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1. Introduction 1.1. General

Significant physiological and pathophysiological processes involve interactions of specific lipoproteins with specific proteoglycans. So far, these interactions fall into two large groups. The first is interactions with heparan sulfate proteoglycans (HSPGs), primarily on the cell surface that lead to cellular uptake of the lipoproteins. These pathways are of interest because they involve endocytic machinery, intracellular itineraries, and regulation that are generally distinct from classic, coated pit-mediated endocytosis (1). Moreover, many important nonlipoprotein ligands, such as infectious agents, growth factors, platelet secretory products, and proteins implicated in Alzheimer's disease, bind to the same HSPGs, and lipoproteins are a convenient model ligand to study HSPG-mediated catabolism. The second group of lipoprotein-proteoglycan interactions involves chondroitin sulfate proteoglycans (CSPGs), primarily in the extracellular matrix, leading to retention of cholesterol-rich lipoproteins. Many lines of evidence now support the concept that retention of lipoproteins within the arterial wall is the key initial step in provoking atherosclerosis, the major killer in Western countries (2,3t).

The following sections contain a brief primer on general lipoprotein biology and methods (see Subheading 1.2.); then essential background for methods used to study lipoprotein-HSPG interactions, with a focus on endocytosis (see Subheading 1.3.); background relevant to molecular methods to study HSPG-mediated endocytosis, with a focus on specific domains within the syndecan-1 core protein (see Subheading 1.4.); and then five detailed, basic protocols (see Subheadings 2-4). Because of space limitations, lipoprotein-CSPG interactions are not covered in detail here, although key methods can be found in the published literature (4-13).

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

1.2. Primer on General Lipoprotein Biology and Methods

Lipoproteins are noncovalent complexes of lipid and protein that allow the body to transport many hydrophobic substances through the aqueous environment of blood. All normally occurring mammalian lipoproteins have the same basic structure, known as the oil-drop model (14): a central core of hydrophobic lipid, chiefly triacylglycerols and esterified cholesterol, surrounded by a layer of amphipathic molecules, chiefly phospholipids, unesterified cholesterol, and proteins known as apolipoproteins or apoproteins (Fig. 1). The most widely used nomenclature defines mammalian lipoproteins by their densities: high-density lipoprotein (HDL, 1.063 g/mL < d < 1.21 g/mL), low-density lipoprotein (LDL, 1.019 < d < 1.063 g/mL), and very low-density lipoprotein (VLDL, d < 1.006 g/mL). Sometimes, intermediate-density lipoproteins are referred to as a separate class (IDL, 1.019< d < 1.019 g/mL). In addition, there are two lipoproteins specifically associated with meals: the chylomicron (d < 0.96 g/mL), which appears in plasma in the post-prandial state and transports lipids, chiefly triacylglycerols, that have been ingested and absorbed; and the chylomicron remnant, which can appear in the VLDL or IDL density ranges and is the particle that remains after peripheral tissues have extracted most of the triglycerides from circulating chylomicrons. An abnormal particle, P-VLDL, which appears in individuals with certain genetic abnormalities in apolipoprotein (apo) E or after prolonged administration of high-cholesterol diets to experimental animals, is commonly used as an experimental substitute for chylomicron remnants. Importantly, the different classes of lipoproteins have distinct lipid compositions and apoprotein constituents (Table 1) and hence distinct metabolic roles.

LDL and P-VLDL are the lipoproteins most commonly studied with proteoglycans. They are abundant, easy to isolate, easy to label either radioactively or fluorescently, and they exhibit important interactions with proteoglycans in vivo. Both contain apoB, a large amphipathic protein that cannot move between lipoproteins and hence serves as an excellent anchor for tags to track entire particles. In contrast, all other known apoproteins readily exchange between lipoprotein particles, and surface and core lipids are moved between lipoproteins by lipid transfer proteins in human plasma. Both LDL and P-VLDL bind cell-surface LDL receptors in vitro, but are also known to undergo substantial catabolism in vitro (1,15) and in vivo (16,17) independent of LDL receptors. Both of these particles have been implicated in atherosclerotic vascular disease.

