INFECrION AND IMMUNITY, OCt. 1992, p. 4213-4220

Vol. 60, No. 10

0019-9567/92/104213-08$02.00/0 Copyright © 1992, American Society for Microbiology

Glycoprotein Receptors for a Heat-Stable Enterotoxin (STh) Produced by Enterotoxigenic Escherichia coli TOSHIYA HIRAYAMA,l* AKIHIRO WADA,2 NAOHITO IWATA,2 SEIICHI TAKASAKI,l YASUTSUGU SHIMONISHI,2 AND YOSHIFUMI TAKEDA3 The Institute ofMedical Science, The University of Tokyo, 4-6-1, Shirokaneda, Minato-ku, Tokyo 108,1 Institute for Protein Research, Osaka University, Yamadaoka 3-2, Osaka 565,2 and Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, 3 Japan Received 3 February 1992/Accepted 14 July 1992

Glycoprotein receptors for heat-stable enterotoxin STh of enterotoxigenic Escherichia colfi in the rat intestinal cell membrane were identified and characterized. Incubation of rat intestinal cell membranes with radioiodinated N-5-azidonitrobenzoyl-STh[5-191 ('251-ANB-STh[5-191) followed by photolysis resulted in specific radiolabeling of two distinct proteins with M,s of 200,000 (designated STR-200A and STR-200B). STR-200A was found to be composed of two molecules of a protein with an Mr of 70,000 (70-kDa protein), whereas STR-200B was composed of two different protein molecules with Mrs of 53,000 (53-kDa protein) and 77,000 (77-kDa protein). These proteins showed no guanylate cyclase activity. The 70-kDa protein was labeled most with 125I-ANB-STh[5-191, suggesting that STR-200A is the main receptor protein in the rat intestinal cell membrane. The carbohydrate moieties of STR-200A and STR-200B were examined by enzymatic deglycosylation. The 70-kDa protein of STR-200A was found to contain N-linked high-mannose-type and/or hybrid-type oligosaccharides, and results suggested that it possesses at least three N glycosylation sites. The 53-kDa protein of STR-200B was found to have an N-linked complex-type oligosaccharide side chain. The deglycosylated 70-kDa protein retained activity for binding to STh, suggesting that the carbohydrate moieties of these receptor proteins are not important for binding with STh.

Heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli are composed of 18 (STp [40]) or 19 (STh [1]) amino acid residues and cause secretory diarrhea in humans and animals (32, 37). In STh, the Cys-6 to Cys-18 region has a cyclindrical structure formed by three disulfide bonds between Cys-6 and Cys-11, Cys-7 and Cys-15, and Cys-10 and Cys-18 (35) and is its active domain (11, 35, 45). There are reports that other enteropathogens, such as Yersinia enterocolitica (42), Vibrio cholerae non-Ol (41), Vibrio mimicus (34), and Citrobacter freundii (14), also produce STs that are structurally similar to STh. These STs probably have biological activities similar to that of STh in the intestinal lumen. Structural details of the STs have been elucidated by protein chemical studies (1, 11, 14, 29, 34, 35, 40-42, 45) and DNA sequencing (28, 38), but the molecular mechanism by which STs induce secretory diarrhea is still not well understood. The initial step in the biological action of ST is its interaction with specific high-affinity receptors (3, 7, 9, 23, 29). The binding of ST to the epithelial cell membranes of intestinal cells of rabbits (7, 30) and rats (6, 13, 15, 23) through these receptors stimulates membrane-bound guanylate cyclase in the cells, leading to an increase in the intracellular concentration of cyclic GMP, followed by activation of cyclic GMP-dependent protein kinase (4, 17), and culminating in the final biological reaction, inhibition of Na+ absorption and stimulation of Cl- secretion (7). Scatchard analysis of stoichiometry by Franz et al. (9) and Cohen et al. (3) indicated a single receptor for STh on rat intestinal epithelial cells and membranes. On the other hand, Kuno et al. (23) demonstrated the presence of three specific, high-affinity receptors for STh with molecular weights of *

