Scavenger Receptor for Aldehyde-modified Proteins*

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THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 261, No. 11, Iesue of April 15, pp Printed in d.s.a. Scavenger Receptor for Aldehyde-modified
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THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 261, No. 11, Iesue of April 15, pp Printed in d.s.a. Scavenger Receptor for Aldehyde-modified Proteins* (Received for publication, December 3, 1985) Seikoh Horiuchi, Masaji Murakami, Kyoko Takata, and Yoshimasa Morino From the Department of Biochemistry, Kumamoto University Medical School, Honjo, 2-2-1, Kumamoto 860, Japan This paper describes an unexpectedly broad ligand least two distinct scavenger receptors seem to be present on specificity of a scavenger receptor of sinusoidaliver plasma membranes of sinusoidal liver cells (15-17) and pericells that is responsible for endocytic uptake of form- toneal macrophages (18, 19). However, the physiological role aldehyde-treated bovine serum albumin (f-alb). Bind- of the f-alb receptor is poorly understood largely due to the ing of 12SI-f-Alb to the isolated cells was effectively argument that the formation of the ligand is unlikely to occur inhibited by bovineserumalbumin (BSA)modified in vivo under physiological conditions. To elucidate its physwith aliphatic aldehydes such as glycolaldehye, DLglyceraldehyde, and propionaldehyde whereas albuiological function, knowledge on the molecular basis of the min preparations modified by aromatic aldehydes such ligand specificity of this receptor appears to be essential. as pyridoxal,pyridoxalphosphate,salicylaldehyde, In the present study we have addressed two questions of and benzaldehyde did not affect this binding process. whether or not the receptor would recognize albumin modified Binding of 1z61-glycolaldehyde-treated BSA to the cells by aldehydes other than formaldehyde, and whether or not exhibited a saturation kinetics with an apparent Kd = this phenomenon could be extended to any proteins other 3.3 pg of the Zigand/ml. This binding process was in- than bovine serum albumin (BSA). The results indicate that hibited by unlabeled f-alb as well as by the antibody the f-alb receptor, originally described as specific for albumin raised against the f-alb receptor. Indeed, 12BI-glycol- modified by formaldehyde, recognizes several proteins modialdehyde-treated BSA underwent a rapid plasma fied by aliphatic aldehydes as its ligand. Thus, this unexpectclearance (& - 2 min) which was markedly retarded edly broad ligand specificity of the receptor suggests its role by unlabeled f-alb. Upon treatment by these aldehydes, as a scavenger receptor for aldehyde-modified proteins. other proteins such as ovalbumin, soybean trypsin inhibitor, and hemoglobin were also converted to active MATERIALS AND METHODS ligands for the f-alb receptor, while no ligand activity was generated with 7-globulin and RNase A. These results clearly show that the f-alb receptor, originally described as being specific for f-alb, exhibits a broad ligand specificity in terms of both aldehydes and proteins and, hence, should be described as a scavenger receptor or aldehyde-modified proteins. Scavenger function of macrophages or macrophage-derived cells for chemically modified proteins has been known with formaldehyde-treated bovine serum albumin (f-alb ) ( 1-4), maleylated albumin (5,6), and malondialdehyde-modified (7) and acetylated low-density lipoprotein (5,8-10). These chemically modified proteins have a common biological feature of being endocytosed via receptor-mediated mechanism, when infused intravenously, by sinusoidal liver cells, major scavenger cells in vivo. Earlier studies from this laboratory have demonstrated the presence of a scavenger receptor for f-alb on the plasma membrane of sinusoidal liver cells (11, 12). Moreover, this receptor was found to be distinct (13) from the scavenger receptor claimed as being