Functional Expression of the Chicken Low Density Lipoprotein Receptor-related Protein in a Mutant Chinese Hamster Ovary Cell Line Restores Toxicity of Pseudomonas Exotoxin A and Degradation of alpha 2-Macroglobulin*

Rita Kohen AvramogluDagger §, Johannes Nimpf, Roger S. McLeodDagger , Kerry W. S. KoDagger , Yuwei WangDagger , David FitzGeraldpar , and Zemin YaoDagger **

From the Dagger  Lipoprotein & Atherosclerosis Group, Departments of Pathology & Laboratory Medicine and Biochemistry, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4E9, Canada, the  Department of Molecular Genetics, Biocenter and University of Vienna, Vienna, Austria, and the par  Laboratory of Molecular Biology, NCI, Division of Cancer Biology, Diagnosis and Centers, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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The low density lipoprotein receptor-related protein (LRP) is responsible for the clearance of several physiological ligands including a complex of proteinase and alpha 2-macroglobulin (alpha 2M) and for the entrance of Pseudomonas exotoxin A (PEA) into cells. We have prepared expression plasmids for the full-length chicken LRP (designated LRP100) and two intermediates encoding 25 and 67% of the receptor (designated LRP25 and LRP67, respectively) using overlapping cDNA fragments. LRP25 and LRP67 encode the N-terminal 22 and 64%, respectively, of LRP100 plus the transmembrane and intracellular domains. Transient transfection of these plasmids into COS-7 cells yielded recombinant proteins of expected molecular mass and immunoreactivity. However, LRP100 was incompletely processed into alpha - (515-kDa) and beta - (85-kDa) chains and was poorly transported from the endoplasmic reticulum to the Golgi compartment. Stable transformants of LRP100, LRP67, and LRP25 were generated in a mutant Chinese hamster ovary cell line that lacked expression of endogenous LRP and was resistant to PEA. All forms of recombinant LRP proteins were transported from the endoplasmic reticulum to the Golgi apparatus in Chinese hamster ovary cells as shown by their sensitivity to endoglycosidase H and resistance to neuraminidase. Cell surface iodination and subcellular fractionation studies indicated that all three LRP variants were expressed on the plasma membrane. Furthermore, expression of the three LRP variants restored, to various degrees, sensitivity to PEA and the ability to degrade methylamine-activated alpha 2M (alpha 2M*). These data suggest that deletion of large internal portions of LRP, including the processing site, does not prevent transport of LRP to the plasma membrane, nor does it abolish the interaction of LRP with alpha 2M* or PEA. This LRP expression system may allow for the characterization of domains within LRP responsible for its multifunctionality.

    INTRODUCTION
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The low density lipoprotein receptor-related protein (LRP)1 of chicken somatic cells has been characterized (1). Comparison of amino acid sequences between the chicken LRP (4522 amino acids) and the human counterpart (4525 amino acids) (2) has revealed 83% sequence identity between the two receptors. LRP is translated as a single polypeptide chain (600 kDa) and subsequently processed into alpha - (515-kDa) and beta - (85-kDa) chains during its transport from the endoplasmic reticulum (ER) to the cell surface (3). Processing of LRP is catalyzed by the trans-Golgi endopeptidase furin (4) that recognizes the RXRR consensus sequence (RHRR in human LRP and RNRR in chicken LRP). The resulting alpha - and beta -chains are noncovalently associated on the cell surface (3). Intracellular transport of the nascent LRP polypeptide from the ER to the Golgi apparatus requires a 39-kDa chaperone called the receptor-associated protein (RAP) (5, 6). Recently, it has been suggested that RAP facilitates LRP folding in the ER and prevents premature association of physiological ligands concomitantly synthesized with the receptor (7, 8).

LRP is a type I membrane protein. Its extracellular alpha -chain contains 31 class A ligand-binding motifs, arranged into four clusters (I through IV), and 22 class B epidermal growth factor type repeats. The beta -chain contains a single membrane-spanning segment followed by two Asn-Pro-X-Tyr motifs at the carboxyl terminus (1, 2). Probably because of the structural similarities between chicken and human LRP, both receptors exhibit identical binding to a number of diverse ligands, including lipid-associated apolipoprotein E (apoE), activated alpha 2-macroglobulin (alpha 2M*), vitellogenin, and RAP (1, 9). The structural basis for the multifunctionality of ligand binding to LRP is not completely understood. Analyses of recombinant LRP minireceptors (7, 11) and proteolytic fragments (10) of human LRP have demonstrated that ligand binding activity is associated mainly with class A motifs in clusters II and IV. Conversely, clusters I and III displayed only weak ligand binding activity. Accumulating experimental evidence suggests that LRP plays an important role in hepatic clearance of chylomicron remnants, a process probably mediated by interplay between many factors including apoE (13, 14), hepatic lipase (15), lipoprotein lipase (16), and heparan sulfate proteoglycans (17), in addition to LRP. With the recognition of the possible link between apoE metabolism and the development of Alzheimer's disease, the potential role of LRP in the pathophysiology of the central nervous system has also been suggested (18).

