Heparan sulfate proteoglycans are primarily responsible for the maintenance of enzyme activity, binding, and degradation of lipoprotein lipase in Chinese hamster ovary cells.

Various aspects of lipoprotein lipase (LPL) metabolism, including cell surface binding, degradation, and enzymatic activity, were compared between Chinese hamster ovary (CHO) cells and two distinct proteoglycan-deficient CHO cell lines. The contribution of low density lipoprotein receptor-related protein in binding LPL was also analyzed by the use of a 39-kDa receptor-associated protein expressed as a glutathione S-transferase fusion protein (GST-RAP). Equilibrium binding data with I-LPL revealed the presence of a class of high affinity binding sites with a K of 7.8 nM in CHO cells, whereas no high affinity binding was observed for proteoglycan-deficient cells. The high affinity binding of LPL in CHO cells appeared to be concentrated in cell surface projections and was not effectively inhibited by GST-RAP. Moreover, degradation of endogenous and exogenous LPL was significantly greater in control CHO cells than in proteoglycan-deficient cells. Degradation of LPL in CHO cells was not affected by GST-RAP, suggesting that proteoglycans and not low density lipoprotein receptor-related protein are responsible for the majority of binding and degradation of LPL in these cells. Our data also show that proteoglycan binding is not essential for the assembly of active LPL homodimers, although proteoglycan binding controls the distribution of LPL activity. Furthermore, LPL produced by CHO cells was more stable than LPL produced by proteoglycan-deficient cells.

Lipoprotein lipase (LPL) 1 is the major enzyme responsible for triglyceride hydrolysis of triglyceride-rich lipoproteins. This hydrolysis controls the rate-limiting step in the removal of triacylglycerol fatty acids from the circulation. Although LPL is synthesized by the parenchymal cells of many tissues, its site of function is at the luminal surface of the capillary endothelium (1). Heparan sulfate proteoglycans (HSPGs) may fulfill a crucial role in the translocation of LPL from sites of synthesis to functional sites and also may control the net amount of LPL that reaches the capillary endothelium.
HSPGs participate in LPL metabolism at several levels.
HSPGs act as cell surface receptors that mediate the binding of LPL to both parenchymal (2,3) and endothelial (4 -7) cell types, although the fate of bound LPL differs dramatically between the cell types. For example, LPL turnover studies in adipocytes have demonstrated that the majority of cell surface lipase is internalized and degraded and that this catabolism is dependent on HSPG binding (2, 8 -10). In endothelial cells, LPL does not undergo a degradative pathway, yet binding to HSPGs is still critical (11). In this case, HSPGs are involved in the transcellular transport of active LPL across endothelial cells (12) and are responsible for maintaining the high concentration of active LPL at the luminal surface of the capillary bed. In addition to the receptor functions of HSPGs, there is some evidence that heparan sulfate binding might also regulate LPL enzymatic activity. A number of conflicting reports show that binding of LPL to heparin, a proteoglycan very similar to heparan sulfate, can stabilize, stimulate, or inhibit catalytic efficiency of the lipase depending on experimental conditions (13)(14)(15)(16). Together, these results stress the importance of HSPG binding in LPL metabolism. In recent years, three additional proteins have been implicated in the cell surface binding of LPL. These proteins are the low density lipoprotein receptor-related protein (LRP) (17,18), gp330, also known as megalin (19), and the amino-terminal fragment of apolipoprotein B (20,21). Both LRP and gp330 are transmembrane proteins that belong to the LDL receptor gene family and have been shown to bind LPL with high affinity in vitro (18,22). The apolipoprotein B fragment is a secreted protein that binds directly to HSPGs and has been proposed to mediate the binding of LPL to HSPGs.
Many questions about the role of HSPGs in LPL metabolism remain to be answered. Do HSPGs function in other aspects of LPL metabolism? For example, are HSPGs necessary for the secretion of newly synthesized LPL? Are HSPGs solely responsible for LPL binding to cell surfaces or do other proteins participate? Are HSPGs or other LPL-binding proteins responsible for the intracellular degradation of LPL? Does HSPG binding influence the enzymatic activity of LPL? In most systems, it is difficult to definitively assign all of these functions to HSPGs, since most experimental systems also contain LRP, gp330, or apolipoprotein B.
