21. Baenziger, J. & Maynard, Y. (1980)J. BioL Chem. 255, 4607-4613. 22. Baenziger, J. & Flete, D. (1980) Cell 22, 611-620. 23. Weigel, P. H., Schnaar, R. L., ...
Proc. Nati Acad. Sci. USA
Vol. 79, pp. 6229-6231, October 1982 Biochemistry
IgA interaction with the asialoglycoprotein receptor (hepatic lectin/erythroagglutination/carbohydrate specificity/circulating asialoglycoproteins/cirrhosis)
RICHARD J. STOCKERT*, MICHAEL S. KRESSNER*, JANNA C. COLLINSt, IRMIN STERNLIEB*, AND ANATOL G. MORELL* *Division of Genetic Medicine and the Liver Research Center, Department of Medicine, and the tDepartment of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461
Communicated by Alex B. Novikoff, July 23, 1982
ABSTRACT- IgA present in normal human serum reacts with the hepatic receptor specific for asialoglycoproteins as demonstrated by'inhibition of receptor-mediated erythroagglutination. Inhibition is reversibly abolished by the oxidation of the galactose or N-acetylgalactosamine residues of IgA with galactose oxidase. The site of receptor recognition appears to be the O-glycosidically linked oligosaccharides present on the hinge region of the IgAl subtype of IgA. The demonstration of a specific binding, in vitro, of IgA by the hepatic receptor suggests that the uptake of polymeric IgA by the liver in vivo may be mediated by this reaction.
An asialoglycoprotein receptor (hepatic binding protein, HBP) mediates the endocytosis of desialylated plasma glycoproteins by the parenchymal cells ofthe liver in vivo (1, 2). The first step in this process is the binding of a plasma glycoprotein to HBP. The binding capacity of HBP for various desialylated glycoproteins can be determined quantitatively in vitro (3, 4), and assays have revealed the presence of binding inhibitors in the serum of patients with cirrhosis or hepatitis (5-7). These inhibitors have been identified as a heterogeneous population of desialylated glycoproteins (5, 6). Because the affinity of HBP for different desialylated glycoproteins varies over several orders of magnitude, we suspect that ligands with low affinities may have remained undetected. Therefore we have developed and applied a more sensitive erythroagglutination assay for inhibitors based on HBP's lectin activity (8). The results of the present investigation demonstrate, first, that the most abundant inhibitor of receptor-mediated erythroagglutination in both normal and pathological sera is IgA; second, that this inhibition is due to the specific recognition by HBP of the carbohydrate moiety of IgA; and third, that polymeric IgA, -present in abnormally high concentration in plasma of patients with cirrhosis (9, 10), is at least an order of magnitude more inhibitory than monomeric IgA, the predominant species in normal human plasma (11).