Corresponding author. 4213

60,000, 68,000, and 80,000 on rat intestinal cell membranes. Gariepy and Schoolnik (12) also reported two different proteins of 57,000 and 75,000 daltons as putative STh receptors in rat intestinal cell membranes. Hugues et al. (19) recently reported affinity purification of a functional ST receptor that has a 74-kDa subunit. Furthermore, a guanylate cyclase-coupled ST receptor has recently been reported by several investigators (5, 33, 36). In this paper we report the identification of two glycoprotein receptors, STR-200A and ST-200B, for STh in rat intestinal cell membranes. One of them, STR-200A, has a dimeric structure consisting of two molecules of a 70-kDa protein with at least three N-linked high-mannose type and/or hybrid-type oligosaccharide chains as a single binding domain protein. The other, STR-200B, seems to be an N-linked complex-type glycoprotein consisting of two binding proteins of 53 and 77 kDa.

MATERIALS AND METHODS Reagents. N-Hydroxysuccinimidyl-5-azido-2-nitrobenzoate (ANB-NOSu) was purchased from Pierce Chemical Co.

Anhydrous N,N-dimethyl-formamide, N-methylmorpholine, acetonitrile, trifluoroacetic acid, and sodium dodecyl sulfate (SDS) were from Wako Pure Chemical Industry (Osaka, Japan). Rat atrial natriuretic peptide (ANP[3-28]) was from the Peptide Institute, Inc. (Osaka, Japan). Dithiothreitol (DTI), phenylmethanesulfonyl fluoride (PMSF), ,B-mercaptoethanol, Coomassie brilliant blue R-250, ,-galactosidase from E. coli, bovine serum albumin (BSA), and hen egg albumin (HEA) were from Sigma. O-Glycanase from Streptococcus pneumoniae, N-glycosidase F from Flavobacterium meningosepticum, and a2-macroglobulin from horse plasma were from Boehringer Mannheim. Lactate dehydrogenase from pig heart and carbonic anhydrase from bovine erythrocytes were from BDH Chemicals. Nonidet P-40 was

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HIRAYAMA ET AL.

from Iwaki Kagaku Co. (Tokyo, Japan). Lubrol PX and neuraminidase from Arthrobacter ureafaciens were from Nakarai Chemicals (Tokyo, Japan). Endo-,3-glycosidase H from Streptomyces griseus and a-mannosidase were from Seikagaku Kogyo Co. (Tokyo, Japan). All other reagents were of analytical grade. Preparation of photoreactive 125I-ANB-STh[5-19]. STh[519] (an STh analog with the sequence from Tyr-5 to Tyr-19) was synthesized by a solid-phase method described previously (20). The deprotected peptide obtained by anhydrous HF treatment of the protected peptide resin was air oxidized to generate intramolecular disulfide bonds, and then STh[519] was purified by reversed-phase high-pressure liquid chromatography (HPLC). N-5-Azidonitrobenzoyl (ANB)STh[5-19] was prepared by the following procedure. ANBNOSu (500 nmol) was added to 50 nmol of STh[5-19] dissolved in 50 of anhydrous N,N-dimethylformamide containing 0.1 of N-methylmorpholine and then incubated in the dark at room temperature. The resulting ANB-STh[519] was purified by reversed-phase HPLC on a YMC-ODS column (4 by 250 mm) with a linear gradient of 10 to 50% acetonitrile in 0.05% trifluoroacetic acid. ANB-STh[5-19] was radioiodinated with Na125I by the lactoperoxidase method (25, 27), using a radioiodination system (New England Nuclear), to a specific activity of 183 to 252 Ci/mmol. Carrier-free Na125I (10 ,u/1 mCi) was added to 500 pmol of ANB-STh[5-19] in 150 pl1 of 0.1 M sodium phosphate buffer (pH 7.2) containing Enzymobeads. lodination was initiated of a 1% glucose solution to the reaction by adding 25 mixture. After incubation for 30 min at room temperature, the mixture was passed through a TOYOPAK ODS column equilibrated with 10 mM ammonium acetate (pH 5.8) containing 10% acetonitrile. The column was washed with equilibration buffer and developed with 10 mM ammonium acetate (pH 5.8) containing 50% acetonitrile. The eluate was diluted with an equal volume of Dulbecco's phosphatebuffered saline (PBS). Biological activities in suckling mice (43) of STh[5-19], ANB-STh[5-19], and 125I-ANB-STh[5-19] were confirmed to be about 1 ng, which was similar to that of native STh. Membrane preparation. Male Sprague-Dawley rats (8 weeks old, 200 to 250 g) were anesthetized with ethyl ether, and their jejuna and upper ilea were removed. All subsequent procedures were carried out at 0 to 4°C unless otherwise stated. Brush border membranes were isolated as described by Kuno et al. (23). Briefly, the small intestine was rinsed with ice-cold membrane preparation buffer (MP buffer) (50 mM Tris-HCl buffer [pH 7.6], 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF) containing 0.25 M sucrose. The mucosa was removed with a glass slide, and preparations from 13 rats were combined, suspended in MP buffer, and homogenized in 180 ml of MP buffer for 3 min in a Polytron homogenizer at setting 10. The homogenate was diluted with distilled water and adjusted to 10 mM Mg2' by the addition of solid MgCl2. After incubation at 4°C for 15 min with mixing, the diluted extract was centrifuged at 1,000 x g for 20 min at 4°C, and the resulting supernatant was recentrifuged at 100,000 x g for 60 min. The supernatant was discarded, and the pellet was suspended in MP buffer and washed twice with MP buffer by resuspension and centrifugation at 200,000 x g for 60 min. The final suspension, which contained 10 mg of protein per ml, as determined by the method of Bradford (2), in 0.1% Lubrol PX, was used promptly or after storage at -70°C. Photoaffinity labeling of ST receptor. Samples (20 p1) of brush border membranes