LRP has also been suggested to serve as a receptor for Pseudomonas exotoxin A (PEA) (19, 21). PEA consists of three functional domains and exerts its toxicity by irreversibly inhibiting protein translation. Following internalization, PEA protein is cleaved by a cellular protease and translocated to the cytosol. The N-terminal domain of PEA mediates cell binding, while the central domain contains the translocating activity and acts as the substrate for proteolytic cleavage. The C-terminal domain of PEA possesses the enzymatic activity and catalyzes ADP-ribosylation of elongation factor 2, resulting in inhibition of protein synthesis and cell death (22, 23). The primary target of PEA is the liver (25, 26). A mutant Chinese hamster ovary (CHO) cell line has been isolated that lacks LRP and is resistant to the toxic effects of PEA (20).

In this study, we prepared expression plasmids for the full-length chicken LRP and two deletion variants using LRP cDNA fragments and expressed them in an LRP-deficient CHO cell line (LRP-null). We used the cytotoxicity of PEA to monitor the function of the recombinant receptor and found that expression of the full-length receptor in the LRP-null cell line restored its sensitivity to the toxin. Furthermore, the expressed chicken LRP also mediated the uptake and degradation of alpha 2M*. This expression system will facilitate investigations of the pathophysiology and cell biology of this enormous cell surface receptor.

    EXPERIMENTAL PROCEDURES
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Materials-- DNA restriction enzymes and endoglycosidase H (Endo H) were purchased from New England Biolabs. Neuraminidase was purchased from Boehringer Mannheim or New England Biolabs. All reagents for cell culture were purchased from Life Technologies, Inc. ProMixTM (a mix of [35S]methionine and [35S]cysteine; 1000 Ci/mmol), carrier-free Na125I, horseradish peroxidase-conjugated goat anti-rabbit IgG antibody, and the enhanced chemiluminescence (ECL) reagents for immunoblotting were obtained from Amersham Corp. Bicinchoninic acid protein assay reagent and D-Salt ExcelluloseTM columns were obtained from Pierce. An expression plasmid encoding human furin (pcDNA3hfurin) was a gift from N. Seidah (Clinical Research Institute of Montreal). The expression plasmid pcDNA3RAP that encodes human RAP (5) and a polyclonal antibody against human LRP (27) were gifts from G. Bu (Washington University, St. Louis, MO). Recombinant Pseudomonas exotoxin A was expressed in Escherichia coli and isolated from the periplasm according to procedures described previously (28).

Preparation of Expression Plasmids-- The expression plasmids pcLRP25, pcLRP67, and pcLRP100 were constructed by combining 12 chicken LRP cDNA fragments that span the total length of 15598 base pairs (Fig. 1A). Plasmids pcLRP25 and pcLRP67 encode the N-terminal 22 and 64%, of the full-length LRP, respectively, plus the transmembrane and intracellular domains. Inserts were cloned into the polylinker region of the pCMV5 vector (29) positioned between the cytomegalovirus promoter and enhancer sequences and the human growth hormone transcription termination and polyadenylation signals (Fig. 1B). A BamHI-EagI fragment (nucleotides 1229-4222), prepared from clones A5, E4, and N3 was ligated together with an EagI-BglII fragment (nucleotides 14524-15248, obtained from clone L-1) into the pCMV5 vector that had been digested with XbaI and BamHI to create pcLRP25. Next, an EagI-EagI fragment (nucleotides 4222-9865) was prepared from clones N1, Z19, and 18 and inserted into pcLRP25 that had been digested with EagI to produce pcLRP67. Finally, an XhoI-XhoI fragment that encoded the C-terminal portion of LRP (nucleotides 9112-15248) plus the hGH region of the pCMV5 vector was prepared by ligation of clones 28, N2, 2B1-2, 112, 2B4, and L-1 and inserted into pcLRP67 to create pcLRP100 encoding the full-length chicken LRP.

Cell Culture and Transfection-- COS-7 cells (100-mm) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Wild-type CHO-K1 and LRP-null CHO cell line (designated 13-5-1) were cultured in Ham's F-12 medium containing 10% fetal bovine serum. Transfection of pcLRP into COS-7 cells was achieved by calcium phosphate precipitation (30) using 10, 20, and 25 µg of pcLRP25, pcLRP67, and pcLRP100 plasmid DNA, respectively. In certain experiments, pcLRP100 (25 µg) was transfected together with plasmids encoding human furin (10 or 15 µg) or RAP (10 or 15 µg). Stable transfection into LRP-null CHO cells (100-mm) was achieved by cotransfection of 15 µg of pcLRP100, 10 µg of pcLRP25, or 20 µg of pcLRP67 with 0.15 µg of pSV2neo. Stable transformants were selected using 700 µM G418 and maintained with 500 µM G418.