MATERIALS AND METHODS HBP was isolated from rat liver by a modification ofthe method described by Hudgin et aL (4). Livers (100 g) were homogenized in 8 vol of 1 mM NaHCO3, pH 9.1, containing 0.5 mM CaC12, -by using a Polytron (Brinkmann) at speed 4 for 30 sec at 4°C and centrifuged at 1,000 x g for 10 min. The supernatant, modified to contain 100 mM Tris'HCl, 7% NaCl, and 1% Triton X-100, was extracted for 30 min, incubated for 15 min with 50 mM cadmium acetate, and centrifuged at 10,000 X g for 10 min at 4°C. The pellet was suspended in 300 ml of 100 mM Tris-HCl/ 7% NaCl/20 mM EDTA to which was added 600 ml of the same buffer without EDTA. Triton X-100 was added to 1% and the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
suspension was centrifuged at 10,000 x g for 10 min. The supernatant was mixed with Sepharose 4B (200 ml bed volume) to which desialylated glycoprotein had been coupled (12), brought to 50 mM CaCl2, and incubated with constant mixing at 40C for 2 hr. This suspension was then centrifuged at 1,000 x g for 10 min, resuspended in half its original volume with 20 mM Tris'HCI/7% NaCI/20 mM CaCI2/1%'Triton X-100, and poured into a chromatography column (6 x 45 cm). After washing with 5 vol of the same buffer, protein was eluted with 20 mnM sodium acetate, pH 5.2/7% NaCI/1% Triton X-100. After the addition of Tris HCI, pH 7.9, to 100 mM and CaCl2 to 50 mM, the eluate was applied to a second affinity column (25 ml). The column was washed with 250 ml of the suspension buffer and the protein was eluted as above. Triton X-100 was removed after dialysis by precipitation of the protein with cadmium acetate as previously described (8). The yield of purified HBP obtained by this procedure was 2-4 mg per 100 g ofliver and represented 20-30%.of the original binding activity present in the homogenate. Orosomucoid was isolated from pooled human serum by the procedure of Whitehead and Sammons (13). Desialylated orosomucoid was prepared by incubation of human orosomucoid (5 mg/ml) in 50 mM sodium acetate, pH 5.6, with insoluble neuraminidase (Sigma) at 0.5 unit/ml at 37C for 20 hr. The extent of desialylation was monitored by the thiobarbituric assay of Warren (14). Desialylated orosomucoid was iodinated by a modification of a chloramine-T method (15) with carrier-free sodium ['"I]iodide (Amersham), and the radiolabeled protein was isolated by Sephadex G-100 chromatography. Protein concentrations were estimated by the Lowry method (16) or by absorbance at 280 nm. Purified dimeric IgA without secretory component was generously provided by Kiron M. Das and samples of serum from patients with myeloma were gifts from Shaul Kochwa, Christine Lawrence, Klaus Mayer, and Barry Wenz. Concentrations of IgA were estimated by immunodiffusion, using commercially prepared plates (Miles) and the Mancini-method (17). The presence of IgA in serum or in purified fractions was demonstrated by double immunodiffusion against antihuman IgA (Cappel Laboratories, Cochranville, PA) in Ouchterlony plates (18). The erythroagglutinating activity of the HBP was determined by a modification of the procedure previously described (8). Applying our earlier finding that HBP possessed lectin activity that could be quantitatively inhibited by desialylated glycoproteins, we assessed levels of inhibitors present in normal and pathologic sera. By using as an endpoint the agglutination of type 0 erythrocytes, which have the lowest affinity for HBP, we were able to detect weak ligands present in serum. Human type 0 erythrocytes, from outdated citrated blood, were washed three times with 150 mM NaCl and a 2% suspension Abbreviations: HBP, hepatic binding protein; ASOR, asialoorosomucoid.
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was made in 150 mM NaCl/20 mM CaCl2. To 0.20' ml of this suspension, HBP, in excess ofthe amount required for erythroagglutination (3-5 pg), was added and the suspension was incubated with constant mixing for 10 min at 220C. The amount of a sample inhibiting agglutination by 90% was determined by adding various quantities to the cell suspension prior to the addition of HBP. The degree of agglutination was estimated microscopically. Inhibitory activity of a sample was expressed as the amount of desialylated orosomucoid required for the same degree of inhibition. The inhibitory glycoprotein of serum was purified by procedures described for immunoglobulins (19). Five to 10 ml of serum was dialyzed against 3 liters of distilled water for 10 hr in the cold and then the solution was subjected to centrifugation at 10,000 X g for 10 min. To the supernatant, ammonium sulfate (3.5 g/10 ml) was added and the precipitate was allowed to form at 40C for 30 min. The suspension was centrifuged at 10,000 X g for 10 min and the pellet was dissolved in water. The solution was reprecipitated with ammonium sulfate and centrifuged as above. The pellet was dissolved in 1.5 ml of water and dialyzed against 4 liters of water for 18 hr. The dialysate was clarified by centrifugation at 10,000 x g for 10 min, adjusted to 15 mM sodium phosphate, pH 8.0, and filtered through 5ml bed volume of DEAE-cellulose (DE52, Whatman) equilibrated with 0.015 M phosphate buffer, pH 8.0. After washing of the column, the protein was eluted with 15 mM phosphate buffer, pH 8.0, containing 150 mM NaCI. The eluate was concentrated to 1.0 ml in an Amicon cell (PM-10 filter) under nitrogen and further resolved by gel filtration on a Sephadex G200 column (1.5 X 110 cm) equilibrated with 100 mM Tris HCl, pH 7.9/150 mM NaCl.
