Interaction of Lecithin:Cholesterol Acyltransferase (LCAT) (cid:1) (cid:1) 2 -Macroglobulin Complex with Low Density Lipoprotein Receptor-related Protein (LRP) EVIDENCE FOR AN (cid:1) 2 -MACROGLOBULIN/LRP RECEPTOR-MEDIATED SYSTEM PARTICIPATING IN LCAT CLEARANCE*

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of criti-cal importance in reverse cholesterol transport, but the structural and functional pathways involved in the reg-ulation of LCAT have not been established. We present evidence for the direct binding of LCAT to (cid:1) 2 -macro-globulin ( (cid:1) 2 M) in human plasma to form a complex 18.5 nm in diameter. Forty percent of plasma LCAT-HDL was associated with (cid:1) 2 M; moreover, most of the LCAT in cerebrospinal fluid and in the medium of cultured human hepatoma cell line was associated with (cid:1) 2 M. Puri- fied recombinant human LCAT (rLCAT) labeled with 125 I bound to native and methylamine-activated (cid:1) 2 M ( (cid:1) 2 M-MA) in vitro in a time- and concentration-depend- ent manner, and this binding did not depend on the presence of lipid. rLCAT bound to (cid:1) 2 M-MA with greater affinity than to (cid:1)

Human lecithin:cholesterol acyltransferase (LCAT) 1 is a 416-amino acid glycoprotein circulating in plasma associated with lipids and apolipoproteins in the high density lipoprotein (HDL) fraction (1). LCAT plays a key role in cholesterol homeostasis by mediating the production of most of the cholesteryl esters in human plasma. It has been suggested (2, 3) that LCAT plays an important role in reverse cholesterol transport (RCT) by creating a concentration gradient for the efflux of free cholesterol from peripheral cells to HDL particles and its conversion to cholesteryl esters. The cholesteryl esters are internalized within the lipoprotein core for ultimate transport to the liver for clearance or recycling. Thus, factors affecting the structure, activity, or concentration of LCAT are likely to affect the homeostasis of plasma cholesterol and the RCT process, one of several proposed mechanisms (4) by which HDL may protect against atherosclerosis.
Recent investigations (5) suggest that LCAT can act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins. In human plasma, LCAT is almost entirely associated with lipoproteins containing apoA-I, its principal physiological activator (6). Francone et al. (1) have shown the presence of LCAT, cholesteryl ester transfer protein, apoD, and a small amount of apoA-I in a complex termed pre-␤ 3 -LpA-I that is involved in the esterification and transfer of cell-derived cholesterol.
We have recently found (7) an association between apoE and ␣ 2-macroglobulin (␣ 2 M) in human plasma, when we observed that LCAT and apoE migrate to the same position in twodimensional electrophoretic gels together with ␣ 2 M, in particles 18.5 nm in diameter. This raises the possibility that LCAT circulates in plasma in association with ␣ 2 M. Human ␣ 2 M, the largest known proteinase inhibitor (M r ϭ 720,000), is found at high concentrations (2-5 M) in plasma and in extravascular spaces (8,9). It plays a pivotal role in the clearance of proteinases from the circulation and in regulating their activity in fibrinolysis, coagulation, and complement activation (10,11). ␣ 2 M is also a carrier of specific cytokines and various nonproteolytic proteins that include the transforming growth factor TGF-␤, the platelet-derived growth factor-BB (12), the ␤-amyloid peptide (13), and recently, apolipoprotein E (7).
