Haptoglobin biosynthesis in rats Immunological identification of polysomes synthesizing haptoglobin and quantitation of haptoglobin in the cytoplasm of liver cells

Biochimica et Biophysica Acta, 696 (1981) 107-113 Elsevier Biomedical Press

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BBA 91005

HAPTOGLOBIN BIOSYNTHESIS IN RATS IMMUNOLOGICAL IDENTIFICATION OF POLYSOMES SYNTHESIZING I-IAPTOGLOBIN AND QUANTITATION OF HAPTOGLOBIN IN THE CYTOPLASM OF LIVER CELLS F.A. BAGLIA *, S.-W. KWAN and G.M. FULLER

Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550 (U.S,A.) (Received September 30th, 1981)

Key words: Haptoglobin biosynthesis; ELISA; Polysome; Inflammatory response; Plasma glycoprotein; Protein synthesis

A quantitative enzyme-linked immunosorbent assay was developed and utilized to study the stimulation of haptoglobin biosynthesis during an acute inflammatory challenge. A 10-fold increase in intracellular haptoglobin was measured at the peak of the inflammatory response. The increase in serum haptogiobin levels was concomitant with the intracellular levels, demonstrating the secretory output is also elevated during the inflammatory period. A monospecific antihaptoglobin was produced and used to detect the specific polysomes involved in haptoglobin synthesis. The amount of radioactively labeled antibody bound to the nascent haptoglobin chain was increased approx. 3-fold during the inflammatory response, indicating that new haptoglobin was being synthesized and suggesting an increase in functional haptoglobin mRNA resulting from the inflammatory signal.

Introduction Haptoglobin is a ptasma glycoprotein that binds free hemoglobin. The haptoglobin molecule has a mass of about 86000 and is composed of two pairs of non-identical polypeptide chains (a/3) [1]. Several lines of evidence suggest that the liver is the primary site of haptoglobin synthesis [2,3], although other organs may be involved [4]. The complete amino acid sequence of the human haptoglobin molecule has now been reported [5]. It is well documented that during an inflammatory challenge the plasma level of haptoglobin is elevated 3-fold [6]. These findings have categorized * Present address: Wistar Institute of Anatomy and Biology, 36th and Spruce Street, Philadelphia, PA 19104, U.S.A. Abbreviation: ELISA, Enzyme-linked immunosorbent assay. 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

this protein as a member of a class of hepaticaUy derived glycoproteins known as the 'acute-phase' proteins. The use of antibodies to identify polysomes synthesizing a specific protein has been reported [7]. If polysomes synthesizing a protein are identified and isolated, this should facilitate the isolation and characterization of the mRNAs coding for a specific protein. In this report, we have used monospecific ~25Ilabeled antibodies directed against haptoglobin to identify polysomes involved in the synthesis of this glycoprotein. Moreover, in order to understand the processes of haptoglobin synthesis and secretion it was important to be able to detect small amounts of this molecule within the soluble fraction of liver cells. A quantitative assay, enzyme-linked ira-

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munoabsorbent, was utilized to determine the amount of haptoglobin in liver cells before and after an inflammatory challenge. Materials and Methods