In this report, we have used proteoglycan-deficient and CHO-K1 cells as a model system to investigate the significance of the LPL-HSPG interaction. CHO-K1 cells are a good model system since they are known to produce endogenous LPL (23). In addition, CHO-K1 cells have two types of proteoglycans: heparan sulfate (70%) and chondroitin sulfate (30%) (24); thus, the major proteoglycan in these cells is the proteoglycan responsible for binding LPL. Two CHO cell mutants, pgsA 745 and pgsB 761, lack both chondroitin sulfate and HSPGs (24,25). We compared cell surface binding, degradation, distribution, and enzymatic efficiency of LPL in these proteoglycan-deficient strains versus the CHO-K1 strain. In addition, we investigated the contribution of LRP in the binding of LPL to these cells.

EXPERIMENTAL PROCEDURES
Cell Culture-CHO-K1 cells were obtained from American Type Culture Collection (CCL-61). Proteoglycan-deficient cells, pgsA 745 and pgsB 761, were a generous gift from Dr. J. D. Esko (University of Alabama, Birmingham). PgsA 745 and pgsB 761 cells were isolated by replica plating and 35 SO 4 colony autoradiography, as described previously (25), and shown to be defective in xylosyltransferase activity and in galactosyltransferase activity, respectively (24,25). For selected experiments, mutant and wild-type CHO-K1 cells were transfected with chicken LPL as described previously (26). All cells were maintained in Ham's F-12 supplemented with 5% (v/v) fetal bovine serum (Atlanta Biological). Cells were grown at 37°C in an atmosphere of 5% CO 2 in air and 100% relative humidity. Cells were subcultured every 3-4 days; after 12 passages, fresh cells were revived from frozen stocks stored at Ϫ140°C (Cryo-fridge TM , Baxter).
Some experiments required special growth media. Methionine-free Ham's F12 and serum-free media, CHO-S-SFM, were obtained from Life Technologies, Inc. According to the manufacturer, serum-free medium contains no heparin or HSPGs. Transfected CHO-K1 cells showed no significant difference in avian LPL production over a 5-h period between CHO-S-SFM and normal media.
Immunofluorescence Microscopy-Cells were grown to approximately 60 -80% confluency on 12-mm round glass coverslips. Coverslips were rinsed thoroughly with PBS and then incubated at 4°C with continuous shaking for 1 h with 1 g/ml purified avian LPL. Coverslips were rinsed thoroughly with PBS. Cells were fixed in 2% formaldehyde/ PBS for 10 min at room temperature and then were rinsed several times with PBS. Cells were incubated with affinity-purified polyclonal antibody to chicken LPL (0.4 g/ml in PBS) (10) for 30 min at room temperature in a humidified chamber. After rinsing several times with PBS, cells were treated with fluorescein isothiocyanate-conjugated donkey anti-goat IgG (Cappell 67-210) diluted 1:250 in PBS. Coverslips were rinsed and then mounted in 90% glycerol, 1% n-propyl gallate, 1 mg/ml phenylenediamine, PBS, pH 8.0. Specimens were viewed with a Ziess Axioskop, and images were recorded on Kodak T-Max 400 film (Eastman Kodak Co.).
Even in the absence of detergent treatment, a few cells apparently were permeable to the immunoreagents. Therefore, we routinely included rhodamine-phalloidin (diluted 1:500, Molecular Probes), which stains intracellular actin filaments, in the secondary antibody incubation. This permitted us to positively identify non-permeabilized cells as those that lacked rhodamine-phalloidin staining.
Studies with Transfected Lipase-For the experiments that examined the effect of heparin on LPL distribution, transfected cells were plated in 60-mm dishes. Cells were first rinsed with room temperature PBS and then incubated for 5 h in media containing 0 or 10 /ml heparin. The medium was then collected. To collect the fraction of LPL associated with the cell surface, a heparin wash was performed as follows. Cells were rinsed with ice-cold PBS, and 1 ml/dish of cold Ham's F12 containing 10 units/ml heparin was added. After a 15-min incubation on ice with shaking, the heparin wash was collected. Cell extracts were collected by first washing the dishes with ice-cold PBS and scraping the cells with a rubber policeman in 1 ml/dish of CHAPS lysis buffer (4 mM CHAPS, 3 units/ml heparin, 50 mM NH 4 OH, pH 8.1). Cell extracts were sonicated at 50 watts for 30 s using a Braun-Sonic 1510 probe sonicator equipped with a 4-mm microprobe.