The binding affinities of ␣ 2 M for different non-proteolytic proteins depend on its conformation, but ␣ 2 M in plasma is present almost entirely in the native conformation. This form of ␣ 2 M is fully functional as a proteinase inhibitor but is not recognized by the cell surface receptor, which is an ␣ 2 M receptor/low density lipoprotein receptor-related protein (LRP) (8,14). This receptor is responsible for the rapid plasma clearance of conformationally transformed ␣ 2 M following its reaction with proteinases or small primary amines that modify the ␣ 2 M thiol ester bonds. Gonias and co-workers have documented that the binding of TGF-␤ isoforms to ␣ 2 M neutralizes the activity of TGF-␤ toward various cells (12,15,16). Indeed, ␣ 2 M may be involved in controlling apoptosis (17), the immune system, and atherogenesis (18,19) via its regulating effects on TGF-␤ activity. Structural and functional evidence for several binding domains in different ␣ 2 M forms led us to hypothesize that ␣ 2 M modulates LCAT activity and concentration in plasma and may be involved in LCAT clearance via an LRP-mediated endocytic process. The physical association of LCAT and ␣ 2 M has not been previously reported. The present study aims at providing evidence for the association of LCAT with ␣ 2 M and to examine how these interactions could be affected by native and activated forms of ␣ 2 M and by apolipoproteins known to bind LCAT. Cellular binding, internalization and degradation assays were used to evaluate the role of activated ␣ 2 M and LRP receptor in mediating the clearance of LCAT by cells.

EXPERIMENTAL PROCEDURES
Materials-Anti-human ␣ 2 M monoclonal antibody, affinity-purified rabbit anti-goat IgG and affinity-purified goat anti-mouse IgG antibody were purchased from Biodesign International (Kennebunk, ME). Methylamine hydrochloride, phosphatidylcholine-specific phospholipase C and sphingomyelinase were purchased from Sigma. Partially purified, anti-human ␣ 2 M polyclonal antibody and anti-human IgG polyclonal antibody were purchased from ICN Pharmaceuticals Inc. (Aurora, OH). HepG2 cells (HB-8065) were purchased from American Type Culture Collection (ATCC). Anti-LCAT goat polyclonal antibody and purified human recombinant LCAT (rLCAT) were a gift from Dr. Henry Pownall, Baylor College of Medecine,s Houston, TX. C-terminal histidinetagged human recombinant lecithin:cholesterol acyltransferase (hrL-CATH6) was a gift from Dr John S. Parks, Wake Forest University School of Medecine, Winston-Salem, NC. Anti-LCAT monoclonal antibody 2H11 was generously provided by Dr. Ross Milne, University of Ottawa Heart Institute. Mouse embryonic fibroblasts (MEF1) that express LRP, and mouse embryonic fibroblasts genetically deficient in LRP (MEF2) were generously provided by Dr. Joachim Herz, Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas.
Blood Sampling-Blood samples were obtained after an overnight fast from male subjects with an apoE3/3 phenotype. Blood was drawn from an arm vein into evacuated tubes containing ethylenediaminetetraacetate (EDTA, final concentration 1.5 mg/ml). Blood collection tubes were immediately placed in ice. Plasma was obtained by centrifugation (3,000 rpm, 15 min) and was kept in ice until electrophoretic separation, which was routinely carried out within 30 min of plasma isolation, or was frozen at Ϫ70°C until analysis of lipids and apolipoproteins.
Gel Electrophoresis-Plasma samples were separated by two-dimensional, non-denaturing gradient gel electrophoresis as described previously (20,21). In some experiments, only the ␣ 2 M-containing pre-␤ 2migrating segment (ϳ2 cm) of agarose gels was separated in the second dimension.
Immunoprecipitation Procedures-LCAT associated with ␣ 2 M was isolated from human plasma by immunoprecipitation. Plasma (120 -500 l) was incubated overnight at 4°C with 50 -200 l of anti-human ␣ 2 M antibody or with control anti-human IgG antibody. The immunoprecipitates were centrifuged at 10,000 rpm for 6 min and washed three times with buffer (20 mM HEPES, pH 7.5, 0.15 M NaCl, 0.1% Triton, and 10% glycerol). They were analyzed by 4 -22.5% SDS-polyacrylamide gel electrophoresis, together with molecular weight standards (Amersham Pharmacia Biotech). The presence of LCAT and ␣ 2 M in immunoprecipitates was detected by immunoblotting. The efficiency of ␣ 2 M immunoprecipitation was assessed as Ͼ95%, based on the absence of ␣ 2 M in the supernatant.