Chemicals. EDTA, ~-aminocaproic acid, aprotinin (trasylol), DEAE-cellulose, Sephadex G100, sodium deoxycholate, Triton X-100, Tween 20, sodium acetate, sodium heparin, cycloheximide and glycine were from Sigma Chemical Co. Nal:5l (3 mCi/mmol) was purchased from New England Nuclear. Ammonium sulfate (enzyme grade) and sucrose (ribonuclease free) were obtained from Schwarz-Mann Chemical Co., acrylamide and bisacrylamide from Eastman Kodak Corp. Turpentine was a product of Rosen Factories of Texas. Sepharose 4B was from Pharmacia and Affigel Blue was obtained from Bio-Rad. Animals. Rats (Sprague-Dawley) weighing approx. 150-200g were purchased from Texas Inbred Mice Co. Rats were injected with 1.0 ml turpentine subdermally on the dorsal surface in order to cause an acute inflammatory response and thus to increase haptoglobin synthesis [6]. Control animals were injected subdermally with 1.0 ml sterile saline. A goat was injected intramuscularly with a total of 9.0 m rat haptoglobin (3 mg purified haptoglobin at weekly intervals for 3 weeks) in order to produce antiserum to the rat protein. Isolation of haptoglobin. Haptoglobin was purified from the plasma of 24-h-turpentine-stimulated rats by a modification of an established procedure [9]. Each rat was anesthetized with diethyl ester. The abdominal cavity was opened and the animal was quickly exsanguinated through the dorsal aorta. The blood was drawn into a syringe containing 0.15 M NaC1/1.2% EDTA, 12 mM caminocaproic acid and 2 units/ml aprotinin. The blood was centrifuged at 10000 rev./min for 10 min at 5°C and the plasma was carefully removed. Haptoglobin was initially precipitated from plasma by adding (NH4)zSO4 to final concentration of 55% (w/v). The precipitate was collected by centrifugation (15000 × g) at 4°C for 15 rain. The pellet containing haptoglobin was redissolved into 0.01 M sodium acetate, pH 5.1, and dialyzed against three changes of the buffer. This preparation was chromatographed on an ion-exchange

column (DEAE-cellulose) according to the meth. ods described by Lombart et al. [9]. In some instances haptoglobin was shown to bind to Affigel-Blue resin. The haptoglobin bound to the resin at pH 5.1 in" 0.08M sodium acetate buffer. Haptoglobin was specifically eluted when the resin was washed with 0.02 M sodium phosphate buffer (pH 7.0)/0.25 M NaC1. Haptoglobin fractions were pooled, dialyzed exhaustively against 0.01 M ammonium bicarbonate, lyophilized and stored at -20°C. Polyacrylamide gel electrophoresis. Routinely, 6% polyacrylamide gels were used to evaluate the purity of haptoglobin. To demonstrate the presence of haptoglobin, hemoglobin was added to sample and this mixture was layered on the polyacrylamide gel. A Tris-glycine (pH 8.6) buffer system was utilized as a running buffer and a gel buffer. Antibody purification. Monospecific antibodies to rat haptoglobin were prepared by passing the goat antiserum through a Sepharose 4B affinity column to which purified rat haptoglobin had been eovalently bound. Goat anti-rat haptoglobin was eluted with glycine-HC1 (pH 2.8) according to an established procedure [10]. Immunoelectrophoretic analysis of the specifically eluted antibody produced a single cross-reactive arc only to haptoglobin. Iodination of antibodies. The monospecific haptoglobin antibodies were radioactively labeled with Na125I according to a reported procedure [7]. The reaction was carried out at room temperature for 30 min and terminated by the addition of cold saturated (NH 4)2SO4. The pellet was resuspended in phosphate buffer (10 mM sodium phosphate/15 mM NaC1, p H 7.2) and dialyzed overnight. Since the radiolabeled antibody was to be used in experiments involving polysomes, the step separating bound vs. free iodine could be combined with making the antibody free of ribonuclease. This was done by molecular sieving on a Sephadex G-100 column equilibrated in the same buffer. The iodinated proteins were ribonuclease free as determined by the integrity of the polysomes in which they were incubated.

Polysomepreparation and sedimentation analysis. Liver polysomes containing both free and membrane-bound polysomes were isolated from normal

109 and 24-h-turpentine-stimulated rats according to a standard procedure [11]. Polysomes from 15-day chick embryo brains were prepared by the same procedure as used to isolate liver polysomes except that heparin (100/~g/ml) and cycloheximide 920 /xg/ml) were added to the polysome buffer. Binding studies. In order to localize haptoglobin-synthesizing polysomes, 5A260 units of total polysomes were mixed with 5/~g 125I-labeled antihaptoglobin at 0°C for 30 min. The labeled antibody polysome mixture was centrifuged through a 17-52% continuous linear sucrose gradient with a 1 ml cushion of 61% sucrose at the bottom and overlaid with 0.5 ml 8% sucrose. Centrifugation was for 90 in at 39000 rev./min at 5°C in a SW-40 rotor. Polysome profiles were obtained by pumping the gradient from the bottom through a LKB Uvicord. Fractions were collected and counted in a gamma counter.