For degradation studies with metabolically labeled 35 S-LPL, transfected CHO-K1 and pgsA 745 cells were plated in 100-mm dishes. Cells were rinsed twice with 5 ml/dish PBS, and 5 ml was added of methionine-free media supplemented with 3 M methionine and with 250 Ci of Tran 35 S-Label. After 1 h at 37°C, pulse medium was removed, and cells were rinsed with 5 ml of PBS. Cells were chased at 37°C with methionine-free media supplemented with 300 M methionine for 0 or 5 h. Medium, heparin washes, and cell extracts were collected and treated as described (10). Radiolabeled chicken LPL was isolated by immunoadsorption essentially as described by Cupp et al. (10). Due to the lower cross-reactivity of the anti-chick LPL antibody to CHO cellular proteins, only a single immunoadsorption was performed, and immunobeads were washed only five times with 0.1% N-lauroylsarcosine in PBS.
For activity assays, media were collected essentially as described by Berryman and Bensadoun (26). For measurement of intracellular and total cell-associated lipase activity, cells were treated with and without 100 units/ml heparin in Ham's F12 for 10 min at 4°C with continuous shaking. Cell surface enzyme activity was calculated as the difference between cell samples treated with and without the heparin wash.
Studies with Exogenous 125 I-LPL-For equilibrium binding studies, untransfected wild-type and mutant cells were plated in 60-mm dishes. Media were changed 1 h prior to the experiment to reduce the amount of endogenous LPL on the cell surface. Cells were placed at 4°C and rinsed twice with cold PBS containing 0.2% BSA. 2 ml of cold, fresh medium containing 0.2% BSA was added to each dish, followed by addition of the appropriate amount of 125 I-LPL and specified competitors. Cells were incubated at 4°C with constant shaking for 3 h. Media were then collected, and cells were rinsed three times with cold PBS containing 0.2% BSA. Bound 125 I-LPL was released by two sequential heparin washes (100 units/ml heparin in Ham's F12), which were pooled. Iodinated LPL was precipitated in 10% trichloroacetic acid.
For degradation studies using 125 I-LPL, cells were plated in 12 well plates (22 mm/well). Media were changed 1 h prior to the start of the experiment. Cells were placed on ice and rinsed twice with PBS containing 0.2% BSA. Media (0.5 ml) containing 0.2% BSA, 0.5-1 g/ml 125 I-LPL, and the appropriate competitors were added. Cells were placed in a humidified CO 2 incubator at 37°C with constant shaking for 5 h. Media were collected, and the amount of degraded lipase was determined by measurement of 125 I-tyrosine as described by Bierman et al. (30). Background degradation in the absence of cells was measured and subtracted from the total degradation. Additional wells, which were treated in the same manner except 125 I-LPL was omitted, were harvested and assayed for protein content by the method of Lowry (31) as modified by Bensadoun and Weinstein (32).
Other Methods-A glutathione S-transferase (GST) 39-kDa expression plasmid, which encodes rat 39-kDa receptor-associated protein (RAP) fused to GST, was provided by Dr. Joachim Herz (Southwestern Medical Center, University of Texas, Dallas). The GST-RAP fusion protein was produced and purified on glutathione-agarose according to the methods of Herz et al. (33). To generate free RAP, the fusion protein was cleaved with thrombin as described by Williams et al. (34). DNA content of cell extracts was determined fluorometrically (35). Chicken LPL protein was quantified by enzyme-linked immunosorbent assay (2). Catalytic activity assays were performed as described by Berryman and Bensadoun (26). Statistical comparisons were made using a twosample t test. RESULTS We have addressed the significance of the LPL-HSPG interaction on the fate of LPL by the use of wild-type and mutant CHO-K1 cells. Both mutant strains employed, pgsA 745 and pgsB 761, have been characterized as having defects in synthesizing the tetrasaccharide linkage necessary for glycosaminoglycan chain initiation, rendering these cells proteoglycan deficient (24,25). We compared some aspects of LPL metabolism in these proteoglycan-deficient cells (pgsA 745 and pgsB 761) versus cells containing their normal complement of HSPGs (CHO-K1).
