Fig. 3). We found that the uptake of E2/E2 VLDL by hLPL-producing COS cells was identical in the presence or absence of heparin pretreatment and was not different from the results obtained with normal VLDL in COS cells pretreated with heparin. Thus, apoE, but not apoB-100, appeared to be involved in LPL-mediated metabolism of VLDL. Additional support for this conclusion is provided by the next series of experiments.

LPL-mediated uptake and degradation of VLDL may be a multistep process involving both LPL and a lipoprotein receptor. We tested the hypothesis that the interaction involves the LDL receptor and LPL may serve either to concentrate VLDL at the cell surface or it may metabolize VLDL to a lipoprotein with a greater affinity for the LDL receptor. Alternatively, it is possible that a VLDL-LPL complex is internalized and degraded. Since LDL is a poorer substrate than VLDL for LPL, but a better ligand than VLDL for binding to the LDL receptor, its ability to compete with VLDL for binding, uptake, and degradation by hLPL-transfected COS cells was evaluated. An 8-fold excess of LDL had little effect on binding of VLDL by the COS cells while a similar excess of VLDL or apoE-rich rabbit -VLDL reduced binding of VLDL to a level observed in heparin-treated cells (Fig. 4a). Therefore, the LPL-dependent VLDL binding appears to be specific to apoE-containing lipoproteins and is competed by excess VLDL and -VLDL; it is not effectively competed by excess LDL which contains only apoB-100 as its ligand. However, excess unlabeled LDL reduced uptake and degradation of VLDL to the same degree as unlabeled VLDL or -VLDL. Pretreatment of COS cells expressing wild-type hLPL with lovastatin to up-regulate the LDL receptor resulted in a 3-4-fold increase in VLDL uptake and degradation in the absence of heparin (Fig. 4b). Binding of VLDL was unaffected by lovastatin treatment. At every VLDL concentration, the difference in uptake or in degradation between heparin-treated and untreated cells was 2-4 times greater in lovastatin-treated cells. These results (Fig. 4, a and b) suggest the involvement of both LPL and the LDL receptor in uptake and catabolism of VLDL by these cells.

a, a comparison of binding (top panel), uptake (middle panel), and degradation (bottom panel) of ¹²⁵I-VLDL in the presence of an 8-fold excess of unlabeled LDL, VLDL, or rabbit -VLDL in COS cells transfected with WT human LPL. The difference between the values obtained with ¹²⁵I-VLDL and those with the other treatments was significant in all cases, with p < 0.05 except for the value obtained with excess unlabeled LDL in the top panel, only where there was no difference between the two. b, comparison of binding, uptake, and degradation of ¹²⁵I-VLDL by COS cells transfected with human LPL cDNA in the presence (D-F) and absence (A-C) of lovastatin pretreatment to up-regulate the LDL receptor. Open symbols represent binding, uptake, or degradation of VLDL measured in the absence of pretreatment with heparin. Closed symbols represent binding, uptake, or degradation of VLDL measured following a 20-min pretreatment with 200 units/ml of heparin. The bars indicate S.D. values. Where no bars are evident, the S.D. values fall within the size of the symbol.

We compared two site-specific mutants, S132A and S132D, with wild-type hLPL expression in COS cells on VLDL metabolism. The two mutants and wild-type hLPL were expressed individually and examined VLDL binding, uptake, and degradation by COS cells (Fig. 5, left and right panels). Although there is some minor experimental variation, the results indicate that the S132A and S132D hLPL mutants were competent in enhancing the metabolism of normal VLDL at all levels (i.e. binding, uptake, and degradation) by COS cells. These catalytic triad mutants are enzymatically inactive (Refs. 17, 18, and confirmed in our studies). They involve single amino acid substitution mutations that produce minimal perturbation of LPL secondary and tertiary structure. Here we show that they are capable of mediating the bridge function of LPL.

A Naturally Occurring Truncated hLPL, Ser⁴⁴⁷Ter, Has Normal Bridge Function

A hLPL variant, Ser-447Ter, is a fairly common polymorphism in normal populations. It is known that this variant hLPL has relatively normal lipolytic activity although reports on its enzymatic activity vary from somewhat diminished to normal to supernormal (17, 36, 37). We have compared the bridge function of Ser-447Ter with wild-type hLPL expressed in COS cells (Fig. 6). It is evident that this variant enhances the binding, uptake, and degradation of normal VLDL as well as wild-type hLPL.

To further explore the structure-function correlation between LPL enzyme catalysis and bridge function, we tested three other hLPL mutants (Fig. 7). We found that the S251T mutant, which has ~25% wild-type activity, had full bridge function. When we tested two substitution mutants involving Ser-172, we found that the inactive S172G mutant hLPL enhanced the uptake and degradation of normal VLDL, whereas the other mutant, S172D, which has ~25% wild-type hLPL activity, had essentially no enhancing activity.