Preparation of Cell Extract and Immunoblot Analysis-- Transfected cells were washed with phosphate-buffered saline (PBS), resuspended in buffer S (200 mM Tris-maleate, pH 6.0, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 µM leupeptin, and 1.4% Triton X-100) and incubated on ice for 30 min. Insoluble protein was removed by centrifugation (14,000 rpm, 40 min, 4 °C) in an Eppendorf microcentrifuge. The Triton X-100-soluble cell proteins were collected from the supernatant, incubated with an equal volume of buffer R (8 M urea, 2% SDS, 10% glycerol, 10 mM Tris-HCl, pH 8.3, and 5% beta -mercaptoethanol) at 70 °C for 15 min, and resolved by electrophoresis on 3-8% gradient polyacrylamide gels containing 0.1% SDS (SDS-PAGE). Proteins were electrophoretically transferred (6 h) onto nitrocellulose membranes for immunoblot analysis. Rabbit antiserum against the carboxyl-terminal 17 amino acids of the beta -chain of chicken LRP or the C-terminal 15 amino acids of the alpha -chain was used as primary antibody (1) for ECL detection.

Fractionation of Subcellular Membranes-- Transfected CHO cells (ten 100-mm dishes) were washed and harvested into PBS. Cells were collected by low speed centrifugation and resuspended in 2 ml of buffer A (20 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.1 mM PMSF, 0.1 mM leupeptin, 40 µg/ml acetyl-leucyl-leucyl-norleucinal, 10 kallikrein-inactivating units/ml aprotinin) and homogenized by 20 passes through a ball bearing homogenizer (H and Y Enterprise, Redwood City, CA). After centrifugation of the homogenate (16,000 × g, 20 min), the supernatant was subjected to another centrifugation (250,000 × g, 1.5 h) in a TLA100.4 rotor to isolate microsomal membranes. The 16,000 × g pellet was resuspended in 1 ml of buffer B (20 mM HEPES, pH 7.4, 1 mM EDTA, 0.1 mM PMSF, 0.1 mM leupeptin, 40 µg/ml acetyl-leucyl-leucyl-norleucinal, 10 kallikrein-inactivating units/ml aprotinin) using a glass homogenizer (10 strokes of a Teflon pestle), layered on a 1.12 M sucrose cushion, and subjected to centrifugation (100,000 × g, 1 h) in an SW41 rotor. The plasma membrane fraction was collected from the interface of the sucrose cushion and pelleted by centrifugation (30,000 × g, 30 min) in a TLA100.4 rotor. The microsomal and plasma membranes were resuspended in buffer B.

Endoglycosidase H or Neuraminidase Digestion of LRP Proteins-- For Endo H digestion, cell extracts (Triton X-100-soluble proteins) or subcellular membrane fractions (in buffer B) were mixed with SDS (final concentration of 0.5%) and beta -mercaptoethanol (final concentration of 1%) and heated to 100 °C for 10 min. The sample was then acidified by the addition of sodium citrate, pH 5.5, to a final concentration of 50 mM. Endo H (500 units) was then added, and the mixture was incubated at 37 °C for 5 h. For neuraminidase experiments, cell extract or membrane fraction was acidified by the addition of sodium citrate buffer, pH 6.2, to a final concentration of 50 mM. Neuraminidase (0.75 units) was added, and the mixture was incubated at 37 °C for 4 h. Both Endo H and neuraminidase reactions were stopped by the addition of 100 µl of buffer R and resolved by SDS-PAGE under reducing conditions. LRP was detected by immunoblotting as described above.

Pseudomonas Exotoxin A Cytotoxicity Assay-- Cells were seeded (1 × 105 cells/well) in 24-well dishes and allowed to adhere for 24 h. PEA was added to the medium at indicated concentrations, and cells were incubated for up to 18 h. The cells were washed with PBS (37 °C) and labeled with [35S]methionine/cysteine (200 µCi/ml) for 1 h in a methionine- and cysteine-free medium. After labeling, cells were washed with cold PBS and lysed with 250 µl of lysis buffer (1 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, 1% SDS, 1 mM dithiothreitol, 0.015% PMSF, 50 mM Tris-HCl, pH 8.0). Complete solubilization of cell protein was achieved by heating the samples at 75 °C for 30 min. Each lysate was then diluted 10-fold with water, and an aliquot was mixed with an equal volume of fetal bovine serum and spotted under gentle vacuum onto 23-mm nitrocellulose membranes (saturated with ice-cold 10% trichloroacetic acid) using a Millipore 1225 manifold system. Membranes were washed twice with ice-cold 10% trichloroacetic acid and once with water and then dried for liquid scintillation counting.

Cell Surface Iodination-- Transfected CHO cell monolayers (80-90% confluent in 60-mm dishes) were washed twice, and the cells were collected into 1 ml of PBS (pH 6.5). Na125I (300 µCi) and two IODO-BEADs (Pierce) were added, and the cell suspension was mixed by rocking for 15 min at room temperature. The cell suspension was separated from the IODO-BEAD, and the cells were pelleted by centrifugation (3000 × g, 2 min). Unincorporated radioiodine was removed by washing the cells three times with PBS. The cell pellet was resuspended in 160 µl of buffer I (200 mM Tris-maleate, pH 6.0, 2 mM CaCl2, 0.5 mM PMSF, 2.5 µM leupeptin) and were lysed with 40 µl of 7% Triton X-100 at 4 °C overnight. The lysate was then diluted to 0.5 ml with buffer I containing 1.4% Triton X-100 and precleared with protein A-agarose beads (40 µl of 50% suspension) for 2 h. After removing the protein A beads, an aliquot of the supernatant was incubated with anti-alpha -chain or anti-beta -chain antibody (overnight at 4 °C) to precipitate LRP. The immune complex was recovered with protein A-agarose beads, washed extensively with buffer I containing 1.4% Triton X-100, and eluted into buffer R. Following separation by SDS-PAGE, the radiolabeled LRP was visualized by autoradiography.

