(Received for publication, June 19, 1995)
From the
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) ()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, 5, 6, 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, 9, 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.
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.
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.
For degradation studies with metabolically
labeled 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
µM methionine and with 250 µCi of
Tran
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 µM 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.
For degradation
studies using 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
µg/ml
I-LPL, and the appropriate competitors were
added. Cells were placed in a humidified CO
incubator at 37
°C with constant shaking for 5 h. Media were collected, and the
amount of degraded lipase was determined by measurement of
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
I-LPL was
omitted, were harvested and assayed for protein content by the method
of Lowry (31) as modified by Bensadoun and
Weinstein(32) .
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).
where represents µg of LPL bound per dish, S represents the concentration of free enzyme at equilibrium (in
µg/ml), n represents the maximum amount of enzyme
specifically bound per dish (in µg/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
in Fig. 1was 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.
Figure 1:
Total binding of LPL to CHO-K1 versus proteoglycan-deficient cells. CHO-K1 and mutant cells
were incubated with medium containing 0.2% BSA for 1 h at 37 °C.
Cells were then placed at 4 °C with medium containing 0.2% BSA and
increasing concentrations of I-LPL. Media were collected,
and 2
1-ml heparin washes were performed. Total binding curves
for CHO-K1, pgsA 745, and pgsB 761 are shown. Each point represents
data from a single dish. Inset shows the nonspecific binding
component for CHO-K1 cells and total binding for pgsA 745 and pgsB 761
cells. Data shown are representative of three separate
experiments.
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.
Figure 2: Immunofluorescence staining of exogenous LPL on the cell surface of mutant and wild-type CHO cells. Untransfected cells were incubated for 1 h at 4 °C with 1 µg/ml purified avian LPL with continuous shaking. Coverslips were rinsed several times with PBS, fixed, and processed without detergent permeabilization as described under ``Experimental Procedures.'' Fielda, CHO-K1 cells; field c, pgsA 745 cells; and field e, pgsB 761 cells. Fieldsb, d, and f are the corresponding phase contrast images. Fieldsa, c, and e were photographed under identical conditions.
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.
Figure 3: Effect of heparin on the distribution of LPL in CHO-K1 and mutant cells. CHO-K1, pgsA 745, and pgsB 761 cells were incubated for 5 h in the presence or absence of 10 units/ml heparin. After 5 h, media, heparin washes, and cell extracts were collected. LPL levels were measured by enzyme-linked immunosorbent assay. Values are the mean ± S.D. from three pools of three 60-mm dishes.***, significant differences with p < 0.001; *, significant differences with p < 0.05.
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 1). 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.
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 µM. This concentration
is much higher than that predicted by the K value
(1.4 nM) determined for inhibition of LPL binding to familial
hypercholesterolemia cells(18) .
Figure 4:
Effect
of GST-RAP on the binding of LPL to mutant and CHO-K1 cells. Cells were
incubated at 4 °C with media containing 1 µg/ml I-bLPL (specific activity, 303,000 cpm/µg of protein)
and specified concentrations of unlabeled competing ligand. Data are
representative of four separate
experiments.
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 I-LPL and 0, 1, or 10 µM GST-RAP for 5 h at 37 °C. We then measured the appearance of
I-tyrosine in the media (Fig. 5). In the absence
of GST-RAP,
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 µM 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 µM 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
µM 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 µM GST-RAP resulted in a 30% decrease in LPL
degradation as compared with no GST-RAP controls.
Figure 5:
Effects of GST-RAP on the degradation of I-LPL in mutant and CHO-K1 cells. Cells were incubated
with medium containing 0.4 µg of
I-cLPL (specific
activity, 420,000 cpm/µg of protein) and 0, 1, or 10 µM GST/RAP for 5 h at 37 °C. The medium was collected and assayed
for the presence of
I-tyrosine as described under
``Experimental Procedures.'' Each bar represents the
data from six separate measurements.***, significant differences (p < 0.001) compared with 0 µM level.
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 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 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 µg/ml) (data not shown), indicating that exogenous heparin does not appear to enhance LPL binding to any cell surface receptor.
Figure 6: Specific activity of transfected LPL in wild-type and pgsA 745 cells. Assays were performed as described under ``Experimental Procedures.'' Results are expressed as mean ± S.D. (n = 9). The units are µeq of free fatty acid released/µg of LPL/h.
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.
Figure 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 ± S.D. (n = 3). The units are µeq of free fatty acid released/µg of LPL/h.
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).
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 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
-macroglobulin(40, 41, 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
-macroglobulin binding studies ( (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 µM) 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 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 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
I-hepatic 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 members (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 proteoglycan-deficient 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. 6correlate 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 µeq of free fatty acid released/µg 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 S-labeled
glycosaminoglycan chains are known to be secreted into the medium in
CHO-K1 cells metabolically labeled with
SO
,
and both of the proteoglycan-deficient 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.