Isolation of these particles from plasma requires sequential ultracentrifugation at accelerations sufficient to overcome Brownian motion. Several companies in the U.S. sell human LDL, such as Sigma Chemical Company (St. Louis, MO), CalBiochem (La Jolla, CA), Molecular Probes (Eugene, OR), and Academy Bio-Medical (Houston, TX), but as with most materials, it can be less expensive to isolate it yourself, particularly for long-term needs [ultracentrifugal isolation is described in detail in (18,19)]. Radioactive labeling is best performed by the iodine monochloride method (19-22), which covalently attaches 125I to tyrosyl residues in apoproteins, with minimal disruption of double bonds in the fatty acyl side chains of lipoprotein lipids. Fluorescent labeling usually employs DiI or DiO, which are generally nontoxic lipophilic compounds that insert into cell membranes or lipoprotein surfaces, but then are poorly

Fig. 1. General schematic of normal lipoprotein structure. All normal human plasma lipoproteins exhibit the same basic structure, known as the oil-drop model, which consists of a single layer of amphipathic molecules (phospholipids, apoproteins, and unesterified cholesterol) surrounding a hydrophobic core (cholesteryl ester or triacylglycerides). The fatty acyl side chains of each phospholipid molecule point inward toward the hydrophobic core, while the polar head group is exposed on the particle surface. Apoproteins are similarly oriented, with a hydrophobic face associating with lipid, and the hydrophilic face directed outward. Owing to its alcohol group, unesterified cholesterol is associated mainly with the phospholipid molecules of the particle surface, although some partitions into the core. This overall arrangement keeps hydrophobic molecules and domains shielded from the surrounding aqueous environment of blood and interstitial fluid. Only one schematic apoprotein has been drawn, although many species of lipoproteins have several protein molecules per particle. Apoproteins are often convenient sites to place radioactive tags, shown here as an 125I label (see Subheading 1.2. for more details).

Table1

Physical Characteristics of the Major Plasma Lipoproteins in Humans

Table1

Physical Characteristics of the Major Plasma Lipoproteins in Humans

Diameter

Core

Principal

Particle

(nm)

lipid

Apoproteins

Chylos

80-1000

Tg (diet)

B48, AI,AII,CII,E

VLDL

30-80

Tg (liver)

B100, CII,E

LDL

18-28

ChE

B100

HDL

5-12

ChE

AI, AII, occasionally E

Abbreviations: Chylos, chylomicrons and remnants; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; diet, originating from dietary intake and secreted by the intestines; liver, synthesized and secreted from the liver; Tg, triacylglycerol; ChE, cholesteryl ester; B100, the full-length apolipoprotein-B, which is originates primarily from the liver; B48, a truncated version of apolipoprotein-B that contains the N-terminal 48% of the full molecule and originates primarily from the intestine.

exchangeable to other particles or surfaces. These moieties emit at 571 nm (orange) and 501 nm (green), respectively, and can therefore be used with standard optical filters for rhodamine and fluorescein during multicolor imaging. Fluorescently labeled lipoproteins are available commercially (Molecular Probes, Eugene, OR, and Leiden, The Netherlands), and so protocols from the literature for preparing these particles (22,23) are not reproduced here. Quantitative methods using fluorescently labeled lipoproteins have been developed (24), but 125I-labeled lipoproteins remain the standard in catabolic studies and will be the focus of the rest of this chapter.