were

mixed with 20

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containing 3 mM cystamine. Then 20 pl of 125I-ANB-STh[519] (100,000 cpm) was added, and the mixtures were incubated at 30°C for 15 min. Samples were then photolyzed at 4°C by exposure to light from a UV lamp (R-52G; UVP Inc.) at 254 nm. The photoaffinity-labeled membranes were then solubilized in 0.1% Lubrol PX and heated at 100°C for 5 mm with 20 mM Tris-HCl buffer (pH 7.6) containing 2% SDS, 4% glycerol, 1.6 mM EDTA, 5.2% DTT, and 0.05 mg of bromphenol blue per ml. They were then subjected to SDSpolyacrylamide gel electrophoresis with a 10% separating gel by the method of Laemmli (24). The gels were stained with Coomassie brilliant blue R-250, vacuum dried, and autoradiographed with Kodak X-Omat film with a Dupon Cronex Lighting-Plus intestifying screen at -70°C. Apparent molecular weights (Mrs) of radiolabeled proteins were determined from a standard curve relating log Mr to the relative mobility of standard proteins on electrophoresis gels. The molecular weight markers used were horse plasma a2-macroglobulin (not reduced, Mr = 340,000; reduced, Mr = 170,000), a-galactosidase from E. coli (Mr = 116,000), pig heart BSA (Mr = 68,000), hen egg albumin (HEA; Mr = 45,000), lactate dehydrogenase from pig heart (Mr = 36,000), and carbonic anhydrase from bovine erythrocytes (Mr = 30,000). Assay for guanylate cyclase. Guanylate cyclase activity was measured according to the method described by Kuno et al. (23). Column chromatographies. Washed membranes were treated with 0.1% Lubrol PX for 2 h at 4°C. The solubilized preparation containing Lubrol PX was applied to a Sephacryl S-300 HR column (16 by 920 mm), and material was eluted with 50 mM Tris-HCl buffer (pH 7.6) containing 1 mM EDTA, 0.1 mM PMSF, and 0.1% Lubrol PX. All procedures were carried out at 4°C. The fractions of eluate were assayed for photoaffinity labeling with 125I-ANB-STh[5-19]. The affinity-labeled protein was purified on a concanavalin A (ConA)-Sepharose column (12 by 10 mm) equilibrated with 50 mM Tris-HCl buffer (pH 7.6) containing 0.1 mM PMSF, 0.1% Lubrol PX, 0.5 M NaCl, 1 mM MnCl2, and 1 mM CaCl2. The column was washed extensively with the equilibrating buffer to remove unbound 1"I-ANB-STh[5-19], and then materials Were eluted with the equilibrating buffer containing first 0.5 M a-methylglucoside and then 1.3 M a-methylmannoside. Enzyme treatments of affinity-labeled proteins. The affinitylabeled proteins purified by ConA-Sepharose column chromatography were digested with a-mannosidase, endo-,Bglycosidase H, neuraminidase, N-glycosidase F, or O-glycanase. For a-mannosidase treatment, jack bean a-mannosidase (0.025 U) in 20 pl of 0.1 M NaCl was added to a mixture of 20 pi of affinity-labeled protein (STR-200A) and 80 pl of 0.07 M citrate-phosphate buffer (pH 5.0) containing 3 mM ZnCl2, 0.14% Lubrol PX, and 0.14 mM PMSF, and the mixture was incubated at 30°C for an appropriate time. For endo-p-glycosidase H treatment, endo-,B-glycosidase H from Streptomyces griseus (0.004 U) in 20 ,ul of 0.1 M NaCl was incubated with 40 ,ul of the affinity-labeled proteins at 30°C for an appropriate time. For neuraminidase treatment, neuraminidase from A. ureafaciens (0.05 U) in 20 pl of 10 mM sodium phosphate buffer (pH 7.3) was incubated with 40 pl of the affinity-labeled proteins for 20 h at 30°C. For N-glycosidase F treatment, N-glycosidase F from F. meningosepticum (2 U) in 10 pl of 20 mM potassium phosphate buffer containing 50 mM EDTA and 0.05% sodium azide (pH 7.2) was incubated with 40 pl of the affinity-labeled protein (STR-200B) for 20 h at 30°C. For O-glycanase treatment, O-glycanase (2.5 U) from Strepto-