Preparation of ␣ 2 M-MA, ␣ 2 M, and binding experiments were performed as previously described (7). The effect of various apolipoproteins and reconstituted HDL particles on the binding of rLCAT to ␣ 2 M was determined by adding these substances (as specified for each experiment) to reaction tubes prior to the addition of 125 I-rLCAT. In certain experiments, samples were treated with phospholipases or trypsin before addition of 125 I-rLCAT. Bound and unbound 125 I-rLCAT was separated by non-denaturing gradient gel electrophoresis 2.5-18% at 60 V (16 h, 15°C). No dissociation of 125 I-rLCAT bound to ␣ 2 M was detected with this electrophoretic system as assessed by reseparating electroeluted 125 I-labeled rLCAT⅐␣ 2 M or 125 I-labeled rLCAT⅐␣ 2 M-MA complexes. In experiments using 125 I-rLCAT, gels were dried and exposed at Ϫ70°C to XAR-2 Kodak film for 4 -72 h. Exposed films were used as a template to identify the position of appropriate bands for excision and counting.
Measurement of LCAT Activity and LCAT Mass-LCAT activity was measured as relative cholesterol esterification achieved during a 5-h incubation of plasma at 37°C as described previously (21). The LCAT activity of the LCAT⅐␣ 2 M complex was assayed as described previously (24) but with minor modifications. The [ 3 H]cholesterol-labeled r(LpA-I) substrate was prepared as previously described (25) and was diluted in buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM NaN 3 , and 0.01% EDTA) to give final apoA-I concentrations ranging from 10 Ϫ7 to 10 Ϫ6 M. Each reaction mixture contained 50 l of 30 mg/ml bovine serum albumin and 50 l of 100 mM ␤-mercaptoethanol in a final volume of 300 l. Incubation was for 3 h at 37°C. LCAT activity was determined as the amount of esterified cholesterol that was incorporated into apoA-I-containing proteoliposomes. LCAT mass was measured by the method of Qu et al. (26), with purified LCAT as standard.
Preparation and Purification of 125 I-rLCAT⅐␣ 2 M-MA Complex-125 I-rLCAT was incubated with ␣ 2 M-MA at 2:1 molar ratio for 24 h at 37°C. Radiolabeled rLCAT⅐␣ 2 M-MA complex was then separated from free 125 I-rLCAT by 500-kDa exclusion filters and isolated from preparative non-denaturing gradient gels by electroelution. The integrity of the complex was evaluated by electrophoresis under non-denaturing conditions on 3-15% gradient gels followed by autoradiography.
Cell Culture-HepG2 cells were cultured under standard conditions. Briefly, HepG2 cells (ATCC HB 8065) were grown in Eagle's minimum essential medium with nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum. After a 24-hour incubation, serum-free medium from HepG2 (3 ml) were collected in the presence of 1 mmol/L phenylmethylsulfonyl fluoride, concentrated by using Centricon filters (Amicon) to a final volume of 200 l and separated by two-dimensional gel electrophoresis.
Cell Association and Degradation Assays-Mouse embryonic fibroblasts (MEF1) that express LRP and mouse embryonic fibroblasts genetically deficient in LRP (MEF2) were used. Cells were incubated with 125 I-␣ 2 M-MA, 125 I-rLCAT, 125 I-LPL and 125 I-rLCAT⅐␣ 2 M-MA complex at the concentrations indicated in the figure legends. Cell-specific binding, association, and degradation were measured after 5-h incubations at 4°C or 37°C. Aliquots of culture medium were taken for degradation of radiolabeled ligand by measurement of non-iodide 125 I (soluble in 10% trichloroacetic acid) as previously described (27).