The parameters most commonly measured after incubation of 125I-lipoproteins with cells are surface binding, intracellular accumulation, and lysosomal degradation of the particles. Typically, monolayers of essentially confluent cells are used, usually fibroblasts, macrophages, or hepatocytes, that have been preincubated overnight in cholesterol-poor medium to stimulate expression of LDL receptors (19). Cells are then incubated at 37°C for 2-6 h in the continuous presence of 125I-labeled LDL or P-VLDL (usually 1-10 ^g lipoprotein protein per millilliter of medium, although wider concentration ranges are used for formal assessment of the Kd and Vmax). During this time, particles bind to the cell surface, become internalized, and are then delivered to lysos-omes. In the lysosomes, 125I-labeled protein is degraded to individual amino acids, but [125I]monoiodotyrosine, the radiolabeled form of tyrosine, does not charge the tyrosine-specific tRNA (25), and so it simply leaks out of the cells back into the media. At the end of the incubation, cells are chilled to 4°C to stop further metabolism. The culture media are harvested for isolation of [125I]monoiodotyrosine as an indication of lysosomal degradation (see Subheading 3.1.) (19,26). The cell monolayers are rinsed twice for 10 min with chilled phosphate- or Tris-buffered saline, pH 7.4, supplemented with 2 mg of fatty acid-free bovine serum albumin per milliliter, then twice rapidly with chilled buffered saline without albumin. Surface-bound particles are released from LDL receptors and other sites by a 30-min incubation at 4°C in buffered saline with 10 mg of heparin/ mL. The cell monolayers are rinsed rapidly once more in buffered saline at 4°C, this rinse is pooled with the heparin wash, and the radioactivity in an aliquot is determined by gamma counting. The cell monolayers are then dissolved in 0.1 M NaOH. One aliquot of dissolved cells is used for gamma counting to assess intracellular accumulation of labeled material, and another aliquot is used to assess total cellular protein content. Radioactivity results are converted to mass of lipoprotein protein using the known specific activity of the particular radiolabeled preparation, and results are expressed as nanograms of catabolized lipoprotein protein per milligram of total cell protein. To verify that particle degradation involves lysosomes, cells are treated with chloroquine (150 ^M), an inhibitor of lysosomal proteases (19), beginning 0-45 min before the incubation with 125I-labeled lipoproteins, and continuing until the end of the experiment. Typically, ligand degradation is inhibited by 85-90%, and there is a corresponding increase in the accumulation of intracellular radioactive material. 1.3. Background for Methods Used to Study Lipoprotein-HSPG Interactions

Several modifications in the general methods outlined above are necessary for the study of lipoprotein-HSPG interactions. First, there are several distinct genetic families of cell-surface HSPGs, prinicpally syndecans, perlecan, and glypicans, and it is most informative to use cellular preparations that have one predominant cell-surface HSPG, or else a limited and known combination of these HSPGs. For the study of syndecans (Fig. 2A, left schema), we have used mainly Chinese hamster ovary (CHO) cells that we transfected with an expression construct for the human syndecan-1 core protein (27). For perlecan, we have used the WiDr colon carcinoma line, which expresses perlecan but no other proteoglycans (American Type Culture Collection [ATCC], Manassas, VA, cat. no. CCL 218, also known as HT-29) (28-32). For glypicans, there are reports of transfected mammalian cells in the literature (33).