When we combine the data from these three mutants with the Ser-447Ter (Fig. 6) and catalytic triad mutants (Fig. 5), we find that there is no simple correlation between enzyme catalysis and bridge function. The most interesting results pertain to substitution mutants involving Ser-172. The two mutants we tested involve a change in only one and the same amino acid residue. Yet, their ability to enhance VLDL uptake and degradation differs markedly.

The catalytic center of LPL is covered by a surface loop which restricts the access of substrate to the active site. We examined the contribution of this loop, which contains 21 amino acid residues flanked by disulfide bonds, to the bridge function of LPL. We tested the following hLPL mutants: (i) LPL-cH, where the hLPL loop was completely replaced by a hepatic lipase loop, (ii) LPL-cP, where it was replaced by a pancreatic lipase loop, (iii) C216S/C239S, a double amino acid mutant hLPL with the Cys residues flanking the loop substituted by Ser resides, and (iv) C418S/C438S, where the C-terminal intramolecular disulfide is removed by substitution of the two Cys residues with Ser residues; this mutant serves as a control for the last double Cys mutant.

Results of the VLDL binding, uptake, and degradation experiments on the three loop mutants and the control double Cys mutant and on wild-type hLPL expressed in COS cells are shown in Fig. 8. It is evident that the two chimeric loop mutants, LPL-cH and LPL-cP, have normal bridge function, although they differ greatly in catalytic activity. Of the two double Cys mutants, the one affecting the disulfide bond in the C-terminal domain has normal bridge function, whereas the other with the disulfide bond delimiting the loop removed has completely lost its bridge function. In terms of LPL catalytic activity, LPL-cH has 60%, and C418S/C438S, 100% wild-type catalytic activity, whereas LPL-cP and C216S/C239/S are totally inactive (19). Again, there is no correlation between bridge function and enzyme catalysis in this group of mutants. The experiments indicate that an intact substrate-binding loop, held in place by disulfide bonds, is required for the noncatalytic function of LPL. However, unlike catalytic activity, there is substantial flexibility in the primary sequence requirement involving the loop itself for the bridge function of LPL.

DISCUSSION

The capacity of LPL to enhance the binding of chylomicrons to LRP was first described by Biesiegel et al. (5). Subsequently, a number of laboratories have shown that in addition to chylomicrons, LPL also enhances the binding of -VLDL, VLDL, and LDL binding to various types of cultured cells, mainly via the LRP (6, 7, 8, 10, 11), and in one study, via the LDL receptor (9). In all cases, the enhancement was demonstrated by observing the difference in binding and catabolism upon the addition of exogenous LPL to the cultured cells. LPL is produced by many different types of cells in the body (3). The amount of circulating LPL compared to the amount at the site of production is miniscule (1). In this study, instead of adding LPL exogenously, we have induced its production in COS cells, a fibroblast-like cell line well suited for transient transfection experiments. Although a similar strategy was used previously by other investigators (38, 39), we have chosen COS cells not only because of the ease of transfection, but also because they do not produce any detectable LPL mRNA or protein prior to transfection. Many other types of cultured cells, including some that have been used for this type of experiments, produce LPL constitutively and the wild-type LPL produced by these cells makes the interpretation of results obtained following the addition of exogenous LPL difficult. We believe that our system more closely mimics the natural situation in which LPL is produced in situ and affects lipoprotein metabolism locally.

Our experiments showed that untransfected COS cells display minimal basal binding to normal human VLDL. Upon transfection with a wild-type hLPL expression vector, these cells would bind to, take up, and degrade VLDL (Fig. 1). The effect of hLPL expression was eliminated by removal of the hLPL by heparin pretreatment. We found that the LPL-enhanced VLDL metabolism is inhibited by a monoclonal antibody ID7 (Fig. 2) that is specific for the LDL receptor-binding region of apoE; furthermore, E2/E2 VLDL gave no specific binding to COS cells expressing hLPL (Fig. 3). These data suggest that the binding and subsequent metabolism of the VLDL particles require a functional receptor-binding domain in apoE. However, the addition of excess LDL did not inhibit normal VLDL binding, indicating that binding is independent of the LDL receptor and is a direct interaction between VLDL and hLPL on the cell surface. Interestingly, LDL inhibited the uptake and degradation of the ¹²⁵I-VLDL to the same extent as unlabeled VLDL or apoE-rich -VLDL (Fig. 4a), indicating that the subsequent metabolism of the VLDL bound on the cell surface via hLPL is mediated by the LDL receptor. The involvement of the LDL receptor in VLDL catabolism in these cells is also supported by the fact that lovastatin treatment to up-regulate the LDL receptor had minimal effect on VLDL binding, but substantially increased uptake and degradation of VLDL in the presence of hLPL (Fig. 4b). Our observations corroborate the data of Mulder et al. (9) who found that VLDL binding to HepG2 cells and fibroblasts is LDL receptor-independent and mediated directly by the cell-surface bound LPL, but the uptake and subsequent degradation of the lipoprotein by these cells is mediated predominantly by the classical LDL receptor pathway.