Lipid and Lipoprotein Assays-Cholesterol and triglyceride concentrations were determined enzymatically on an autoanalyzer (Cobas Mira, Roche Molecular Biochemicals). HDL-cholesterol concentration was determined by measuring cholesterol in the supernatant after heparin-manganese precipitation of apoB-containing lipoproteins in the d Ͼ 1.006 g/ml fraction of plasma prepared by ultracentrifugation where d is density. Plasma apoB and apoA-I concentrations were measured by nephelometry (Behring Nephelometer 100 Analyzer). ApoE phenotypes were determined by immunoblotting of plasma separated by minigel electrophoresis (28). Low density lipoprotein (LDL) (1.019 Ͻ d Ͻ 1.063) and HDL 3 (1.125 Ͻ d Ͻ 1.21 g/ml) were isolated from normolipidemic plasma by sequential ultracentrifugation using a Beckman ultracentrifuge. The total HDL fraction was isolated from plasma by automated gel filtration chromatography (FPLC).

RESULTS
We used two-dimensional gel electrophoresis to separate plasma apolipoprotein complexes in the HDL size range (Fig.  1A). LCAT protein co-migrated in approximately equal proportions with ␣-HDL and ␣ 2 M plasma protein. LCAT was consistently found to co-migrate with plasma ␣ 2 M protein, having a molecular diameter of 18.5 nm. (Fig. 1A, right panel), detected with 125 I-labeled anti-␣ 2 M monoclonal antibody. LCAT migrated in the same position as ␣ 2 M (Fig. 1A, left panel). LCAT associated with ␣ 2 M was also detected in human cerebrospinal fluid (CSF) and in the medium of cultured HepG2 (Fig. 1B). Most of the LCAT in CSF and secreted by HepG2 cells was associated with ␣ 2 M and had an apparent diameter of 18.5 nm, as was the case for LCAT⅐␣ 2 M in the plasma. ␣-LCAT species present in CSF and the medium of cultured HepG2 were smaller than ␣-LCAT in human plasma. The control sample in this experiment (Fig. 1B, right panel) was purified plasma ␣ 2 M, which contained bound LCAT.
To provide evidence for a direct association of LCAT with ␣ 2 M, we used a purified anti-␣ 2 M IgG fraction to immunoprecipitate ␣ 2 M from the plasma of three normolipidemic subjects. The amount of LCAT associated with ␣ 2 M immunoprecipitate was assessed by immunodetection of LCAT and ␣ 2 M ( Fig. 2A, upper panel) after separation by SDS-polyacrylamide gel electrophoresis. In lane a the nonspecific IgG immunoprecipitation revealed one IgG band detected by the second antibody used in the LCAT detection. Lanes b, c, and d demonstrate, in addition, an LCAT band with molecular mass of ϳ66 kDA. The absence and presence of ␣ 2 M in respective samples are shown in the ( Fig. 2A, lower panel). Removal of ␣ 2 M from plasma by affinity chromatography reduced most of the LCAT associated with ␣ 2 M but not ␣-migrating LCAT (data not shown). The amount of plasma LCAT associated with ␣ 2 M was estimated to be ϳ40% of total plasma LCAT in the HDL size range (see later, Fig. 6A).
LCAT was also present in commercial preparations of ␣ 2 M, purified by metal chelate chromatography. Lane a of the left panel of Fig. 2B shows that a significant amount of LCAT was immunodetectable in commercial ␣ 2 M separated, in this case, by non-denaturing gradient gel electrophoresis; the complex had an apparent diameter of 18.5 nm. The specificity of anti-LCAT antibody was confirmed by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by immunodetection of LCAT in commercial preparations of ␣ 2 M.
LCAT was present with molecular masses of 66 kDa, as shown in lanes a and b of Fig. 2C. In this experiment purified rLCAT was used as control, lane c. The difference in apparent molecular mass between plasma LCAT dissociated from native purified ␣ 2 M and rLCAT (Fig. 2C) was because of different degrees of glycosylation as previously reported (29).