Preparation of RAP-GST Fusion Protein-- The Salmonella japonicum glutathione S-transferase (GST)/39-kDa expression plasmid containing human 39-kDa protein (RAP) cDNA was obtained from D. Strickland. The purification of GST/39-kDa protein from E. coli (DH5alpha ) and of the 39-kDa protein after thrombin cleavage were carried out essentially as described by Herz et al. (31).

Purification, Iodination, and Uptake/Degradation of alpha 2M*-- Human alpha 2M was purified from fresh plasma (obtained from the Blood Bank of the Ottawa Civic Hospital) by Zn2+-chelate affinity chromatography as described previously (32). Preparation of alpha 2M* was performed according to previously described procedures (12). The purified alpha 2M* (100 µg in 250 µl of PBS, pH 7.3) was iodinated with one IODO-BEAD and 1 mCi of Na125I for 15 min. Unincorporated 125I was removed by passing the reaction mixture over a D-Salt Excellulose column equilibrated with PBS containing 0.1% bovine serum albumin. 125I-alpha 2M* (8.9 × 103 cpm/ng) was collected in the void volume of the column effluent. LRP-null and LRP-transfected cells (confluent in 12-well dishes) were incubated with 125I-alpha 2M* (2.5 nM, 0.4 ml/well) in the presence or absence of RAP in F-12 medium containing 5 mM CaCl2 and 6 mg/ml bovine serum albumin for up to 4 h at 4 °C for alpha 2M* binding or 37 °C for alpha 2M* degradation. For degradation, the cells were then placed on ice, and the medium was collected into tubes containing ice-cold trichloroacetic acid (final concentration, 20%). After 30 min, trichloroacetic acid-insoluble material was pelleted by centrifugation, and the supernatant was removed for determination of trichloroacetic acid-soluble, non-iodide radioactivity as described previously (33). For alpha 2M* binding, the cells were washed twice with cold PBS containing 6 mg/ml bovine serum albumin and twice with cold PBS. The cells were then solubilized with 0.1 N NaOH, and radioactivity was quantified.

Protein Assays-- Proteins were determined by the bicinchoninic acid method (Pierce) according to the manufacturer's instructions.

    RESULTS
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Transient Expression of LRP in COS-7 Cells-- The expression plasmid encoding the full-length chicken LRP (LRP100) was prepared by combining 12 LRP cDNA fragments as indicated in Fig. 1A. Two intermediate plasmids, LRP25 and LRP67, representing ~25 and ~67%, respectively, of the full-length receptor, were also generated during the preparation of LRP100 (Fig. 1B). Fig. 2 shows immunoblot analysis of the LRPs treated with and without Endo H (Fig. 2A) or neuraminidase (Fig. 2B), and Fig. 2C represents the domain structures of LRP. LRP25 encodes the N-terminal 984 amino acids (including the 21-residue signal peptide, cluster I, and the first three class A repeats of cluster II) plus the C-terminal 125 amino acids (including the transmembrane domain and the intracellular domain) of LRP100. LRP67 contains the N-terminal 2865 amino acids (including clusters I and II and nine class A repeats of cluster III) plus the transmembrane and intracellular domains.


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Fig. 1.   Construction of chicken LRP expression plasmids. A, structures of the full-length (top line) and 12 fragments (bars) of the chicken cDNA spanning the total length of 15,598 base pairs. The translational start (ATG at position 1270 of the LRP cDNA) and stop (TAG at position 14899 of the LRP cDNA) sites are indicated. B, cloning strategy for assembly of the minigenes pcLRP25, pcLRP67, and pcLRP100. The cross-hatched portions of the bar denote coding regions derived from the corresponding LRP cDNA fragments. B2/B1 denotes the jointed sequences between BglII and BamHI sites. The numbers under the bars are based on the published cDNA sequence (1). CMV, cytomegalovirus promoter-enhancer sequences; hGH, human growth hormone transcription termination and polyadenylation signals; kb, kilobase pairs.


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Fig. 2.   Endoglycosidase H or neuraminidase digestion of LRP variants expressed in COS-7 cells. COS-7 cells were transfected with LRP25, LRP67, or LRP100, and cell extracts were prepared 48 h after transfection. A, the cell extracts were denatured followed by incubation with (+) or without (-) Endo H. B, the cell extracts were incubated with (+) or without (-) neuraminidase. Samples were resolved by SDS-PAGE under reducing conditions, and LRP proteins (indicated by downward arrows) were detected by immunoblotting using the anti-beta -chain antibody. CLM, chicken liver membrane extracts. C, schematic representation of the domain structures of the chicken LRP variants. The length of each variant (in amino acids) is indicated in parentheses. Filled rectangles, ligand-binding repeats; open ovals, epidermal growth factor type repeats; double vertical lines, transmembrane domain. The proteolytic processing site is indicated by the downward arrow, and the antibody epitopes are shown by filled arrowheads. Roman numerals denote positions of the four clusters of the postulated ligand-binding domains.