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monospecific antibody to rat haptoglobin was utilized to establish a quantitative immunological assay for haptoglobin. The method used in developing the assay has previously been described in detail using rat fibrinogen [8]. The sensitivity of the assay is such that haptoglobin levels as low as 20 n g / m l could be detected. Postmicrosomal supernatant and plasma samples were analyzed for haptoglobin content using this sensitive and specific assay. Preparation of postmicrosomal supernatants. The soluble fraction of liver ceils or postmicrosomal supernatants (S3) of rat liver was prepared according to procedures described by Kwan and Webb [12]. To the postmitochondrial supernatant ($2) was added Triton X-100 and deoxycholate to a final concentration 1%. The postmitochondrial supernatant was centrifuged at 12000 × g for 50 min and the postmicrosomal supernatant was the soluble fraction. The fractions were diluted 1 : 100 with phosphate-buffered saline/Tween 20 and were stored at 5°C until analyzed for haptoglobin content. Total protein of the postmicrosomal supernatant was determined by the procedure of Lowry et al. [13]. Results

Haptoglobin purity and electrophoretic analysis The purification of rat haptoglobin by affinity

chromatography using the Affigel-Blue procedure is shown in Fig. 1. It should be pointed out that the ion-exchange procedure described by Lombart [9] was also employed in the studies. Peak II of the chromatogram shown in Fig. 1 is the purified haptoglobin (Hp). The results from Fig. 1 gave an apparently pure protein (second peak) when analyzed on polyacrylamide gels. Electrophoretic analysis of peak II in alkaline gels revealed a single band (Fig. 2) that when incubated with hemoglobin formed a complex of slower migration representing the Hp-Hb complex. In addition, the Hp-Hb complex exhibited a positive reaction when stained with benzidine, indicating its peroxidative activity.

Specificity of antibodies Antibodies to rat haptoglobin purified by immunosorbent chromatography were tested by immunoelectrophoresis as well as crossimmunoelectrophoresis. A n t i - r a t haptoglobin exhibited single cross-reactivity with rat plasma, demonstrating the antibodies' monospecificity.

Plasma haptoglobin levels and haptoglobin biosynthesis 24 h after a subdermal injection of turpentine in rats, plasma samples were taken and the concentration of haptoglobin determined using the

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Fig. 1. Affinitychromatographyof a DEAE-cellulosefraction of rat haptoglobin on an Affigel-Blueresin. The haptoglobin binds to the resin at pH 5.1 in 0.08 M sodium acetate buffer. Haptglobinis specificallyeluted, when the resin is washedwith 0.02 M sodium phosphate buffer at pH 7.0 containing0.25 M NaC1.

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Fig. 2. Polyacrylamide (6%) gel electrophoresis of rat haptoglobin (Hp) (lane 1); rat haptoglobin complexes with hemoglobin (HpHb) (lane 2); and hemoglobin (Hb) (lane 3). A gel buffer of 0.1 M Tris-glycine, p H 8.7, was used,

enzyme-linked immunoassay procedure. An approx. 3-fold increase in the plasma concentration of hapt0globin was observed after a 24-h stimulation with turpentine (Fig. 3). Plasma levels of haptoglobin returned to control levels (approx. 1.2 mg/ml) 6 days (144h) after stimulation. Approx. 8 hours after turpentine stimulation there is a significant drop in the plasma concentration of haptoglobin. This has been observed for other acute-phase globulins [5]. While there is no direct information on the cause of this decrease, it seems reasonable that some hemolysis at the site of inflammation could cause a decrease in the level of free haptoglobin by first binding to hemoglobin and then removing through the Hp-Hb clearance mechanism.