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FIG. 1. Effect of STh[5-19] on binding of l25I-ANB-STh[5-19] to intestinal membrane proteins. Rat intestinal membranes (20 ,ug of protein) suspended in MP buffer were incubated with 3 nM 'IANB-STh[5-19] (100,000 cpm) in the presence or absence of the indicated amounts of either STh[5-19] or ANP[3-28] at 30°C for 15 min in the dark. After photolysis for 45 min, radioactivities incorporated into membrane proteins (90,000 cpm/18 ,ug of protein) were examined by SDS-polyacrylamide gel electrophoresis as described in the text. Numbers at the left or right of panels show molecular weights (103) of markers. Lane 1, absence of STh[5-19]; lanes 2 to 5, presence of 10'6 1o-7, 10-8, and 10-9 M STh[5-19], respectively; lane 6, absence of ANP[3-28]; lane 7, presence of 10-5 M ANP[3-

28].

pneumoniae in 5 ,ul of buffer containing 15 mM sodium cacodylate, 10 mM sodium azide, 0.15 M NaCl, 25% glycerol (pH 6.0), and 0.01% BSA was incubated with 40 ,ul of affinity-labeled protein (STR-200B) and 15 ,ul of 0.1 M NaCl for 20 h at 30°C. After these enzyme treatments, the

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samples were analyzed by SDS-polyacrylamide gel electrophoresis. RESULTS Photoaffinity labeling with 125j-ANB-STh[5-191 of proteins of rat intestinal brush border membranes. Photoaffinity labeling of rat intestinal membranes with 1"I-ANB-STh[5-19] resulted in incorporation of radioactivity into five proteins with molecular weights of 70,000, 53,000, 52,000, 45,000, and 42,000 (Fig. 1). The 70- and 52-kDa proteins were labeled most strongly and were detectable after incubation for 20 s. The levels of radiolabeling of the 53- and 42-kDa proteins were low but were consistently seen. On the other hand, the labeling of the 45-kDa protein was very weak and was not always observed. To determine the specificity of incorporation of 125I-ANB-STh[5-19] into these proteins, we added excess STh[5-19] at various concentrations (10-6 to 10-9 M) during the initial period of incubation of the membranes with 125I-ANB-STh[5-19] (Fig. 1, lanes 2 to 5). STh[5-19] inhibited the incorporation of 125I-ANB-STh[5-19] into the proteins of 70, 53, and 45 kDa but not into those of 52 and 42 kDa. STh[5-19] at a concentration of 10-8 M reduced the photoaffinity labeling of the 70-kDa protein by more than 50% (Fig. 1, lane 4), whereas even at a concentration of 10-6 M it did