All three LRP variants exhibited the expected molecular mass in transfected COS-7 cells and reacted with a specific antibody raised against the C-terminal 17 amino acids of the chicken LRP beta -chain (Fig. 2, anti-beta chain). Recognition of the full-length recombinant LRP by the anti-beta -chain antibody (right two lanes in Fig. 2, A and B) and the absence of the 85-kDa subunit indicated that the precursor protein was not processed into alpha - and beta -chains. Processing was not expected for LRP25 or LRP67, since they do not contain the furin recognition site (Fig. 2C). However, the native LRP in the chicken liver membrane (cLM lanes in Fig. 2, A and B) and endogenous LRP in COS-7 cells (probed with an antibody against human LRP; data not shown) were fully processed into alpha - and beta -chains. Incomplete proteolytic cleavage of the recombinant LRP100 is most likely attributable to the impaired intracellular transport from the ER to the trans-Golgi network (where furin resides) based upon analysis of the carbohydrate moiety. Thus, while the native LRP in the chicken liver membrane was fully resistant to Endo H and sensitive to neuraminidase (left two lanes in Fig. 2, A and B), all three recombinant LRPs were sensitive to Endo H and resistant to neuraminidase. The high molecular mass species (~200 kDa) found in the LRP25-transfected cells (Fig. 2A) were probably self-associated dimer. Dimerization of LRP25 and LRP67 was also observed when PAGE was performed under nonreducing conditions (data not shown). We attempted to improve LRP processing by cotransfection of LRP100 with RAP (to enhance ER-to-Golgi transport) or furin. Although RAP and furin expression was increased, processing of LRP100 was not significantly improved (data not shown). In ligand blotting studies, the unprocessed full-length LRP100 and LRP67 expressed by COS cells could bind to Ca2+, vitellogenin, and RAP (data not shown). However, to demonstrate LRP function at the cellular level, we sought a cell culture system in which proteolytic processing and transport to the plasma membrane could be demonstrated.

Expression of LRP100 in CHO-K1 Cells-- We tested several different cell lines and found that CHO-K1 cells were a suitable host for functional expression of recombinant LRP. Stably transfected cells expressing LRP100 were generated in a mutant CHO-K1 cell line that lacked expression of endogenous LRP (Fig. 3, A and B, LRP-null). This LRP-null cell line was resistant to PEA toxicity and was unable to internalize activated alpha 2M (20). In LRP100-transfected cells, the majority of LRP protein was processed into alpha - and beta -chains that could be detected in both the microsomes (Fig. 3A) and plasma membrane (Fig. 3B). The unprocessed LRP100 was found in the microsome fraction but was not detectable in the plasma membrane (middle two lanes in Fig. 3, A and B), indicating that the majority of the surface-presented LRP is proteolytically processed. Surface iodination experiments with intact LRP100-transfected cells also demonstrated that the processed alpha -chain was the predominant form presented on the cell surface, although a small amount of unprocessed LRP was iodinated in these experiments (data not shown). As expected, the LRP alpha -chain was sensitive to neuraminidase (right two lanes in Fig. 3, A and B). The LRP beta -chain, like the native beta -chain (left two lanes in Fig. 2B), was also sensitive to neuraminidase (middle two lanes in Fig. 3, A and B), indicating that the LRP beta -chain is sialylated.


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Fig. 3.   Neuraminidase digestion of LRP100 in stably transfected mutant CHO (13-5-1) cells. Triton X-100-soluble extracts of microsomal (A) or plasma (B) membranes were prepared from the mutant 13-5-1 cells (LRP-null) and cells stably transfected with LRP100 (LRP100). Membrane samples were treated with neuraminidase as in Fig. 2, and LRP100 proteins (indicated by downward arrows) were detected by immunoblotting using anti-alpha -chain or anti-beta -chain antibody.

Functional Analysis of Recombinant LRP in Transfected Mutant CHO Cells-- We tested if expression of LRP100 in LRP-null cells would restore the toxicity of PEA. Preliminary time course experiments indicated that at 200 ng/ml, PEA exerted maximal inhibitory effect on protein synthesis after a 12-h incubation (data not shown). The effect of PEA dose was assessed in PEA toxicity assays using cells that had been treated with PEA for 18 h (Fig. 4A). In wild-type CHO-K1 cells, incorporation of [35S]methionine/cysteine into cell protein decreased with increasing PEA dose, while LRP-null cells were insensitive to the toxin. In LRP100-transfected cells, expression of the full-length LRP restored the toxicity of PEA to LRP-null cells. The PEA dose required to reduce protein synthesis to 50% of untreated cells (IC50) decreased from >500 ng/ml in LRP-null cells (19) to 50 ng/ml in the LRP100-transfected cells (Fig. 4A). The IC50 for wild-type CHO-K1 cells was approximately 25 ng/ml, similar to an earlier observation (19). When the apparent PEA toxicity observed in wild-type CHO-K1 cells and in LRP100-transfected cells was corrected for the number of receptor molecules on the surface (as determined by 125I-alpha 2M* binding at 4 °C), the chicken LRP gave a value that was two-thirds of the endogenous LRP (Table I, sixth column).