The effect of turpentine on the intracellular content of haptoglobm The effect of turpentine on the quantity of haptoglobin within the soluble fraction of liver cells was investigated. Postmicrosomal supernatants of rat liver were prepared as described

above. The total intracellular level of haptoglobin before and after turpentine treatment was determined using the ELISA method. The haptoglobin content per mg of total postmicrosomal supernatant is shown in Fig. 3. A stimulated level 10-times that of control was obtained 28 h after turpentine treatment. At this peak period of the inflammatory reaction the amount of haptoglobin within the hepatocyte represents 1% of the total cytosol protein. A rapid drop in the level of haptoglobin is seen thereafter, with near normal levels by 48 h. The amount of haptoglobin in the unstimulated animal is 1/~g/ml hepatic protein, thus comprising 0.1% of the soluble protein. Data shown in Fig. 3 also relate the plasma level of haptoglobin and the hepatic intracellular content of haptoglobin at various times after the injection of turpentine. The rise in the hepatic concentration and plasma level of haptoglobin occurs concomitantly, indicating that the transport of this glycoprotein out of the cell is rapid.

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Binding of 12SI-labeled anti-haptoglobin to rat fiver polysomes Several different studies have shown that specific antibodies can be used to bind nascent polypeptides [7,11]. This procedure permits one to identify and quantitate those polyribosomes involved in the synthesis of specific proteins. In order to identify and gain information concerning the polyribosomes involved in the synthesis of haptoglobin, several experiments were performed using monospecific anti-haptoglobin. A significant amount of antibody binding to polysomes of both normal and stimulated rats was observed. Polysomes derived from chick brain were also incubated with I25I-labeled anti-haptoglobin from Fig. 4, middle panel. This control demonstrates that nonspecific antibody binding to polysomes is insignificant. The radioactivity fails into the mid-range of polysomes, suggesting that the nascent chains are within a size range that could account for a protein of about 35000-50000 daltons [11]. The radioactivity at the top of the gradient represents unbound labeled antibody. In the turpentine-stimulated rat, a substantial increase in antibody binding to polysomes occurred when compared to the normal control

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Specificity of s25I-labeled antibody binding The binding characteristics with six different quantities of 12SI-labeled anti-haptoglobin (0.5-35 #g) are shown in Fig. 5. When a constant amount of polysomes from turpentine-stimulated rats (5 A26o) were incubated with increasing amounts of labeled antibody there was an increase in the amount of antibody bound. The same quantitative pattern distribution of radioactivity is seen along the profile with different concentrations of 125Ilabeled anti-haptoglobin. This essential control experiment demonstrates the antibody is in fact consistently binding to the nascent haptogiobin polypeptide and is not a result of antibody entrapment in the polysomal structures.

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Discussion

The purification of rat haptoglobin has been achieved by two procedures. In the previous studies at least three different chromatographic steps were necessary to obtain electrophoretically pure haptoglobin. The Affigel-Blue procedure simplifies the purification procedure [9]. It is well established that during an acute inflammation the circulating level of haptoglobin, as well as of other proteins, is significantly elevated [5,11]. In order to understand the synthesis and secretion of haptoglobin at the cellular level, the quantity of haptoglobin within the soluble fraction of liver cells and plasma was determined during an inflammatory challenge. The results presented here indicate that about 10 h after turpentine injections into rats the hepatic synthesis of haptoglobin is stimulated (Fig. 3). The intracellular content of haptoglobin reaches a peak at 20h, indicating maximal synthesis of haptoglobin within the liver cell. However, approx. 20 h later the intracellular