4215

not affect the incorporation of radioactivity into the proteins of 52 and 42 kDa (Fig. 1, lane 2), suggesting that the proteins of 70, 53, and 45 kDa were specifically labeled with 15IANB-STh[5-19]. Rat ANP[3-28], which has been shown to activate membrane-bound guanylate cyclase (GC-A and GC-B) selectively by binding to the enzyme (10, 21), did not inhibit the binding of 125I-ANB-STh[5-19] to these proteins (Fig. 1, lanes 6 and 7). These results suggest that the proteins of 70, 53, and 45 kDa of rat intestinal membranes are distinct from the ANP

receptor. Solubilization and purification of membrane proteins that bind to STh. To characterize the 70-kDa protein, which was the main protein labeled with 1"I-ANB-STh[5-19], we treated rat intestinal brush border membranes with 0.1% Lubrol PX and applied the solubilized preparation to a Sephacryl S-300 HR column. Chromatography was carried out as described in Materials and Methods, and the A280 of the eluate is shown in Fig. 2A. The binding of each fraction to 125I-ANB-STh[5-19] was assayed by photoaffinity labeling. The autoradiogram of photoaffinity-labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis showed the presence of three bands of labeled proteins of 53, 70, and 77 kDa in fractions 90 to 97 (Fig. 2B). The photoaffinity labeling of the 77-kDa protein was weak but was consistently seen. The peak of incorporation of radioactivity into the proteins was eluted in fraction 95. The molecular weight of the protein in fraction 95 was estimated to be 200,000 from chromatograms of several standard proteins on a Sephacryl S-300 HR column (Fig. 2C). These results suggest that the protein that binds STh is a 200-kDa protein that is separated into proteins of 53, 70, and 77 kDa on SDS-polyacrylamide gels. Each fraction from the Sephacryl S-300 HR column chromatography was also assayed for guanylate cyclase activity. It was found that guanylate cyclase activity applied to the column was exclusively eluted around fractions 77 to 83. Photoaffinity labeling of these fractions with 125I-ANBSTh[5-19] yielded no specifically labelled proteins. These results suggest that the 53-, 70-, and 77-kDa proteins are distinct from a protein(s) that contains guanylate cyclase activity. Characterization of the 200-kDa protein. The solubilized membrane preparation was chromatographed on a Sephacryl S-300 HR column as for Fig. 2A, and fractions 92 to 97 were pooled, designated as 200-kDa protein, and examined further. Confirming the result mentioned in the previous section, no guanylate cyclase activity was detected for the 200-kDa protein even at a high concentration (data not shown). The 200-kDa protein was photoaffinity labeled with 125IANB-STh[5-19] and then applied to a ConA-Sepharose column previously equilibrated with 50 mM Tris-HCl buffer (pH 7.6) containing 0.1 mM PMSF, 0.1% Lubrol PX, 0.5 M NaCl, 1 mM MnCl2, and 1 mM CaCl2. As shown in Fig. 3, 125I-ANB-STh[5-19] which did not label the 200-kDa protein was eluted with the equilibration buffer. About 6.9 and 3.5% of the total radioactivity applied eluted with equilibration buffer containing 0.5 M a-methylglucoside and 1.3 M a-methylmannoside, respectively. The radioactivity retained by the column after elution with a-methylmannoside was less than 0.4% of the total radioactivity applied. The fractions eluted with 0.5 M a-methylglucoside and 1.3 M a-methylmannoside were pooled separately, concentrated, and applied to a Sephacryl S-300 HR column to determine the molecular weight of each photoaffinity-labeled