Methylamine activation of commercial ␣ 2 M did not dissociate LCAT from ␣ 2 M, as shown by the similar amount of LCAT  a and b, respectively). C, commercial ␣ 2 M separated by SDS polyacylamide gradient gel electrophoresis under nonreducing conditions and in the presence of a reducing agent (␤-mercaptoethanol) (lanes a and b, respectively). Purified rLCAT (5 g) (lane c) was used as control. 125 Ilabeled molecular mass standards are also shown in panels A, B, and C. associated with ␣ 2 M under non-denaturing conditions (Fig. 2B,  lanes a and b, respectively). Under non-reducing conditions, SDS gel electrophoresis caused a part of LCAT to dissociate from commercial ␣ 2 M, as shown in lane a of Fig. 2C. LCAT⅐␣ 2 M complex was resistant to boiling in the presence of SDS. In addition, the in vitro formation of 125 I-rLCAT⅐␣ 2 M was not inhibited by iodoacetic acid, indicating that free sulfhydryl groups are not required for complex formation, providing evidence for the non-covalent association of LCAT with ␣ 2 M (data not shown).
The binding of LCAT to activated (␣ 2 M-MA) and native ␣ 2 M was investigated in vitro by incubation of 125 I-rLCAT at 37°C with different concentrations of ␣ 2 M for various times. Bound and unbound 125 I-rLCAT were separated by nondenaturing gel electrophoresis, and 125 I-rLCAT was quantified by direct scintillation counting. LCAT binding was found to occur in a timeand concentration-dependent manner. Maximum binding was reached after 6 h for both ␣ 2 M and ␣ 2 M-MA and remained constant for the remaining 18 h of the experiment (data not shown). ␣ 2 M-MA had a 2-fold greater capacity to bind LCAT relative to ␣ 2 M (Fig. 3, upper and left panels). The affinity of LCAT binding to both forms of ␣ 2 M was assessed from a double-reciprocal plot of the concentration-dependent binding data (right panel). Dissociation constants (K D ) calculated from the slope of the regression lines were 3.93 and 1.9 M for the nonactivated and activated forms of ␣ 2 M, respectively, indicating that LCAT had a greater affinity to bind to ␣ 2 M-MA than to ␣ 2 M.
We have previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) activate native ␣ 2 M (7). To verify that LCAT does not bind to ␣ 2 M through its well characterized proteinase-trapping mechanism, we determined whether the conformation of native ␣ 2 M could be affected by LCAT treatment. Native ␣ 2 M isolated by metal chelate chromatography (having detectable quantities of bound LCAT (Fig.  2B, lane a) experiment. PC-PLC (but not SM-ase or rLCAT) caused ␣ 2 M to become "activated" (as evidenced by the smaller size and increased mobility of ␣ 2 M separated by nondenaturing gradient gel electrophoresis) as shown (Fig. 4).
To verify that the association of LCAT does not depend on the presence of lipid, we investigated whether the binding of 125 I-rLCAT could be prevented by prior treatment of ␣ 2 M with phospholipases (sphingomyelinase and phosphatidylcholinespecific phospholipase C) or delipidating solvents. No decrease was observed in the binding of 125 I-rLCAT to native ␣ 2 M (data not shown).
We next investigated whether HDL particles would affect the association of LCAT with native ␣ 2 M. Native ␣ 2 M (50 g) was incubated with increasing amounts of reconstituted HDL, r(LpA-I), or native HDL 3 (Fig. 5). 50 g of r(LpA-I) or HDL 3 inhibited the association of LCAT with native ␣ 2 M by 50 and 30% respectively. At all concentrations used in this experiment, r(LpA-I) particles were more efficient inhibitors than native HDL 3 .