Binding Studies-The first experiments examined the surface binding of LPL to each cell type. This was addressed initially by performing equilibrium binding studies using 125 I-LPL. Equilibrium binding data were fit to a function, , which includes a term representing a class of high affinity binding sites and a term representing nonspecific binding as follows: where represents g of LPL bound per dish, S represents the concentration of free enzyme at equilibrium (in g/ml), n represents the maximum amount of enzyme specifically bound per dish (in g/dish), and a represents the slope of the linear function describing nonspecific binding. Assuming a molecular mass of 120 kDa for the homodimeric enzyme, the calculated K D in Fig. 1 was 7.8 nM for CHO-K1 cells. This dissociation constant is comparable to the published values for adipocyte and endothelial cell surface binding (2,4). Curve fitting with binding data obtained with pgsA 745 and pgsB 761 failed to identify a class of high affinity binding sites. Interestingly, the binding data yielded linear plots with slopes of 0.0998 and 0.0999 for pgsB 761 and pgsA 745, respectively, which were very similar to the slope, 0.1075, of the calculated nonspecific binding observed for wild-type cells (Fig. 1, inset). Therefore, with the sensitivity of the techniques employed, no high affinity binding to mutant cells could be identified.
To determine the distribution of LPL on the surface of mutant and wild-type cells, we utilized immunofluorescence microscopy to detect bound, exogenous LPL. Cells were incubated with purified avian LPL at 4°C for 1 h, washed extensively in cold buffer, fixed immediately, and then processed for immunofluorescence in the absence of detergent permeabilization. This treatment resulted in an LPL staining pattern, which correlated with the results of the in vitro binding study; cell surface staining was readily detected in CHO-K1 cells, whereas no significant staining was detected in either mutant cell line (Fig. 2). Furthermore, LPL on wild-type cells appeared to be concentrated in cell surface projections, such as microvilli and retraction fibers. We also examined the distribution of endogenous LPL on the surface of cells transfected with chicken lipase (data not shown). The transfected lipase was bound only to the cell surfaces of CHO-K1 cells but not to the surface of either mutant cell line, consistent with the data in Fig. 2.
Distribution and Degradation of Transfected LPL-Our next goal was to determine quantitatively the distribution of transfected LPL in CHO-K1 and mutant cells. Our current model of LPL turnover in cultured adipocytes suggests that the majority of secreted lipase binds to the cell surface through association with HSPGs, eventually resulting in internalization and deg-radation of the HSPG-LPL complex (2). Any molecule that competes with HSPGs for binding to LPL (such as heparin) or that inhibits binding to HSPGs causes a marked decrease in cell surface binding, internalization, and degradation of LPL. To validate our model cell system and the proposed function of HSPGs in lipase metabolism, we first had to establish that LPL has similar fates in CHO-K1 cells and in adipocytes.
Initially, we compared the distribution of transfected lipase in the absence and presence of heparin in CHO-K1 and mutant cells. Media, cell surface, and cell extracts were collected and assayed for LPL protein by enzyme-linked immunosorbent assay (Fig. 3). The results with CHO-K1 cells were as expected and as observed previously by Rojas et al. (23). In the presence of heparin, CHO-K1 cells had a 2.5-fold increase in the media pool and a 5.5-fold decrease in the cell surface pool of LPL. In the absence of heparin, CHO-K1 cells had similar levels of LPL on the cell surface and within the cell extract, showing that approximately half of the cell-associated enzyme was bound to the cell surface. Since the mutant cells have no HSPGs for heparin to "compete" with, one would expect very little difference in LPL distribution in the presence or absence of heparin. Indeed, no significant differences between treatments were observed for the cell extract and cell surface pools in mutant cells. For both mutant cell types, media did show statistically significant differences between the two treatments, although the differences were not as great as those observed with CHO-K1 media. A possible reason for the LPL increase in media of mutant cells upon heparin administration is that heparin causes the release of nonspecifically bound LPL from culture dish surfaces. Another interpretation is that heparin in the media protects LPL from proteolytic degradation. In this regard, heparin is known to bind and activate a variety of protease inhibitors (36). Overall, the distribution of LPL between the mutant and CHO-K1 cells was dramatically different, suggesting that HSPGs play a significant role in the cellular partitioning of the lipase.