Second, the binding of lipoproteins to HSPGs in vivo is facilitated by bridging molecules, such as lipoprotein lipase (LpL), apoE, defensins, and hepatic lipase, each of which has a hydrophobic face that adheres to the lipoprotein surface and a cationic face that binds HS. Thus, studies in vitro typically involve examination of cellular catabo-lism of lipoproteins in the absence and in the presence of one of these molecules, and the arithmetic increase in each of the catabolic parameters is calculated (27,32,34). We have preferred to use LpL because, unlike apoE, it does not bind LDL receptors, and unlike hepatic lipase, it is relatively easy to isolate in large quantities [see Subheading 3.2., adapted from references (35,36)]. Typically, we add 5 ^g of 125I-labeled lipoprotein protein per milliliter of medium, without or with 5 ^g LpL/mL. Alternatively, cells that naturally secrete these bridging molecules (37) or cells transfected to express them [e.g., (38,39)] can be used. The role of HSPGs in the increased catabolism upon addition of LpL is verified by heparitinase digestion of the cells, which typically abolishes ~90% of LpL-dependent catabolism (27,34,37,40,41); by the use of HS-negative CHO mutants (34,40,42,42a); by addition of very low concentrations of heparin (<100 ^g/mL) that are insufficient to interfere with LDL receptor binding but are able to displace surface-bound LpL and other bridging molecules (34,42a); or by pre-incubation of cells in chlorate to block sulfation of glycosaminoglycan side chains (43-45). Heparitinase digestion usually involves preincubation of cells in serum-free medium at 37°C for several hours to allow the cells to clear surface-bound serum-derived molecules, then an initial digestion with heparitinase for 60-90 min at 37°C before addition of ligand, and finally an incubation of cells with ligand, but in the continuous presence of heparitinase, to avoid rapid regeneration of side chains (34). Apoproteins normally found on LDL, VLDL, and P-VLDL include apoB and apoE, both of which bind HS, although lipoprotein concentrations around 100 ^ig/mL are usually required before this binding becomes a significant contributor to total cellular catabolism in the absence of added bridging molecules (15,45). These higher concentrations may be physiological, and LDL receptor-independent clearance of lipoproteins is substantial in vivo (16,17). Interestingly, two common, naturally occurring polymorphisms of apoE, one of which has been associated with Alzheimer's diease (46), show substantial differences in their catabolism by cells through a pathway mediated by HSPGs (47), particularly syndecan HSPGs (48).

Third, careful attention must be paid to catabolic contributions by members of the LDL receptor family. These contributions take the form of direct internalization, which appears simply as background in measurements of lipoprotein catabolism in the absence of LpL, and synergistic interactions, in which cell-surface HSPGs and LDL

Fig. 2. The FcR-Synd1 chimera, a molecular tool to study the syndecan transmembrane and cytoplasmic domains. (A) Comparison of the syndecan-1 HSPG (left schema) and the FcR-Synd1 chimera (right schema). As indicated, the chimera contains the ectodomain of the IgG Fc receptor-Ia linked to the highly conserved transmembrane and cytoplasmic domains of the human syndecan-1 core protein (27). (B) Clustering of FcR-Synd chimeras. Two chimeric molecules are shown, each bound to an 125I-labeled nonimmune human IgG. Clustering is accomplished by adding the clustering agent, that is, goat F(ab')2 fragments against the Fab domain of human IgG. This clustering agent cannot interfere with the binding of whole IgG to the chimeras, because it has no Fc domain to bind to the chimeras, nor does it interact with the Fc domain of the IgG ligands.

Fig. 2. The FcR-Synd1 chimera, a molecular tool to study the syndecan transmembrane and cytoplasmic domains. (A) Comparison of the syndecan-1 HSPG (left schema) and the FcR-Synd1 chimera (right schema). As indicated, the chimera contains the ectodomain of the IgG Fc receptor-Ia linked to the highly conserved transmembrane and cytoplasmic domains of the human syndecan-1 core protein (27). (B) Clustering of FcR-Synd chimeras. Two chimeric molecules are shown, each bound to an 125I-labeled nonimmune human IgG. Clustering is accomplished by adding the clustering agent, that is, goat F(ab')2 fragments against the Fab domain of human IgG. This clustering agent cannot interfere with the binding of whole IgG to the chimeras, because it has no Fc domain to bind to the chimeras, nor does it interact with the Fc domain of the IgG ligands.