specific for the negatively charged proteins (5,14) such as acetylated, molondialdehyde-modified low-density lipoprotein and maleylatedalbumin. Thus, at * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked adue~tisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: f-alb, formaldehyde-treated BSA; BSA, bovine serum albumin; glycol-alb, glycolaldehyde-treated BSA; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. Chemicals-BSA (Fraction V) from Sigma was chromatographed on a Sephacryl S-200 column and the monomeric fraction was used as an albumin source for preparation of chemically modified ligands. RNase A, ovalbumin, soybean trypsin inhibitor, human hemoglobin A, and bovine y-globulin were purchased from Sigma. Formaldehyde, DL-glyceraldehyde, proprionaldehyde, benzaldehyde, dihydroxyacetone, and collagenase were purchased from Wako Chemical Co. (Osaka, Japan). Glycolaldehyde, dihydroxyacetone phosphate, pyridoxal 5 -phosphate, and salicylaldehyde were obtained from Sigma. Na =I (15.8 mci/pg of iodine) was from Amersham. All reagents used were of the best grade available from commercial sources. Preparation of Aldehyde-modified Proteins-The f-alb was prepared by a slight modification of the method reported previously (11). BSA was treated with 0.33 M formaldehyde at 37 C for 1 h. The modification of BSA by other aldehydes was performed as follows. The reaction mixture contained, in a total volume of 1 ml of 0.1 M sodium carbonate buffer (ph 10.0), 7.2 mg of BSA and either of mm glycolaldehyde, mm glyceraldehyde, 0.1 M acetaldehyde, 0.1 M propionaldehyde, 0.1 M dihydroxyacetone, 20 mm dihydroxyacetone phosphate, 0.1 M pyridoxal, 0.1 M pyridoxal 5 -phosphate, 0.1 M salicylaldehyde, or 0.1 M benzaldehyde. Each reaction mixture was incubated at 37 C for 5 h in the dark, followed by extensive dialysis against 20 mm sodium phosphate buffer (ph 7.4) containing 0.15 M NaCl at 4 C. Under the same conditions, soybean trypsin inhibitor (3.6 mg), hemoglobin (6.2 mg), ovalbumin (5.4 mg), y-globulin (12.5 mg), and RNase A (2.6 mg) were treated with glycolaldehyde, DLglyceraldehyde, or propionaldehyde. Under the identical conditions, these proteins were treated with glycolaldehyde, DL-glyceraldehyde, or propionaldehyde in the presence of mm NaBH4. After extensive dialysis, all these modified proteins were determined both for the extent of lysine modification and for their ligand activity by the binding assay described below. The extent of lysine modification by these aldehydes was determined according to the method of Habeeb (20) with trinitrobenzenesulfonic acid as the difference in lysyl residues of modified and unmodified protein preparations. The extent of lysine modified was expressed as percentage of modification. %f- Alb and 261-glycol-Alb was prepared as described previously (11) by iodination with %I to a specific radioactivity of 5400 and 3200 cpm/ 4962 Scavenger Receptor for Aldehyde-modified Proteins 4963 ng, respectively. The protein concentrations were determined by the method of Lowry et al. (21). Binding Assay-Sinusoidal liver cells were prepared from male Wistar rats ( g) as described previously (11) by a modification of the collagenase perfusion method (22). The isolated cells were suspended in Eagle's minimal essential medium containing 3% BSA buffered with 20 mm Hepes to ph 7.4 (buffer A). Binding assay was performed in a 1.6-ml polyethylene centrifuge tube (Eppendorf) as described previously (13). The reaction mixture contained, in a total volume of 0.1 ml of buffer A, 3 X lo6 cells and a fixed amount of '%Ilabeled ligand in the absence or presence of an unlabeled ligand to be tested. The reaction was initiated by the addition of lz5i-f-alb or lz5iglycol-alb and incubated on ice for 1 h with several intervals on a Vortex mixer. Upon termination of the reaction, 1 ml of ice-cold buffer A was added to each reaction tube, followed by centrifugation at 12,800 X g for 25 s at 4 C. The supernatant was discarded and the