To examine the degradation of transfected lipase in this cell system, we examined the disappearance of metabolically labeled enzyme. Enzyme degradation was measured in triplicate by a pulse-chase protocol. After 5 h at 37°C, only 32.9% of the LPL radioactivity was recovered from CHO-K1 cells, whereas 68.5% was recovered from mutant cells (Table I). This corresponds to 67.1% degradation of LPL in CHO-K1 cells versus 31.5% in mutant cells. In adipocytes, Cupp et al. (10) reported that 76% of newly synthesized LPL is degraded. Interestingly, when 100 units/ml heparin was added to either CHO-K1 or pgsA 745 cells, the entire cellular pool of labeled lipase at time 0 is recovered after 5 h. Altogether, the results for degradation and distribution studies show that LPL has similar fates in CHO-K1 cells and adipocytes.
LPL Binding to LRP-Recent studies have implicated a role for the LRP, in addition to HSPGs, in the cell surface binding of LPL. As demonstrated by the equilibrium binding studies (Fig.  1), there is little if any specific binding of LPL to cell surfaces in the absence of proteoglycans. On the other hand, degradation studies with metabolically labeled lipase did show that approximately 31.5% of transfected lipase in pgsA cells is degraded. Although the amount of lipase degraded in proteoglycan-deficient cells was much less than that observed for CHO-K1 cells (67.1%), a proportion (31.5%) of LPL was de-graded in an HSPG-independent manner. Consequently, we wanted to determine whether or not LRP was responsible for this HSPG-independent degradation.
To address the potential role of LRP in this system, we utilized GST-RAP. RAP and GST-RAP have been shown to interact directly with LRP and to inhibit the binding of LPL (18), as well as other ligands (33,34), to LRP. Therefore, we repeated the binding and degradation studies in the presence or absence of GST-RAP. Initially, we needed to establish the appropriate concentration of GST-RAP to use in this cell system. We performed competitive binding studies with increasing concentrations of unlabeled LPL or GST-RAP (Fig. 4). Surprisingly, GST-RAP was only effective at reducing the binding of LPL to CHO-K1 cells at the relatively high concentration of 10 M. This concentration is much higher than that predicted by the K I value (1.4 nM) determined for inhibition of LPL binding to familial hypercholesterolemia cells (18).
The significance of LPL degradation via the LRP pathway was also examined with exogenous iodinated lipase. We incubated each cell line (CHO-K1, pgsA 745, and pgsB 761) with 125 I-LPL and 0, 1, or 10 M GST-RAP for 5 h at 37°C. We then measured the appearance of 125 I-tyrosine in the media (Fig. 5). In the absence of GST-RAP, 125 I-LPL was degraded most efficiently by CHO-K1 cells, whereas pgsA 745 and pgsB 761 cells degraded only 7.5 and 16%, respectively, of the observed values for CHO-K1 cells. In the presence of 1 and 10 M GST-RAP, there was a small but significant decrease in degradation with both mutant cell lines, as compared with values in the absence of GST-RAP. In contrast, only a minor decrease was seen in CHO-K1 cells even in the presence of 10 M GST-RAP. Collectively, these data demonstrate that HSPG binding accounts for the majority of LPL degradation, since over 85% more lipase was degraded in the cell system containing HSPGs and since 10 M GST-RAP did not cause a statistically significant decrease in degradation of LPL in CHO-K1 cells. However, data with mutant cells disclose a potential minor role for LRP since the addition of 10 M GST-RAP resulted in a 30% decrease in LPL degradation as compared with no GST-RAP controls.
Several control experiments were performed to confirm the above results. For competitive binding experiments, the cleaved fusion product, RAP, was used instead of the fusion protein, GST-RAP, as an inhibitor. As previously reported (33), the removal of the glutathione S-transferase did not alter the cell binding or degradation results observed with GST-RAP (data not shown). We were also concerned that the results we obtained may be due to the source of the lipase; in all experiments using transfected cells, avian LPL was monitored. However, all studies with 125 I-LPL were performed with radiolabeled chicken and bovine LPL, and the results were similar regardless of the source of LPL (data not shown). Finally, the   35 S-label for 1 h and then chased for 0 or 5 h with label-free medium containing 3 M L-methionine. Radiolabeled LPL was isolated by immunoadsorption followed by electrophoresis and fluorography. The data shown are from triplicate measurements, and each measurement is from a single pool of two 100-mm dishes. Data are presented as percentages (total cpms at 5 h in cells, on cell surface, and in medium divided by the total cpm at time 0 in cells and on cell surface multiplied by 100).