The structural requirements for the bridge function of LPL with respect to VLDL uptake via the LDL receptor have not been addressed in previous publications. If LPL simply acts as a bridge binding to the circulating lipoproteins and subsequently transferring them to the neighboring lipoprotein receptors, our analysis of the structural requirements for LPL-mediated enhancement of LDL receptor binding will likely also be applicable to binding and catabolism by LRP and possibly other lipoprotein receptors such as the VLDL receptor (40, 41, 42, 43).

The data generated by the use of 10 well defined site-specific mutant hLPLs are summarized in Table I. Wild-type hLPL controls were always included in individual experiments to ensure that the observed impaired bridge function of some hLPL mutants was not the result of experimental variation. The normal enhancement of VLDL metabolism in COS cells by the catalytic triad mutants S132A and S132D (Fig. 5) indicates that enzyme catalysis is not required for bridge function. The results obtained with three other mutants, S251T, S172G, and S172D (Fig. 5), confirm that there is complete lack of correlation between bridge function and LPL enzymatic activity. The fact that S172G, which has normal bridge function, and S172D, which has markedly impaired bridge function, gave divergent results suggests that subtle changes in structure and possibly protein folding can lead to loss of this noncatalytic function of LPL, perhaps partly mediated by its binding to VLDL. We note that Rumsey et al. (12) demonstrated increased uptake of triglyceride emulsions by fibroblasts and macrophages in the presence of LPL. The relevance of their observations to our experiments is unclear because they used an emulsion phospholipid concentration over 20-fold that in our LDL experiments and no LPL mutants were examined.

Table I. Enzymatic and non-enzymatic function of wild-type and mutant hLPL Mutant Lipolytic activity Bridge function Comment Wild-type +++ +++ Mediates VLDL metabolism in COS cells 1 S132A 0 +++ Catalytic triad mutant 2 S132D 0 +++ Catalytic triad mutant 3 S447Stop +++ +++ Natural genetic variant 4 S251T + +++ Highly conserved S 5 S172G 0 +++ Highly sonserved S 6 S172D + ± Highly conserved S 7 LPL-cH ++ +++ Loop mutant-1 8 LPL-cP 0 +++ Loop mutant-2 9 C216S/C239S 0 0 Loop mutant-3 10 C418S/C438S +++ +++ C-terminal disulfide mutant

A common polymorphism in hLPL amino acid sequence involves a S447Ter mutation (17, 44, 45). It is a consequence of a CG transversion at nucleotide 1595 in exon 9, converting the Ser-447 codon (TCA) to a termination codon (TGA). The hLPL protein encoded by this genetic variant contains 446 amino acid residues and is shorter than wild-type hLPL by two residues at its C terminus. The truncated hLPL has been found to have normal enzymatic activity (17), although there are also reports suggesting that it has either partially impaired (37) or actually enhanced (36) lipolytic activity. The allelic frequency of the Ser-447Ter mutation (i.e. the G allele) is reported to be 0.11 in one study involving caucasians (45). Interestingly, this allele has been found to be associated with lower serum triacylglycerol and higher high density lipoprotein levels (45, 46). Because of this association, we tested whether the Ser-447Ter mutant hLPL may have altered bridge function which might cause a change in VLDL metabolism and hence plasma triacylglycerol and high density lipoprotein levels in vivo. Our data (Fig. 6) indicate that the capacity of this truncated hLPL to enhance VLDL binding and metabolism is similar to that observed for the wild-type enzyme, and altered bridge function is not a cause of the observed statistical association between the G allele and plasma lipoproteins.

The three-dimensional structure of pancreatic lipase and fungal lipases has several conserved structural motifs (14, 15, 47). One common feature is that the catalytic center of the lipase molecule is covered by an amphipathic loop which serves as a lid that must be ``opened'' before the lipid substrate can have access to the center. Experiments from several laboratories suggest that, for LPL, the loop does not have a rigid sequence requirement (19, 20, 21) and can be replaced by a loop from hepatic lipase with only minor impairment in enzyme activity (19, 20). Replacement of the loop by the pancreatic lipase loop, however, completely inactivates the enzyme (19). We have performed experiments to examine the role of this loop in LPL bridge function. We found that hLPL mutants having this loop replaced with hepatic or pancreatic loop have normal bridge function. However, with the loop sequence left intact but the disulfide bonds anchoring the loop removed by substituting the two flanking Cys residues with Ser residues, the mutant hLPL completely loses its capacity to enhance VLDL binding and catabolism. These studies indicate that although the actual primary sequence of the loop is relatively unimportant (note that the pancreatic lipase loop sequence has little sequence similarity to the LPL loop (discussed in Refs. 15, 19, and 47)), the intact folding of this region, ensured by the flanking disulfide bonds, must be maintained for LPL to express its bridge function. In analyzing the LPL-mediated catabolism of VLDL by LRP, Chappell et al. (7) found that a C-terminal fragment of hLPL (residues 313-448) mimics many of the effects of intact LPL. Here we show that a proper folding of the loop, which is situated entirely in the N-terminal domain, plays just as an important role in the LPL-mediated catabolism of VLDL via the LDL receptor, and that this function is not dependent on catalytic activity of LPL.