content of haptoglobin returns to control levels. This suggests that the hepatic synthesis ot haptoglobin is stimulated for a short time -enough, however, to increase significantly the plasma level of haptoglobin. Furthermore, the rises in the hepatic concentration and in the plasma level of haptoglobin occur concomitantly, indicating that transport of this glycoprotein from the cell is rapid. The results are somewhat disparate from previously reported data [5]. In this latter study, immunofluorescence of liver slices was used to detect haptoglobin synthesis in turpentine-treated rats. Maximal stimulation of haptoglobin biosynthesis in the liver cell was determined to occur 4 h after the injection of turpentine. The intensity of the immunofluorescence of liver slices was used as a criterion to judge the degree to which the liver cell was engaged in haptoglobin biosynthesis. Unfortunately, immunofluorescence is at best semiquantitative and subject to some interpretation. The increase in the intracellular level of haptoglobin within the liver cell during the acutephase condition is obviously due to enhanced biosynthesis, as shown here as well as in other studies [5]. The maximum circulating level of plasma haptoglobin is attained 24-72 h following turpentine stimulation. Total polysomes were prepared from rat liver 24h after the injection of 1.0 ml turpentine, since maximum haptoglobin biosynthesis was observed at this time. When polysomes from normal vs. turpentine-stimulated rats were compared, a substantial increase in the synthesis of haptoglobin was observed. The increase in the quantity of haptoglobin-synthesizing polysomes reflects a greater availability of functional m R N A for this glycoprotein. This increase in mRNA may come about by either a decrease in m R N A degradation or an increase in the number of functional mRNAs. In either case, enhanced haptoglobin synthesis was apparently brought about by translation of more haptoglobin mRNA molecules. The relative position of the radioactive peak in the polysomal complex is of some interest. It has been suggested that the alpha chain and beta chain could be synthesized from a single mRNA species [14-16]. If this were true then a single radioactive peak in the region of a 42000-dalton protein would

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be expected, which is consistent with the data reported here. More recent and direct data have shown that the alpha and beta chains are synthesized as a single propolypeptide [17]. Acknowledgments This work was supported in part by grants HL 16445 and HL 01662 (G.M.F.) and an NIH Training Grant DHEW 5T 32GM 07204 (F.A.B.).

References 1 Putnam, F.W. (1973) Haptoglobin in the Plasma Proteins, Vol. 21-50, 2nd edn., Academic Press, New York 2 Kauss, S. and Sarcione, E.I. (1964) Blood 26, 705-719 3 Mouray, H., Moretti, J. and Jayle, M.F. (1964) C.R. Acad. Sci. (Paris) 258, 5095-5098 4 Wada, T., O'Hara, H., Watanabe, K., Kinoshita, H. and Nishio, H. (1970) J. Reticuloendothel. Soc. 8, 195-197 5 Kurosky, A., Barnett, D.R., Lee, T.H., Touchstone, B., Hay, R.E., Arnott, M.S., Bowman, B.H. and Fitch, W.M. (1980) Proc. Natl. Acad. Sci. USA 77, 3388-3_392 6 Lombart, C., Nebut, M., Oilier, M.P., Jayle, M.F. and Hartmann, I. (1968) Rev. Franc. Etudes Clin. Biol. 13, 258-266

7 Palacios, R., Palmiter, R.D. and Schimke, R.T. (1972) J. Biochem. 247, 2316-2321 8 Kwan, F., Fuller, G.M., Krauter, M.A., Van Bavel, J.H. and Goldbloem, R.M. (1977) J. Anal. Biochem. 83, 589-596 9 Lombart, C., Moretti, J. and Jayle, M.F. (1965) Biochim. Biophys. Acta 97, 262-269 10 Cooper, T.G. (1977) The Tools of Biochemistry, p. 421, John Wiley, New York 11 Bouma, H., Kwan, S.-W. and Fuller, G.M. (1975) Biochem. 14, 4787-4792 12 Kwan, S.-W. and Webb, T.E. (1967) J. Biol. Chem. 242, 5542-5548 13 Lowry, O.H., Rosebrough, H,J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 14 Barnett, D.R., Kurosky, A., Fuller, G.M., Han-Hwa, K., Rasco, M.A. and Bowman, B.H. (1975) in The Protides of the Biological Fluids, 22nd Colloquium (Peeters, H., ed), pp. 589-595, Pergamon Press, Oxford 15 Doolittle, R.F. (1979) in The Proteins (Neurath, H. and Doolittle, R.F., eds.), 3rd edn., vol. 4, pp. l-118, Academic Press, New York 16 Kurosky, A. (1980) in The Protides of the Biological Fluids (Peeters, H., ed.), vol. 28, pp. 99-102, Pergamon Press, Oxford 17 Haugen, T.H., Hanley, J.M. and Heath, E.C. 91981) J. Biol. Chem. 256, 1055-1057