RESULTS AND DISCUSSION Sera obtained from patients with cirrhosis were more inhibitory than those of normal volunteers (Table 1), in agreement with the findings of Marshall et al (5) and Lunney and Ashwell (6), who used different inhibition assays. To determine whether immunoglobulins were uniquely inhibitory, we assayed sera obtained from patients with different types of myeloma. Marked inhibitory activity was found only when their sera contained high concentrations of IgA. Furthermore, sera containing IgAl myeloma proteins were more inhibitory than those containing IgA2 (Table 2). IgAl, the predominant IgA subtype in human serum (11), contains O-glycosidically linked N-acetylgalactosamine and galactose residues Table 1. Inhibition of erythroagglutination by sera obtained from normal volunteers and patients with cirrhosis Specific activity, ASOR equiv./mg Inhibition, IgA, Serum ASOR equiv.*/ml mg/ml IgA Normal AM 3.8 2.5 9.5 MK 2.4 2.1 5.0 RS 5.0 1.4 3.6 JJ 3.5 3.7 13.0 Cirrhotic JM 21.7 65 3.0 JS 13.6 5.8 79 FA 4.5 13.3 60 HM 4.4 16.1 71 MM 5.5 37.8 208 3.5 18.0 SD 63 * Micrograms of asialoorosomucoid (ASOR) required for the same level of inhibition.
Proc. Natl. Acad. Sci. USA 79 (1982) Table 2. Inhibition of erythroagglutination by myeloma serum Specific activity, IgA, Myeloma Inhibition, protein ASOR equiv./ml mg/ml* ASOR equiv./mg IgA 6.2 0.4 IgG 2.5 0.7 5.1 IgG 3.6 2.7 1.0 IgM 2.7 10.0 IgAl 280 28.0 12.8 IgAl 100 7.8 0.9 IgA2 27 30.0 2.8 IgA2 27 9.5 * The IgA concentration measured is total IgA (IgAl + IgA2).
(20). The oxidation of these residues by galactose oxidase reversibly abolishes their recognition by the receptor protein (1, 2). Moreover, treatment of purified IgAl, or of inhibitory serum, with galactose oxidase reduces the inhibitory activity of either by over 80%, an effect substantially reversed by subsequent reduction with sodium borohydride (Table 3). These results indicate that the inhibition of HBP-mediated erythroagglution by serum is predominantly-if not exclusively-due to IgAl because the oligosaccharides essential for the reaction, and present at the hinge region of IgAl, are deleted in IgA2, and specific binding of the hinge-region glycopeptide to HBP has been previously demonstrated by Baenziger and co-workers (21, 22). When inhibitory activities ofnormal and pathologic sera were compared to their total concentrations of IgA (Table 1), it appeared that inhibition was not simply proportional to the concentration of IgA. Therefore, inhibitory activities of fractions enriched in monomeric IgA or polymeric IgA and IgA prepared from normal sera and from sera of patients. with cirrhosis were compared (Table 4). While the total inhibitory activities varied widely from patient to patient, in every case the specific activity of the fraction enriched in polymeric IgA was at least an order of magnitude greater than that enriched in monomeric IgA. Moreover, when 200 ,ug of anti-IgA was added to the -polymeric IgA fractions isolated from normal sera and from sera ofcirrhotic patients the inhibitory activity was reduced by at least 80%, providing further evidence that polymeric IgA is the main component responsible for inhibitory activity (Fig. 1). Finally, a purified dimeric IgA preparation (without secretory component) was 50-fold more inhibitory than the same amount of monomeric IgA purified from an IgA myeloma serum. These experiments demonstrate an enhanced activity of polymeric IgA (including dimeric IgA) over that of monomeric IgA. This may be due to conformational changes that expose cryptic sugar residues, or to polymerization of IgA that may simply provide the greater density of carbohydrates required for high-affinity binding by HBP (21, 23). They suggest, too, at the hinge region
Table 3. Galactose oxidase treatment Purified
IgAl,* Treatment
None
ASOR equiv./ mg IgA 22
Normal serum., ASOR equiv./ml 15 3 12
4 Galactose oxidaset 20 Galactose oxidase + NaBH4* * Purified IgAl from myeloma serum. t Galactose oxidase from Polyporus circinatus (Sigma; 45 units) was added either to' 100 lg of IgA in 0;5 ml of 50 mM sodium acetate, pH 6.0/150 mM NaCl or to 0.5 ml of serum and incubated at 220C for 18 hr. t Two hundred micrograms of NaBH4 added.,
Biochemistry: Stockert et aL
Proc. Natl. Acad. Sci. USA 79 (1982)
Table 4. Inhibitory activity of IgA fractions isolated from sera obtained from normal volunteers and patients with cirrhosis ASOR equiv./mg IgA Polymeric Monomeric Serum IgA* IgAt Normal MK 163 2.7 JJ 277 4.5 RS 500 5.9 Cirrhotic HM 190 7.9 SD 11.4 135 MM 266 12.3 FA 354 12.0 * Over 300-kilodalton fraction obtained from Sephadex G-200, containing both dimers and higher-order polymers of IgA. t The 150- to 180-kilodalton fraction from Sephadex G-200, which contained monomeric IgA.
that the high level of inhibitory activity in sera from patients with cirrhosis is due to an abnormally high concentration of polymeric IgA. Polymeric IgA has a short survival in the circulation compared to monomeric IgA (24, 25). Light and electron microscopic studies of intrahepatic transport ofradiolabeled IgA have revealed its selective endocytosis by the parenchymal cells and 100 90 80
>
70
U
>60 0
92 50 40
in\ C
30
20 _
1
40 80 120 160 Anti-IgA Antibody (1tg/tube)
FIG. 1. Neutralizing antibody titration. The high molecular weight fraction isolated from a cirrhotic patient's serum was diluted 1: 3 with 10 mM Tris HCl, pH 7.9/150 mM NaCl. To 0.3 ml, increasing amounts of anti-human IgA antibody (dialyzed against Tris/NaCl to remove phosphate, which interferes with the erythroagglutination assay) were added and the mixtures were allowed to stand at 4°C for 18 hr. After centrifugation, the inhibitory activity present in the supernatant was determined. The vertical bars indicate the magnitude of increments in sequential determinations. Addition of nonspecific antibody (goat IgG) did not reduce the inhibitory activity of the fraction.