We measured the ability of LCAT⅐␣ 2 M complex to esterify cholesterol in vitro by examining the effect of removing the LCAT⅐␣ 2 M complex from plasma by immunoprecipitation of ␣ 2 M using a purified anti-␣ 2 M IgG fraction and nonspecific anti-human IgG. The fractional esterification rate of cholesterol in plasma was the same in ␣ 2 M-depleted plasma samples as in IgG-depleted plasma samples (Fig. 6B). As ϳ40% of the plasma LCAT was associated with ␣ 2 M (Fig. 6A), this result shows that LCAT associated with ␣ 2 M in plasma is inactive in the esterification of cholesterol. To investigate this observation further, we assayed for LCAT activity using a proteoliposome substrate. The same amount of LCAT (1 g) present in commercial ␣ 2 M preparation (native ␣ 2 M and ␣ 2 M-MA), purified plasma ␣ 2 M (isolated by ultracentrifugation (d Ͼ 1.25) and separated from others plasma proteins by FPLC, thus reducing the possibility of inactivating LCAT by the purification process) and the IgG-depleted HDL fraction (isolated by FPLC) was tested for LCAT activity. Under these conditions, there was no significant LCAT activity in either conformation of commercial and purified plasma ␣ 2 M in contrast to the activity associated to HDL fraction (Fig. 6C).
To evaluate the ability of 125 I-rLCAT⅐␣ 2 M complex to interact with the LRP receptor, we combined 125 I-rLCAT with unlabeled ␣ 2 M-MA, and the resulting complex was purified. The To determine whether 125 I-rLCAT alone might interact with LRP-receptor, we added equivalents amount of 125 I-rLCAT to cultured MEF1 (LRPϩ/ϩ) and control MEF2 (LRPϪ/Ϫ), and the level of specific binding was measured. As shown in Fig. 7C, minimal LCAT binding to LRP ϩ/ϩ and LRP Ϫ/Ϫ cells is observed. The control protein in this experiments was purified LPL, which bind to LRP ϩ/ϩ but not significantly to LRP Ϫ/Ϫ (Fig. 7D).
To evaluate the ability of 125 I-rLCAT⅐␣ 2 M-MA complex to interact with the LRP receptor, equivalent amounts of 125 I-rLCAT⅐␣ 2 M-MA were incubated with LRP (ϩ/ϩ) and control LRP (Ϫ/Ϫ) cells, and the level of cell association and degradation of this complex was measured. As shown in Fig. 8, incubation with cells expressing the LRP receptor resulted in the internalization and degradation of the complex, but cells deficient in this receptor (LRP Ϫ/Ϫ) were unable to internalize or degrade significant amounts of 125 I-rLCAT⅐ ␣ 2 M-MA complex. DISCUSSION We have shown that LCAT, co-migrates with ␣ 2 M in human plasma in an intermediate-sized complex (18.5 nm in diameter) between LDL and HDL lipoproteins (Fig. 1A). Plasma LCAT associated with ␣ 2 M could be specifically immunoprecipitated using antibodies directed against ␣ 2 M. Another experiment demonstrated that purified native ␣ 2 M (isolated by metalchelate chromatography) consistently contained detectable amounts of LCAT (Fig. 2B). LCAT became bound to ␣ 2 M in a concentration-dependent manner (Fig. 3). Such binding does not depend on the presence of lipid molecules and, for LCAT, is apparently noncovalent (Fig. 2C, right panel, lane a). LCAT may be recognizing a protein motif, such as an amphipathic ␣-helix, that is common to apolipoproteins and ␣ 2 M and is independent of lipid content. The association of LCAT with apolipoprotein complexes may thus be due to direct proteinprotein interaction, suggesting that LCAT binding and activation by apolipoprotein are independent events. It was documented that the association of LCAT to lipoprotein surfaces essentially was independent of their composition (31).
We have previously shown (7) that the binding of apoE to ␣ 2 M depends on the apoE phenotype. Charge or conformational differences in LCAT mutants might similarly affect their binding to ␣ 2 M species in plasma. Adimoolam et al. (32) have shown that two naturally occurring mutants of human LCAT, T123I and N228K, expressed in COS-1 cells, bind to a lipoprotein particle (rHDL) with about half the affinity of wild-type LCAT. Again, activated ␣ 2 M-MA binds two times as much LCAT as native ␣ 2 M (Fig. 3), and various cytokines and growth factors (e.g. TGF-␤1, NGF-␤, PDGF, bFGF, and TNF-␣) also bind to ␣ 2 M-MA with greater affinity than to native ␣ 2 M (12).