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Fig. 4.   Functional analysis of LRP100 in transfected 13-5-1 cells. A, restored sensitivity to Pseudomonas exotoxin A. Wild-type (CHO-K1), mutant 13-5-1 (LRP-null), and LRP100-transfected cells (LRP100) were plated at 105 cells/well in 24-well plates and cultured for 24 h. PEA was added to the medium, and the cells were cultured for an additional 18 h before metabolic labeling (1 h) with [35S]methionine/cysteine. Incorporation of [35S]methionine/cysteine into cell protein (trichloroacetic acid (TCA)-insoluble) was quantified by scintillation counting. The data (mean ± S.D., n = 3) are presented as the percentage of the 35S-labeled cell protein synthesized in the absence of PEA. B, degradation of alpha 2M*. Top, cells (~230 µg of protein/well) were incubated with 125I-alpha 2M* (5.8 × 103 cpm/ng, 20 µg/ml) at 37 °C, and the trichloroacetic acid-soluble, non-iodide radioactivity in the medium was determined at the indicated times. Bottom, the inhibitory effect of RAP on alpha 2M* degradation was determined by co-incubation of 125I-alpha 2M* with increasing concentrations of RAP for 4 h at 37 °C.

                              
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Table I
Degradation of 125I-alpha 2M* and PEA toxicity index in CHO cells transfected with wild-type or truncated mutant chicken LRP cDNAs
The wild-type CHO-K1 cell, mutant LRP-null cell (13-5-1) and stably transfected cells expressing LRP100 (P2B3), LRP67 (P2B4, P2A2, P5A3), or LRP25 (P7B3, P4B2, P6B4) were incubated with 125I-alpha 2M* (8.9 × 103 cpm/ng) for 2 h at 4 °C to determine surface binding or for 4 h at 37 °C to determine degradation. The same cells were incubated with PEA (200 ng/ml) for 18 h and labeled with [35S]methionine/cysteine for 1 h to determine protein synthesis.

Expression of LRP100 also restored the ability to bind, internalize, and degrade alpha 2M*. While LRP-null cells were unable to degrade 125I-alpha 2M*, LRP100-transfected cells released trichloroacetic acid-soluble, non-iodide radioactivity at a rate similar to the wild-type CHO-K1 cells (Fig. 4B, top). Measurement of the cell-associated radioactivity (at 37 °C) revealed that the failure of LRP-null cells to degrade 125I-alpha 2M* was attributable to their inability to bind or internalize the ligand (data not shown). Degradation of 125I-alpha 2M* by CHO-K1 or LRP100-transfected cells could be effectively prevented, in a dose-dependent manner, by RAP (Fig. 4B, bottom) or unlabeled alpha 2M* (data not shown). Analysis of the alpha 2M* binding and degradation data indicated that the ability of the chicken LRP to degrade alpha 2M* was equivalent (87%) to that of the endogenous receptor (Table I, fourth column).

Functional Analysis of Two Deletion LRP Variants-- We next tested whether LRP25 and LRP67 could function as receptors for PEA and alpha 2M*. In three LRP67-transfected cell lines, two bands representing the sialylated LRP67 (mature form) and the asialyl LRP67 proteins were observed (Fig. 5A). The mature LRP67 was resistant to Endo H digestion (Fig. 5B) and sensitive to neuraminidase digestion (Fig. 5C) and was the major species (in comparison with the asialyl form) in the plasma membrane fraction (Fig. 5, B and C, bottom). Cell surface presentation of the mature form of LRP67 was also demonstrated by surface iodination experiment (data not shown). In contrast, the asialyl form of LRP67 was found predominantly in the microsomal membrane fraction (Fig. 5, B and C, top) and was sensitive to Endo H digestion (Fig. 5B).


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Fig. 5.   Endoglycosidase H or neuraminidase digestion of LRP67 expressed in 13-5-1 cells. A, immunoblots of LRP67 stably expressed in transfected 13-5-1 cells. 5 µg of cell extract protein was loaded in each lane. B, Endo H digestion of LRP67 associated with microsomal (top) or plasma (bottom) membranes. C, neuraminidase digestion of LRP67. Glycosidase digestion experiments were performed as in Figs. 2A and 3, respectively.