receptor family members co-operate in ligand catabolism. Direct internalization via LDL receptor family members is easier to deal with. LDL can be 35% reductively methylated (mLDL; see Subheading 3.3.), which abolishes its ability to bind LDL receptors (49) while having no effect on the binding to cell-surface HSPGs in the presence of LpL (27,32). In an experimental tour de force, a series of transgenic mice were created that express different site-directed mutants of human apoB, including one variant with defective LDL receptor binding but unaltered affinity for proteoglycans, and another that binds LDL receptors but not proteoglycans (9,10,10a). Monoclonal antibodies against specific domains of apoB and apoE have been reported and used in studies of lipoprotein-proteoglycan interactions (50,51) (many of these antibodies are available for purchase from the University of Ottawa Heart Institute, Ottawa, Ontario, Canada). Cellular LDL receptors can be blocked with a polyclonal antibody (52) or with a monoclonal antibody produced by a clone available from the ATCC (#CRL 1691), but this monoclonal antibody must already be bound to cells before the lipoproteins are added (53,54). Cellular LDL receptors are readily suppressed in most cell types by an overnight incubation in medium supplemented with 1-2 ^g of 25-hydroxycholesterol, which potently downregulates LDL receptors, plus 20-40 ^g of cholesterol per milliliter to protect the cells from the toxicity of 25-hydroxycholesterol (19), although cholesterol-induced alterations in glycosaminoglycan synthesis have been reported (55). Suppression of LDL receptors must be verified by a substantial reduction in 125I-LDL binding: some macrophage-like cell lines (56) and cells expressing cholesterol 7a-hyroxylase (57), such as hepatic paren-chymal cells, maintain significant LDL receptor expression despite the presence of abundant sterol. Unlike proteoglycans, LDL receptor family members depend on calcium ions for binding (19), which can be chelated by EDTA (45,51). Finally, LDL receptor-negative human fibroblasts (the ATCC has several lines) (19) and CHO cells (58), as well as an LDL receptor knockout mouse (Jackson Laboratory, Bar Harbor, ME, cat. no. 002207) (59), are available. Owing to their simplicity, our methods of choice for controlling ligand binding to the LDL receptor in vitro have been LDL methylation or cellular supplementation with sterols.

The LDL receptor-related protein (LRP), a cell-surface LDL receptor family member that has been reported to bind LpL, apoE, and hepatic lipase, is readily blocked by the 39-kDa receptor-associated protein (RAP), which is available as a recombinant protein

(60). Unfortunately, RAP is the human homolog of mouse heparin-binding protein 44

(61), readily binds heparin (61), and therefore might also compete for binding to HS side chains, particularly ones that are rich in heparin-like domains. In the experience of our laboratory (27,32), RAP has had small or no effects on the binding of LpL-enriched 125I-labeled mLDL to the HSPGs of CHO or WiDr cells, although the binding of RAP to cell-surface HSPGs remains controversial (62,63). To our knowledge, no studies have systematically examined the binding of RAP to hepatic parenchymal cells, which synthesize HS with abundant heparin-like domains (64). LRP-deficient cell lines (65-67) and liver-specific knockout mice (68) have been reported, although the status of their proteoglycans is unknown. 125I-labeled RAP is prepared using Iodobeads (Pierce Chemical Company, Rockford, IL) instead of the iodine monochloride method, owing to the lack of unsaturated lipid in RAP, and this radiolabeled molecule can serve as a convenient ligand for coated pit-mediated internalization, provided it is known to bind poorly to the particular HSPGs that are expressed by the cell type of interest (27). 125I-RAP can be released from cell-surface LRP by a protease cocktail, to distinguish surface-bound from internalized material (27,69).

Synergistic interactions between cell-surface HSPGs and LDL receptor family members are thought to involve either the transfer of ligand from HSPGs to LDL receptor family members or the formation of ternary complexes of HSPGs, HSPG-bound ligands, and LDL receptor family members (40,41,70,71). No direct evidence to date has distinguished these possibilities. Quantification of synergy between cell-surface HSPGs and LDL receptors requires measurement of the catabo-lism of 125I-labeled native LDL and 125I-mLDL, each in the absence and presence of LpL. Four catabolic components can then be computed (32): (1) the LDL receptor-independent, LpL-independent component, which is usually referred to as nonspecific uptake or assay background and is measured by the catabolism of 125I-mLDL in the absence of LpL; (2) the LDL receptor-dependent, LpL-independent component, which is the classical LDL receptor pathway and is computed by the difference between the catabolism of 125I-LDL vs 125I-mLDL; (3) the LDL receptor-independent, LpL-depen-dent component, which involves cell-surface HSPGs and is computed by the difference between the catabolism of 125I-mLDL in the presence vs the absence of LpL; and (4) a synergistic component, which requires cooperation between LDL receptors and LpL and is computed as the increase in 125I-LDL catabolism upon addition of LpL minus the increase in 125I-mLDL catabolism upon addition of LpL. Total catabolism of 125I-labeled native LDL in the presence of LpL equals the sum of these four components, reflecting the ability of LpL-enriched 125I-LDL to participate in these four potential uptake mechanisms (32). Similar information can be obtained using cellular sterol enrichment instead of LDL methylation to manipulate binding to the LDL receptor, or heparitinase digestion instead of omission of LpL to manipulate binding to cell-surface HSPGs. These alternative methods tend to be more cumbersome and somewhat less effective and, as noted above, cellular sterol enrichment has been reported to affect glycosaminoglycan synthesis (55). In WiDr cells under our experimental conditions, we have found that the four components respectively account for approximately 4%, 15%, 57%, and 23% of total LpL/125I-LDL catabolism. In other words, most LpL/125I-LDL enters these cells via perlecan directly (component 3) without any assistance from LDL receptors (31,32).