The binding studies shown in Fig. 3 predict that LCAT bound with low affinity to native ␣ 2 M may dissociate from the complex in the presence of LpA-I particles. We postulate that native discoidal HDL particles affect the availability of active LCAT, which is crucial for their maturation, by modulating its dynamic distribution between ␣-migrating HDL and ␣ 2 M. When 125 I-labeled rLCAT was incubated with ␣ 2 M in the presence of a 4-fold excess of r(LpA-I) over ␣ 2 M, the association of rLCAT with ␣ 2 M was almost completely inhibited. Actually, r(LpA-I) inhibited the binding of 125 I-rLCAT with ␣ 2 M more efficiently than did native HDL 3 (Fig. 5).
Studies by Adimoolam et al. have documented that normal LCAT dissociates from rHDL after one catalytic cycle (32). It is possible that the fraction of LCAT bound preferentially to ␣ 2 M reflects a catabolic compartment. We may say that an important proportion of HDL-sized LCAT (40%) is sequestered into in an inactive pool by ␣ 2 M, which does not react with cholesterol of lipoprotein origin (Fig. 6) and leads to the concept that plasma ␣ 2 M inhibits the enzymatic activity of LCAT. It is not possible to use a water-soluble substrate such as p-nitrophenylvalerate to provide evidence that the LCAT associated with ␣ 2 M is catalytically active; the latter being a substrate for serine proteases and esterases, it might also interact with chymotrypsine (30), which binds to native purified ␣ 2 M.
LCAT with low activity has been demonstrated in CSF (35). LCAT may be synthesized in the brain (36). Recently, Collet et al. (37) reported that cultured nerve cells secreted a functional LCAT protein. The present study shows that most of the LCAT in CSF was associated with ␣ 2 M (Fig. 1B). Although the functional significance of LCAT binding to ␣ 2 M in CSF remains to be determined, this complex may be involved in mediating the clearance of LCAT by the LRP receptor in the brain.
One can also speculate that native ␣ 2 M could be involved in the stabilization, the decreased plasma clearance, and the resistance of plasma LCAT to proteinase cleavage. Indeed, we have observed that significant amounts of rLCAT resisted trypsin digestion when bound to ␣ 2 M, whereas unbound rLCAT was readily degraded (data not shown).
The results presented in this study provide direct evidence that LRP is a receptor for the LCAT⅐␣ 2 M-MA complex (Figs. 7 and 8), thus indicating that it could mediate LCAT⅐␣ 2 M-MA binding, endocytosis, and degradation in any tissue that ex-  Fig. 1, LCAT was detected by anti-LCAT monoclonal antibody. Individually labeled membrane areas were excised and identified using the autoradiograph as template and their radioactivity were determined in a ␥-radiation spectrometer. Values given are means Ϯ S.D. of five different experiments. Radioactivity in the ␣-migrating HDL region is the sum of that in the multiple punctate reaction areas. B, ␣ 2 M-depleted plasma or IgG-depleted plasma (200 l) were tested for their capacity to esterify cholesterol. Cholesterol esterification was expressed as the difference in esterified form before and after incubation of plasma. C, LCAT activity of purified native commercial ␣ 2 M(C), ␣ 2 M-MA(C), purified ␣ 2 M(P) from fresh plasma, and IgGdepleted plasma HDL fraction isolated by FPLC (1 g LCAT protein) was measured. FER, fractional esterification rate. presses LRP. This work provides evidence for the first time that the LRP receptor may play an important role in the catabolism of LCAT.
Further study of the interaction between LCAT and ␣ 2 M may provide new insights into plasma factors affecting HDL particles remodeling, the mechanism of reverse cholesterol transport, and the clearance of LCAT via an ␣ 2 M/LRP receptor that is dependent on the conformation of ␣ 2 M in vivo.