Among the three LRP67-transfected cell lines, clone P2A2 (a high expressor) was as sensitive to PEA as CHO-K1 cells, whereas in clones P2B4 and P5A3 (low expressors) the toxicity of PEA was only partially restored when compared with LRP-null cells (Fig. 6A). The difference in toxicity of PEA between the cell lines is attributable to the differing levels of expression and cell surface presentation of LRP67 among the clones (Fig. 5, A-C). When the high expressor P2A2 and low expressor P2B4 were tested for their ability to degrade alpha 2M*, we found that both clones degraded 125I-alpha 2M* and that the ability to degrade 125I-alpha 2M* was correlated closely to the level of LRP67 expression (Fig. 6B, left). The LRP67-mediated 125I-alpha 2M* degradation could be prevented by RAP (Fig. 6B, right) and unlabeled alpha 2M* (data not shown). When the alpha 2M* degradation data were corrected for the differing level of expression, the LRP67-transfected cells gave results (64 and 124% in two clones) that were comparable with that of LRP100 (87%) (Table I, fourth column). Similarly, the sensitivity to PEA observed in LRP67-transfected cells (31 and 56% in two clones) was comparable with that in LRP100-transfected cells (66%) (Table I, sixth column). The low values observed for corrected alpha 2M* degradation and corrected PEA toxicity in clone P2A2 are attributable to the exceedingly high level expression of the variant (see 125I-alpha 2M* binding data).


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Fig. 6.   Functional analysis of LRP67 in transfected 13-5-1 cells. A, PEA toxicity assay, as described in Fig. 4A. B, degradation of alpha 2M* (left) and the effect of RAP on alpha 2M degradation (right), as described in Fig. 4B. TCA, trichloroacetic acid.

Similar analyses were performed with the LRP25-transfected cells. Three stable transformants that expressed different levels of LRP25 were analyzed (Fig. 7A). In each cell line, the mature form of LRP25 (sialylated) exhibited Endo H resistance (Fig. 7B) and neuraminidase sensitivity (Fig. 7C). In addition, LRP25 was expressed on the plasma membrane as determined by subcellular fractionation (Fig. 7, B and C) and cell surface iodination experiments (data not shown). Two of the LRP25 clones (P4B2 and P7B3) displayed modest sensitivity to PEA, whereas one clone (P6B4) remained resistant to the toxin (Fig. 8A). Expression of LRP25 also restored the ability to degrade 125I-alpha 2M*, and the extent of alpha 2M* degradation correlated with the level of LRP25 expression (Fig. 8B). The LRP25-mediated binding of 125I-alpha 2M* could be abolished by RAP (Fig. 8C). The efficiency of alpha 2M* degradation (~30% of normal) and the cytotoxicity of PEA (<10% of normal) were much lower in cells expressing LRP25 than in cells expressing LRP100 or LRP67 (Table I).


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Fig. 7.   Endoglycosidase H or neuraminidase digestion of LRP25 expressed in 13-5-1 cells. The experiments (A, B, and C) were performed in essentially the same manner as in Fig. 5, except that the LRP25-transfected cells were used.


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Fig. 8.   Functional analysis of LRP25 in transfected 13-5-1 cells. The experiments (A and B) were performed in essentially the same manner as in Fig. 6, except that LRP25-transfected cells were used. PEA toxicity assay (A) for clone P7B3 was repeated with similar results. C, effect of RAP on the cell surface binding of alpha 2M*. Cells were incubated with 125I-labeled alpha 2M* at 4 °C for 2 h in the presence of the indicated concentration of RAP, and the cell-associated radioactivity was determined. TCA, trichloroacetic acid.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this study, the full-length chicken LRP was stably expressed in a mutant CHO-K1 cell line that lacks endogenous LRP. Biochemical experiments, together with functional analyses, indicated that the recombinant LRP protein was glycosylated, proteolytically processed into alpha - and beta -chains, and presented on the cell surface as a functional receptor. Thus, we have provided conclusive experimental evidence using expressed recombinant protein that LRP indeed serves as a receptor for alpha 2M (34) as well as confirmed that it serves as a gate for receptor-mediated entrance of PEA into cells (18). In addition, analysis of two receptors with internal deletions has revealed that at least part of the sequence elements responsible for the binding of alpha 2M, RAP, and PEA may be located within the amino terminus of the receptor.

It has been proposed that LRP contains multiple binding sites for RAP. Existence of RAP-binding sites in clusters II and IV of LRP has been shown by studies using anchored minireceptors (11), soluble LRP fragments (35), or proteinase and CNBr digests of LRP (10). Using anchor-free, soluble LRP fragments that contained each of the four clusters of the class A ligand binding repeats, Bu and co-workers (7) have shown that RAP binds avidly to clusters II and IV and less avidly to cluster III but does not bind to cluster I. They have also shown that there are at least five independent RAP-binding sites within LRP (two in cluster II, one in cluster III, and two in cluster IV) and that RAP binding activity seems to be conferred primarily by the class A motifs (8). The current studies with the anchored minireceptor LRP25 (Fig. 8B) have provided new evidence that a RAP-binding site may reside within the first three class A repeats of cluster II.

The alpha 2M binding site has been assigned to cluster II of LRP, but the binding activity may not be conferred solely by the class A repeats. Studies with proteolytic fragments of LRP have suggested that some epidermal growth factor type repeats flanking cluster II of class A motifs may also contribute to binding of alpha 2M-proteinase complexes (10). A membrane-anchored minireceptor that contained all eight class A repeats of cluster II but not the neighboring fourth epidermal growth factor repeat did not show binding to alpha 2M (11). Our observations of binding and degradation of alpha 2M* by LRP25-transfected cells indicate that structural determinants essential for binding of alpha 2M* are encoded by the amino-terminal 22% of the LRP molecule, a region that also contains sequence determinants for binding of RAP.