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vesicular transport to the bile (26-28). This process has been considered by many investigators as being mediated primarily by secretory component (29-32). Our demonstration of binding of IgA by HBP suggests a role for this receptor in the endocytosis of IgA by hepatocytes. This work was supported in part by grants AM-17702 and AM-07218 from the National Institutes of Health and by grants from the Foundation for the Study of Wilson's Disease, Inc., and the Gail I. Zuckerman Foundation. 1. Ashwell, G. & Morell, A. G. (1974) Adv. Enzymol. 41, 99-128. 2. Neufeld, E. F. & Ashwell, G. (1980) in The Biochemistry of Glycoproteins and Proteoglycans, ed. Lennarz, W. (Plenum, New York), pp. 241-266. 3. Pricer, W. E. & Ashwell, G. (1971) J. Biol. Chem. 246, 4825-4833. 4. Hudgin, R. L., Pricer, W. E., Jr., Ashwell, G., Stockert, R. J. & Morell, A. G. (1974) J. Biol. Chem. 249, 5536-5543. 5. Marshall, J. S., Green, A. M., Pensky, J., Williams, S., Zinn, A. & Carlson, D. M. (1974) J. Clin. Invest. 54, 555-562. 6. Lunney, J. & Ashwell, G. (1976) Proc. Natl. Acad. Sci. USA 73, 341-343. 7. Arima, T. (1979) Gastroenterot Jpn. 14, 349-352. 8. Stockert, R. J., Morell, A. G. & Scheinberg, I. H. (1974) Science 186, 365-366. 9. Delacroix, D. & Vaerman, J. P. (1981) Clin. Exp. Immunol. 43, 633-640. 10. Kutteh, W. H., Prince, S. J., Phillips, J. O., Spenney, J. G. & Mestecky, J. (1982) Gastroenterology 82, 184-192. 11. Grey, H. M., Abel, C. A., Yount, W. J. & Kunkel, H. B. (1968) J. Exp. Med. 128, 1223-1236. 12. Pricer, W. E., Hudgin, R. L., Ashwell, G., Stockert, R. J. & Morell, A. G. (1974) Methods Enzymol. 34, 688-691. 13. Whitehead, P. H. & Sammons, H. G. (1966) Biochim. Biophys. Acta 124, 209-211. 14. Warren, L. (1959) J. Biol Chem. 234, 1971-1975. 15. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem. J. 89, 114-123. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol Chem. 193, 265-275. 17. Fahey, J. L. & McKelvey, E. M. (1965) J. Immunot 94, 84-90. 18. Ouchterlony, 0. (1958) Prog. Allergy 5, 1-78. 19. Fahey, J. L. & Terry, E. W. (1973) in Handbook of Experimental Immunology, ed. Weir, 0. M. (Blackwell Scientific, Oxford), pp. 7.1-7.16. 20. Baenziger, J. & Kornfeld, S. (1974) J. Biod Chem. 249, 7270-7281. 21. Baenziger, J. & Maynard, Y. (1980)J. BioL Chem. 255, 4607-4613. 22. Baenziger, J. & Flete, D. (1980) Cell 22, 611-620. 23. Weigel, P. H., Schnaar, R. L., Kuhlenschmidt, M. S., Schmell, E., Lee, R. T., Lee, Y. C. & Roseman, S. (1979) J. Biod Chem. 254, 10830-10839. 24. Peppard, J., Orland, E., Payne, A. W. R. & Andrew, E. (1981) Immunology 42, 83-89. 25. Jackson, G. D. F., Lemaitre-Coelho, I. & Vaerman, J. P. (1978) Eur. J. Immunot 8, 123-126. 26. Birbeck, M. S. C., Cartwright, P., Hall, J. G., Orlans, E. & Peppard, J. (1979) Immunology 37, 477-482. 27. Mullock, B. M., Jones, R. S. & Hinton, R. H. (1980) FEBS Lett. 113, 201-205. 28. Renston, R. H., Jones, A. L., Christiansen, W. D., Hradek, G. T. & Underdown, B. J. (1980) Science 208, 1276-1278. 29. Mullock, B. M., Hinton, R. H., Dobrota, M., Peppard, J. & Orlans, E. (1979) Biochim. Biophys. Acta 587, 381-389. 30. Fisher, M. M., Nagy, B., Bazin, H. & Underdown, B. J. (1979) Proc. Natl Acad. Sci. USA 76, 2008-2012. 31. Orlans, E., Peppard, J., Fry, J. F., Hinton, R. H. & Mullock, B. M. (1979) J. Exp. Med. 150, 1577-1581. 32. Brandtzaeg, P. (1978) Scand. J. Immunol 8, 39-52.