Recovery of 35  results from competitive binding experiments were replicated with different GST-RAP preparations, and each preparation gave similar results. Basic fibroblast growth factor has been shown to bind to high affinity cell surface receptors only in the presence of cell surface HSPGs or exogenous, soluble heparin (37). Therefore, we asked whether or not LPL would bind more effectively to pgsA 745 cell surface receptors in the presence of exogenous heparin. Binding studies were performed with the heparin concentration (40 ng/ml) known to enhance basic fibroblast growth factor binding (37). LPL had no apparent increase in affinity for mutant cells at 40 ng/ml or at higher or lower levels of heparin (0 -50 g/ml) (data not shown), indicating that exogenous heparin does not appear to enhance LPL binding to any cell surface receptor.
LPL Activity-Besides controlling the rate of internalization and degradation of LPL, another characteristic often attributed to heparin binding is stabilizing the active dimeric form of LPL (38). To address whether HSPG binding, like heparin binding, also affected catalytic activity, we employed a lipolytic activity assay, which was performed on transfected lipase recovered from the media, associated with the cell, or associated with the cell following a heparin wash (Fig. 6). The activity recovered from cells treated with the heparin wash was assumed to represent LPL activity from intracellular pools since a heparin wash at 4°C would remove cell surface lipase. Thus, the difference between cell-associated activity (includes intracellular and cell surface activity) and intracellular activity data would represent the cell surface enzyme activity. The specific activity in the intracellular pool was significantly lower than cell-associated values in CHO-K1 cells, whereas specific activities were similar for these two pools in pgsA 745 cells. Specific activity of LPL in the media was greater for pgsA 745 cells than CHO-K1 cells. The differences in observed specific activity among the various pools can be explained by the preferential binding of catalytically active LPL dimers to the cell surface of CHO-K1 cells, the efflux of active LPL in pgsA 745 cells, and the presence in both cell types of an immature pool of lipase. Indeed, the specific activity for the surface-bound LPL in CHO-K1 cells, derived from the activity and mass data of cell-associated and intracellular pools, was 30.8 Ϯ 9.2 g of free fatty acid released/g of LPL/h. Activity assays were also performed with media and cell extracts from pgsB 761 cells; the trend was the same with pgsB 761 cells as with pgsA 745 cells.
Proteoglycan-deficient cells produce and secrete catalytically active LPL. However, is LPL produced by these cells as stable over time as LPL produced in medium containing heparan sulfate chains? To address this question, we collected medium from mutant and wild-type cells and examined the loss of enzyme activity over time at 37°C (Fig. 7). Both cell types produced an enzyme that exhibited a loss in specific activity over time, although the loss with pgsA cells was much greater than that observed for CHO-K1 cells. Linear regression analysis of this data yielded a slope 2.8-fold larger for pgsA 745 cells as compared with CHO-K1 cells.
With the methods used to measure LPL stability and activity, it is possible that heparan sulfate chains within the fetal bovine serum could affect the results. We controlled for this possibility by the use of a defined serum-free media, which is known to be devoid of heparin and heparan sulfate proteoglycans. This defined media gave similar specific activity values as experiments using CHO-K1 media containing 5% fetal bovine serum (data not shown).

DISCUSSION
There is substantial evidence that secreted LPL interacts with HSPGs on endothelial and parenchymal cell surfaces. Because HSPGs are ubiquitous molecules found on most cell surfaces, it has been difficult to assess the specific role of HSPG binding on the metabolism of LPL. We are able to address the significance of proteoglycan binding by using CHO cell mutants, which lack both heparan sulfate and chondroitin sulfate proteoglycans. Using CHO-K1 and pgsA 745 cells, Sehayek et al. (39) found no evidence for an interaction of LPL with chondroitin sulfate proteoglycans. It is therefore the lack of HSPGs and not chondroitin sulfate proteoglycans in these proteoglycan-deficient cells that presumably renders these cells defective in LPL metabolism.
By every means tested, we were unable to detect any significant interactions between LPL and the surface of mutant cells. This was demonstrated by binding studies using 125 I-LPL, immunofluorescence microscopy, enzyme activity assays of cell surface lipase, and LPL distribution studies in the presence and absence of heparin. Furthermore, degradation studies with metabolically labeled enzyme or exogenous, iodinated enzyme revealed that HSPGs are responsible for a large proportion of LPL degradation in this cell type. These results support the role for HSPGs in the cell surface binding and degradation of LPL.