pelleted cells were resuspended in 1.0 ml of ice-cold buffer A. After the cells were washed twice more, the cell-associated radioactivity was measured as described previously (13). Unless otherwise specified, each value in the figures and table represents the mean value of duplicate assays. The Scatchard analysis was performed as described (12). Plasma Clearance-Effects of unlabeled glycol-alb and f-alb on the plasma clearance of 'z51-glycol-alb were performed as described previously (23). The injected solution contained 2 pg of '251-glycol- Alb (1800 cpm/ng) alone or with either 2.6 mg of unlabeled glycol- Alb or 3.6 mg of unlabeled f-alb. Each sample dissolved in buffer A (0.1 m1/ g of body weight) was injected intravenously via the femoral vein of a male Wistar rat ( ). Blood samples ( pl) were withdrawn at various times from the jugular vein into heparinized tubes, followed by centrifugation at 0 X g for 5 min at 4 C. Each plasma aliquot (30 pl) was measured for trichloroacetic acid-precipitable radioactivity. The amount of acid-precipitable radioactivity in plasma was expressed as percentage of the injected dose, assuming a plasma volume of3.13m1/ g body weight as described previously (23). Each plasma clearance curve represents a typical pattern obtained from three separate experiments. Anti-f-Alb Receptor Antibody-The antiserum was raised in rabbits against the f-alb receptor purified from rat liver as described previously (12). IgG fraction was prepared from both the antiserum and the preimmune serum by chromatography on a DEAE-Sephacel column as described previously (24). RESULTS Effect of BSA Modified with Glycolaldehyde and DL-Glyceraldehyde on f-alb Receptor-To test whether the f-alb receptor also recognizes BSA modified by aldehydes other than formaldehyde, effects of glycol-alb and DL-glyceraldehydetreated BSA on the f-alb receptor were examined. As Fig. 1 shows, glycol-alb inhibited effectively the binding of lz5i-f- Alb to sinusoidal liver cells: % inhibition was achieved at a concentration as low as 10 pg/ml of glycol-alb (Fig. 1B). Comparison with the data for f-alb (Fig. LA) clearly shows that glycol-alb serves as an excellent ligand for the f-alb receptor. Although less effective, BSA treated by DL-glyceraldehyde was also similarly recognized by the f-alb receptor (Fig. 1C). However, BSA treated by these aldehydes in the simultaneous presence of NaBH4, a reagent known to reduce the aldimine bond (Schiff base) formed between aldehyde and lysine residue(s) of the protein, failed to compete with 12'I-f- Alb for the f-alb receptor. Thus, it is likely that the reaction of these aldehydes with BSA via its peptidyl lysine(s) may lead to the generation of the ligand activity, and that the reduced form of aldimine may not be recognized by the f-alb receptor. Since previous studies (13, 25) have shown that the f-alb receptor binds polyanionic compounds such as dextran sulfate and poly(l-glutamic acid), a possible role of increase in negative net charges of BSA for the specific recognition by the receptor was examined. Unlike f-alb, modification of BSA with glycolaldehyde or glyceraldehyde resulted in only slight increase in negative net charges as judged from their electro- Glycolaldehyde 0 5Q Hx) 0 UnlabeledModified Albumin(pg/ml) FIG. 1. Effect of BSA modified by glycolaldehyde and DLglyceraldehyde on '2'I-f-Alb binding to sinusoidal liver cells. BSA was treated with formaldehyde, glycolaldehyde, or DL-glyceraldehyde in 0.1 M sodium carbonate buffer (ph 10.0) for 3 h at 37 C in the absence (0) or presence (0) of mm NaBb as described under Materials and Methods. After extensive dialysis, each sample was tested for the ligand activity by the binding assay. Each tube contained, in a total volume of 0.1 ml of buffer A, 3 X lo6 cells, 0.37 pg of '251-f-Alb(40 cpm/ng), and indicated amounts of unlabeled f- Alb (A), glycol-alb (B), or DL-glyceraldehyde-treated BSA (C). After incubation on ice for 1 h with several intervals on a