Fourth, careful attention must be paid to the unusual characteristics of HSPG-medi-ated ligand internalization. To assess the efficiency of endocytosis, we calculate ligand internalization as the sum of intracellular accumulation plus degradation (27). Internal-ization calculated in this fashion takes into account ligand still within the cells, as well as ligand that had been taken up by cells but then degraded into amino acids, which the cells release to the culture medium. To examine in detail the kinetics of ligand internal-ization and degradation, we incubate labeled ligands with cells in serum-free medium for 1 h at 4°C, to allow surface binding without further catabolism, and then the cells are washed at 4°C to remove unbound material. Fresh media at 37°C

with no ligands are added, and incubations are continued at 37°C for various times, usually 15 min to 24 h (27,32). Assays for surface-bound, intracellular, and degraded ligand are then performed, and ligand internalization is calculated. In some experiments, TCA-precipitable radioactivity in the media is also quantified (Subheading 3.1.), as an indication of retroendocytosis or desorption from the cell surface during the incubation at 37°C. In addition, because the principle method under Subheading 3.1. for assessing lysosomal degradation requires complete breakdown of labeled apoproteins into individual amino acids, supplementary tests for partial breakdown products can also be informative (also given under Subheading 3.1.). From these studies, we have found that syndecan-mediated endocytosis of LpL/125I-mLDL proceeds with a t1/2 of ~1 h (27), and that perlecan-mediated internalization exhibits a t1/2 of ~5 h (31,32). By contrast, the t1/2 for coated pit-mediated internalization is ~10 min (27,32,72,73).

HSPG-mediated pathways for ligand internalization can also be distinguished through the use of metabolic inhibitors, particularly genistein (0-400 [M), a tyrosine kinase inhibitor (74), and cytochalasin D (0-2 [M), which disrupts the actin cytoskeleton (75). Typically, ligands are bound to the cell surface at 4°C, unbound ligand is washed away, and then specific inhibitors are added simultaneously with fresh medium at 37°C. Cells are incubated at 37°C until ~30-50% of surface-bound ligand has been internalized in the absence of inhibitors, that is, 45 min for syndecan and 2 h for perlecan (incubations longer than 2 h can encounter fading of the genistein effect) (27,76). To allow comparison with coated pit-mediated internalization, we employ a 30-min pre-incubation at 37 °C in the presence of these inhibitors then a 10- to 15-min incubation in the presence of inhibitors plus 125I-RAP (27) or surface-bound 125I-LDL (32). Coated pit-mediated endocytosis is insensitive to genistein, whereas both syndecan- and perlecan-mediated internalization are inhibited (27,32). Coated pit endocytosis exhibits limited sensitivity to cytochalasin (27,32,77), whereas syndecan-mediated endocytosis is readily inhibited (27), and perlecan-dependent internalization is slightly enhanced by this agent (32). Thus, the three pathways exhibit distinctive kinetics of intenalization and different dependence on tyrosine kinases and the actin cytoskeleton.

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