One aspect of LPL metabolism, which has been difficult to address until now, is whether or not HSPG/LPL interactions within secretory vesicles were necessary for proper secretion of the lipase. Our results demonstrate that secretion of LPL does not require HSPGs since significant levels of functional lipase were recovered from the medium of mutant cells. The possibility that LPL and HSPGs interact within such vesicles is not excluded by our studies; however, our results do indicate that if there is an interaction, it is not obligatory for the proper trafficking of LPL or acquisition of enzymatic activity.
Our results suggest that the majority of binding and degradation of LPL in CHO-K1 cells is due to HSPG binding and offers only a minor role, if any, for LRP. Possible explanations for the minor role of LRP in LPL metabolism include the following: 1) LRP is not present in CHO-K1 or pgsA 745 cells; 2) defects in the mutant cells indirectly cause structural defects in either LRP or LPL; 3) there are insufficient LRP binding sites on CHO-K1 cell surfaces; and 4) binding to LRP requires or is facilitated by the presence of HSPGs. Most of these explanations have been eliminated by this and other studies. CHO-K1 cells express LRP at levels sufficient for binding other ligands, such as ␣ 2 -macroglobulin (40 -42), RAP (43), and apoE-enriched lipoproteins (42). Furthermore, LRP is present and functional in the proteoglycan-deficient cells (42,44). In fact, mutant and wild-type cells possess similar amounts of functional LRP, as determined by ligand blot analysis and by ␣ 2 -macroglobulin binding studies (Ref. 42 and data not shown). Therefore, it is unlikely that the mutations in proteoglycan synthesis affect the integrity of LRP. Because LPL is catalytically active in mutant cells, the active site, dimerization, and proper glycosylation of LPL appear to be maintained in pgsA 745 and pgsB 761 cells. Thus, this cell system produces functional LRP and LPL.
Another interpretation of our data is that LRP binding sites may be masked. In this regard, RAP has been proposed to be a common inhibitor of ligand binding to the entire LDL receptor gene family (33,34). Based on this proposed function of RAP, it seems reasonable to assume that the levels of RAP produced by a cell might influence the binding of ligands to this family of receptors. Thus, although CHO-K1 cells have functional LRP, the number of accessible LRP binding sites would be dependent on the levels and distribution of RAP in these cells. CHO-K1 cells do produce their own RAP (43), although the distribution of RAP remains to be determined for these cells. Western blotting using a polyclonal antibody against recombinant rat RAP has shown that RAP levels in CHO-K1 and both mutant strains used in this study are similar (data not shown). However, CHO-K1 cells have relatively large levels of RAP as compared to HEPG2 and familial hypercholesterolemia cells (data not shown), two cell lines commonly used for studying LPL-LRP interactions. Also in support of this interpretation, we found that a relatively large level of RAP (10 M) was necessary to compete for cell surface binding of LPL. This level was much greater than that observed for familial hypercholesterolemia cells (18).
Our data do not support a role for HSPGs in facilitating the binding of LPL to LRP. The degradation studies with 125 I-LPL performed in the presence and absence of GST-RAP revealed only a minor decrease in degradation of the labeled lipase. If there was cooperation between HSPGs and LRP, one would expect to see a dramatic decrease in LPL degradation in the presence of GST-RAP. Moreover, LPL showed no apparent increase in affinity for mutant cell surfaces in the presence of a wide range of heparin concentrations. Therefore, a heparin enhancement of LPL binding, as seen with basic fibroblast growth factor (37), was not apparent. Thus, the most reasonable interpretation of our data is that LRP does not play a prominent role in binding LPL in this cell type and that HSPGs by themselves can bind and target the lipase for degradation. Eisenberg et al. (45) and Mulder et al. (46) reached similar conclusions when they demonstrated that the LPL enhancement of lipoprotein uptake occurs by HSPG binding rather than by an interaction with LRP. Furthermore, Sehayek et al. (39) have shown recently that HSPGs are required for the proper metabolism of LPL in several different cell types.