Vortex mixer, the cell-associated radioactivity was determined as described under Materials and Methods. The extent of lysine modification of each competing ligand was as follows: f-alb, 46.2%; glycol-alb, 58.3%; and glyceraldehyde-treated BSA, 62.3%. phoretic mobility. However, acetylated BSA which was much more negative did not affect the binding of 1251-f-Alb to the cells (data not shown). Thus, the increase in negative net changes per se may not play a major role in the receptor recognition. Effect of BSA Modified by Other Aldehydes on the Binding of lz5i-f-alb to the f-alb Receptor-To further probe into the ligand specificity of the f-alb receptor, BSA derivatives prepared by treating with various aldehydes were examined for their effects on the binding of lz5i-f-alb to sinusoidal cells. Treatment with aliphatic aldehydes such as propionaldehyde, acetaldehyde, dihydroxyacetone, and dihydroxyacetone phosphate converted BSA to active ligands for the f-alb receptor (Table I). In sharp contrast, treatment with aromatic alde- hydes such as pyridoxal, pyridoxal phosphate, salicylaldehyde, and benzaldehyde failed to produce active ligands. With all of these aldehyde-treated samples, the extent of modification of lysyl residues was similar to or even higher than that by formaldehyde (Table I). These findings indicate that derivatives ofbsa modified by aliphatic aldehydes but not by aromatic aldehydes could be active ligands for the f-alb receptor. Binding of1251-glycol-alb to Sinusoidal Liver Cells-The binding of '251-glycol-Alb to sinusoidal cells reached an equilibrium within 80 min at 0 C. In the presence of -fold excess of unlabeled glycol-alb, the binding of '251-glycol-Alb was reduced by more than 90% (data not shown), indicating that the unlabeled glycol-alb and radioactive glycol-alb were competing for a limited number of common binding sites. The binding of '251-glycol-Alb to the cells at 0 C as a function of the concentration of 1251-glycol-Alb in the incubation medium exhibited a typical saturation curve (Fig. 2). The Scatchard analysis revealed a straight line indicating the involvement of a single binding mode with an apparent Kd = 3.3 pg of protein/ml and B, = 2.3 ng of the ligand/1o6 cells (Fig. 2, inset). The binding of '251-glycol-Alb to the cells was also effectively inhibited in a dose-related manner by unlabeled f- Alb (Fig. 3A). This finding, together with the data shown in Fig. lb, provides evidence that both f-alb and glycol-alb bind to a single site of the f-alb receptor. 4964 Scavenger Receptor for Aldehyde-modified Proteins TABLE I Effect of BSA preparations treated with various aldehydes on lz5i-f- Alb binding to sinusoidal liver cells Each assay contained, in a total volume of 0.1 ml of buffer A, 3 X 10 cells and 0.37 pg of lz5i-f-alb (40 cpm/ng) in the presence or absence of indicated amounts of albumin preparations treated with various aldehydes. After incubation on ice for 1 h, the amount of cellassociated radioactivity was determined as described under Materials and Methods. Nonspecific binding was determined by parallel incubations in the presence of 1.0 mg of unlabeled f-alb. The specific binding was determined by subtracting nonspecifk binding fram the total binding. Procedures for modification by these aldehydes was described under Materials and Methods. Each value represents the mean of duplicate assays. Figures in parentheses show the percentage of the control value for the specific binding. None f-alb Extent of Aldehydes lysine modification Ligand Total concentration binding binding Propionaldehyde Acetaldehyde Dihydroxyacetone Dihydroxyacetone phosphate Pyridoxal Pyridoxal phosphate Salicylaldehyde Benzaldehyde % d n l I I I I cpmlsystem () (10.4) (5.3) (12.6) (6.5) (18.5) (10.6) (15.7) (8.7) (22.8) (14.4) (98.0) (96.7) (98.6) (95.7) (96.5) (96.0) (95.3) (93.7) 1251-Glycol-Alb (pg1ml) FIG. 2. Binding of 2sI-glycol-Alb to sinusoidal liver cells as a function of its concentration. Each tube contained, in