This study is the first attempt to define sequence elements within LRP that are required for the entrance of PEA. Evidence that at low concentrations PEA might enter cells via LRP-mediated endocytosis includes (i) binding of PEA to LRP alpha -chain and inhibition of binding by RAP (19) and (ii) resistance to PEA toxicity of cells lacking LRP expression (20, 21). The LRP-null cell line (13-5-1) used in the present study was initially selected for its increased (100-fold) PEA resistance and inability to internalize the alpha 2M-proteinase complex (20). The current transfection experiments have confirmed that the receptor responsible for PEA entrance certainly is the receptor for alpha 2M, and they have also demonstrated that deletion of a significant portion of the LRP molecule including the processing site (in LRP67) does not impair the ability of PEA to exert its toxicity. It is noted that even in cells that express an extremely high level of LRP25 (Fig. 8A), the toxicity of PEA is only restored slightly (<10%). Therefore, the affinity of PEA for LRP may be a function of the receptor length. The LRP expression system established in the current study should be a suitable model to further define the retrograde trafficking of PEA in cells.

Among all members of the low density lipoprotein receptor gene family, LRP is unique in its post-translational processing into two subunits. Similar processing occurs in the insulin receptor. Defective processing of the insulin receptor is associated with impaired insulin binding (<10% of normal) in transfected mutant CHO cells (36) and in some insulin-resistant diabetes mellitus patients (37). However, the functional expression of the membrane-anchored LRP25 and LRP67 demonstrated in this study suggests that the proteolytic cleavage of LRP may not be essential for the receptor's function. Abolishing the LRP processing in the two deletion variants does not seem to adversely affect their post-translational modification (e.g. N-glycosylation and sialylation; although in the mutant cell lines screened there appear to be two glycosylated forms of LRP25 and LRP67), ER-to-Golgi transport, cell surface presentation, or endocytosis. Willnow et al. (11) have previously expressed a functional membrane-anchored minireceptor that contained cluster II plus the transmembrane and intracellular domains but lacked the processing site of LRP in ldlA7 cells. The minireceptor bound and degraded RAP and the tissue-type plasminogen activator/plasminogen activator inhibitor-1 complex (11). To date, the physiological significance of post-translational processing of LRP remains unknown.

An important finding of this study is that the chicken LRP cannot be expressed on the cell surface in COS-7 cells. Biochemical analysis of the carbohydrate moiety indicated that the recombinant LRP was retained within the ER and was not processed into alpha - and beta -chains, suggesting that the transport of the chicken receptor to the distal Golgi was impaired. Previous transfection studies of human low density lipoprotein receptor showed that the recombinant receptors could be functionally expressed in COS cells (38, 39). The inability of COS cells to transport recombinant LRP onto the cell surface may be attributable to the failure of the cellular trafficking machinery to correctly recognize the chicken receptor, since endogenous LRP was shown to be fully processed into alpha - and beta -chains and was presented on the cell surface. Since the molecular chaperone RAP is essential for LRP folding and also to prevent premature binding of the receptor to its ligands (7, 8), we have considered the possibility that RAP might be limiting in COS-7 cells. However, co-expression of human RAP with LRP did not improve trafficking of the receptor. It is unlikely that the lack of an effect of the human RAP expression on LRP trafficking is the result of its inability to react with the chicken LRP, since our in vitro studies demonstrated clearly that the human RAP can effectively abolish uptake and degradation of alpha 2M. Currently, the reason for the inability of COS-7 cells to transport recombinant LRP onto the cell surface is unexplained.

In summary, we have established an expression system for LRP, a multifunctional cell surface receptor involved in the catabolism of proteinases and lipid-associated proteins. The availability of an in vitro LRP expression system will assist in the identification of structural determinants that are responsible for the multiligand binding activity of LRP and will also facilitate investigations of the involvement of LRP in the development of premature atherosclerosis and Alzheimer's disease.

    ACKNOWLEDGEMENTS

We thank N. Seidah for the furin expression plasmid; G. Bu for the RAP expression plasmid and anti-LRP and anti-RAP antibodies; and D. Strickland for the GST/RAP expression plasmid. Work in J. N. laboratory is supported by the Austrian Science Foundation (Grant F-0606).

    FOOTNOTES

* This work was supported by Medical Research Council of Canada Grant MT-12931.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a studentship from the Medical Research Council of Canada.

** Research Scholar of the Heart & Stroke Foundation of Canada. To whom correspondence should be addressed. Tel.: 613-798-5555 (ext. 8711); Fax: 613-761-5281; E-mail: zyao{at}heartinst.on.ca.

1 The abbreviations used are: LRP, low density lipoprotein receptor-related protein; ER, endoplasmic reticulum; RAP, receptor-associated protein; apo, apolipoprotein; alpha 2M, alpha 2-macroglobulin; CHO, Chinese hamster ovary; PEA, Pseudomonas exotoxin A; alpha 2M*, methylamine-activated alpha 2M; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; Endo H, endoglycosidase H; GST, glutathione S-transferase.

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Abstract
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Results
Discussion
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