Hepatic lipase also binds to LRP in vitro (44,47). The metabolism of hepatic lipase has been studied with these proteoglycan-deficient cells and with LRP-deficient CHO cells (44). Interestingly, these authors were unable to detect any internalization or degradation of 125 I-hepatic lipase by proteoglycan-deficient cells despite the fact that these cells express LRP (44). They also report that a small, significant amount of 125 Ihepatic lipase was degraded in an LRP-independent pathway in proteoglycan-deficient cells, consistent with the results presented here. They conclude that HSPGs are necessary for the initial binding of hepatic lipase to cell surfaces. Likewise, our data show that HSPG binding appears to be crucial in binding, internalization, and degradation of LPL in CHO-K1 cells.
This study does not address the role of gp330 or apolipoprotein B in LPL metabolism. CHO-K1 and mutant cells do not produce gp330 (44). Furthermore, our use of GST-RAP eliminates any major role for other LDL receptor gene family mem- FIG. 7. Stability of LPL in CHO-K1 and pgsA 745 media at 37°C. Transfected cells were incubated with fresh medium at 37°C for 10 h. The medium was collected and concentrated 8-fold and then placed at 37°C for the indicated times. LPL activity was determined as described (28). Results are expressed as mean Ϯ S.D. (n ϭ 3). The units are eq of free fatty acid released/g of LPL/h. bers (LDL receptor and very low density lipoprotein receptor). The apolipoprotein B fragment presumably interacts with LPL and the cell surface via HSPGs (20,21). The proteoglycandeficient cells therefore lack the proposed binding sites (HSPGs) for this apolipoprotein, precluding any distinction between the function of this protein and HSPGs in LPL metabolism using this cell system.
Previous studies suggest a role for proteoglycans in regulating the catalytic activity of LPL. In earlier investigations, heparin was reported to stimulate LPL activity (48). However, subsequent studies have shown that heparin can activate (15), stabilize (13,16), inhibit (49,50), or have no effect on enzyme activity (14). All of these studies used heparin and not HSPGs to evaluate the effect of proteoglycans on LPL activity. In vivo, the major proteoglycan responsible for binding LPL is HSPGs, yet the role of HSPG binding on LPL activity has not been established. The mutant cell system offers a unique opportunity to address the significance of HSPG binding on LPL activity. Clearly, HSPG binding is not absolutely required for the acquisition or maintenance of enzyme activity, as demonstrated by activity assays of lipase produced by mutant cells. On the other hand, our results do indicate that HSPGs play a prominent role in the distribution of functional lipase. Our biochemical data show that the secreted LPL has several fates in CHO-K1 cells (attach to cell surface proteoglycans, internalize for degradation or recycling, or release to the media) but only one fate in mutant cells (release to the media). The specific activity data in Fig. 6 correlate well with the biochemical and distribution data. For example, a greater proportion of active enzyme was recovered from the media of mutant than CHO-K1 cells. Moreover, no lipase activity was detected on cell surfaces of mutant cells as compared to significant amounts of lipase with a specific activity of 30.8 eq of free fatty acid released/g of LPL/h in wild-type cells. The observed difference in specific activity among the different cell compartments directly reflects the distribution of HSPG-bound LPL.
LPL activity released into the medium of CHO-K1 cells did appear to be more stable than that produced by mutant cells. This difference in stability suggests that some component of the CHO-K1 medium helps to maintain lipolytic activity. The medium component is most likely glycosaminoglycan chains. A large fraction of 35 S-labeled glycosaminoglycan chains are known to be secreted into the medium in CHO-K1 cells metabolically labeled with 35 SO 4 , and both of the proteoglycandeficient cell lines have been characterized as having marked decreases in secreted, sulfated glycosaminoglycan chains (25). Hence, proteoglycans play an important role not only in binding and distributing LPL activity but also in the maintenance of that activity outside the cell.
In summary, the current study establishes the central role of HSPGs in the binding and degradation of LPL. This is in contrast to previous reports (51), which suggested that HSPGs were important for the high capacity binding of LPL but offered a low capacity for ligand degradation. Although LRP may play a more prominent role in LPL binding and degradation in other cell systems, our results directly demonstrate that proteoglycans by themselves can bind and lead to the degradation of a substantial amount of lipase. Finally, these results provide the first evidence that HSPGs are not necessary for the assembly of functional LPL but that secreted heparan sulfate chains may contribute to the stability of secreted lipase.