a final volume of 0.1 ml of buffer A, 3 X lo6 cells and the graded amounts of 1251-glycol-Alb (3200 cpm/ng). After incubation for 1 h at 0 C, the total binding (0) was determined as described under Materials and Methods. Nonspecific binding (0) was determined by parallel incubation in the presence of 200 fig of unlabeled glycol-alb. Specific binding (- - -) was obtained by subtracting the nonspecific binding from the total binding. The inset shows the Scatchard plot for the specific binding: F, free 251-glycol-Alb; B, bound 1251-glycol Alb. - s lea Unlabeled Liind IgG Concentration FIG. 3. A, effect of unlabeled f-alb on 251-glycol-Alb binding to sinusoidal cells. Each assay tube received 0.1 ml of buffer A containing 3 X lo6 cells, 0.31 pg of 12sI-glycol-Alb (3200 cpm/ng), and indicated amounts of unlabeled f-alb (0) or glycol-alb (0). The cell-associated radioactivity was determined as described under Materials and Methods. B, effect of anti-f-alb receptor antibody on 251-glycol-Alb binding to sinusoidal cells. Each tube received 0.1 ml of buffer A containing 3 X lo6 cells and increasing amounts of either anti-f-alb receptor IgG (0) or preimmune rabbit (0). After incubation for 20 min at 0 C, the binding reaction was initiated by adding 10 pl of 12sIglycol-Alb (3.1 pg/ml, 3200 cpm/ng), followed by incubation for 1 h at 0 C. The cell-associated radioactivity was determined as described under Materials and Methods. Based on the previous finding that the antibody raised against the f-alb receptor has a capacity to block specifically the binding of lz5i-f-alb to both sinusoidal cells (13) and their plasma membranes (12), we tested for the effect of the antireceptor antibody on the binding of 1251-glyco1-Alb to sinusoidal cells. As Fig. 3B shows, the binding of 1251-glycol-Alb was effectively blocked by treatment with the antibody whereas IgG purified from a preimmune serum had no effect on this binding process. This result lends further support to the contention that glycol-alb serves as an active ligand for the f-alb receptor. The conclusion drawn from the in vitro experiments was also supported by the following in vivo observation. Intravenous injection of a trace amount of 1251-glycol-Alb resulted in a rapid disappearance from the blood stream within a few minutes. The plasma clearance was significantly retarded by simultaneous injection of a loading amount of unlabeled f- Alb as well as unlabeled glycol-alb (Fig. 4). Reciprocally, the plasma clearance of lz5i-f-alb was also significantly retarded by the simultaneous injection of a loading amount of unlabeled glycol-alb in a manner similar to that observed above (data not shown). Ligand Activity of Other Proteins Treated with Aliphatic Aldehydes-Since no ligand activity was generated when the reductively methylated BSA was further treated with aliphatic aldehydes (data not shown), it is likely that BSA becomes an active ligand by reacting with aliphatic aldehydes via its lysyl residue(s). Then, one may ask a question whether or not any protein containing lysyl residues could be converted to an active ligand for the f-alb receptor when its peptidyl lysine(s) is modified by aliphatic aldehydes. This possibility was investigated by examining the effect of several proteins treated with glycolaldehyde, glyceraldehyde, and propionaldehyde on the binding of lz5i-f-alb to sinusoidal cells. To our surprise, ovalbumin, soybean trypsin inhibitor, and hemoglobin treated with these aldehydes were found to inhibit the binding of f-alb to the cells whereas y-globulin and RNase A similarly Time (min) FIG. 4. Effect of f-alb on plasma clearance of la'i-glycol- Alb. The samples containing 2 fig of '251-glycol-Alb (1800 cpm/ng) (O), 2 pg of 1251-glycol-Alb plus 2.6 mg of unlabeled glycol-alb (O), or 2 pg of '251-glycol-Alb plus 3.6 mg of f-alb (0) were prepared in buffer A. Each sample was injected (0.1 m1/ g of body weight) intravenously into
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