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FIG. 2. Separation of proteins labeled with 1"I-ANB-STh[5-19] of solubilized rat intestinal brush border membranes by column chromatography. (A) Rat intestinal brush border membranes solubilized with 0.1% Lubrol PX (96 mg of protein) were subjected to gel filtration on a Sephacryl S-300 HR column as described in the text. Fractions (940 p.l) were collected, and theirA2. was measured after fivefold dilution. (B) Each fraction was assayed for labeling with 1"I-ANB-STh[5-19] as described in the text. Numbers at the top indicate fraction numbers, and those on the left indicate molecular weights (103) of markers. (C) Estimation of the molecular weight of the labeled protein in fraction 95 by gel filtration on a Sephacryl

protein (Fig. 4). On the Sephacryl S-300 HR column, the 0.5 M a-methylglucoside eluate showed a peak of radioactivity in fraction 95, and the molecular weight of this material was estimated to be 200,000 (Fig. 4A). As shown in Fig. 4B, the material with radioactivity in the 1.3 M a-methylmannoside eluate was also recovered in fraction 95 and had an estimated molecular weight of 200,000. We named the a-methylmannoside eluate and a-methylglucoside eluate STR-200A and STR-200B, respectively. Further examinations of STR-200A and STR-200B by SDS-polyacrylamide gel electrophoresis under nonreducing and nonheating conditions revealed that the molecular weights of STR-200A and STR-200B were both about 140,000 (data not shown). Thus, the apparent molecular weights of STR-200A and STR-200B were both concluded to be about 140,000, although they were estimated to be 200,000 by molecular sieving on a Sephacryl S-300 HR column. The discrepancy in the molecular weights of STR200A and STR-200B estimated by gel filtration and by nonreducing SDS-polyacrylamide gel electrophoresis was probably due to the contribution of the micelle size of Lubrol PX, which increased the actual molecular sizes of STR-200A and STR-200B by about 50 kDa in the gel ifitration. Radiolabeled STR-200A and STR-200B were further analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 5). S-300 HR column. Gel filtrations of standard marker proteins (1, fibrinogen [Mr = 340,000]; 2, fraction 95; 3, glucose oxidase [Mr = 160,000]; 4, BSA [Mr = 68,000]; 5, HEA [Mr = 45,000]) were carried out in 50 mM Tris-HCl buffer (pH 7.6) containing 1 mM EDTA, 0.1 mM PMSF, and 0.1% Lubrol PX. Fraction 95 was chromatographed as described for panel A.

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120 70 80 90 100 110 90 100 110 120 Fraction number Fraction number FIG. 4. Column chromatographies on Sephacryl S-300 HR of the a-methylglucoside eluate and the a-methylmannoside eluate from the ConA-Sepharose column. Eluates with either 0.5 M a-methylglucoside (A) or 1.3 M a-methylmannoside (B) from the ConA-Sepharose column were concentrated with a Centricon 10 microconcentrator and subjected to gel filtration on a Sephacryl S-300 HR column equilibrated and developed with 50 mM Tris-HCl buffer (pH 7.6) containing 1 mM EDTA, 0.1 mM PMSF, and 0.1% Lubrol PX. Fractions (940 PI) were collected, and the radioactivity of 100 ILI of each fraction was counted in an auto--y-counter.

After being heated in the presence of DfT, STR-200A migrated as a single band of 70 kDa while STR-200B migrated as two bands of 53 and 77 kDa. The dissociation of STR-200A into 70-kDa proteins and of STR-200B into 53and 77-kDa proteins was observed in the presence of DTT even without heating (data not shown). From these results, we concluded that STR-200A consists of two molecules of a single binding peptide of 70 kDa, whereas STR-200B con-

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sists of two binding peptides of 53 and 77 kDa. The dimer formations in STR-200A and STR-200B may be either through a hydrophobic interaction or by a disulfide bridge(s). Characterization of STR-200A and STR-200B. The findings that both STR-200A and STR-200B bound to ConA-Sepharose (Fig. 3) suggested that carbohydrate side chains with a-linked glucose or mannose residues were involved in their binding. For determination of whether STR-200A and/or STR-200B is a glycoprotein, the 200-kDa protein was photoaffinity labeled with la¶I-ANB-STh[5-19], and STR-200A and STR-200B were fractionated as shown in Fig. 3. As seen in Fig. 6, a-mannosidase digestion of photoaffinity-labeled STR-200A increased the electrophoretic mobility of the 70-kDa protein on SDS-polyacrylamide gel electrophoresis, suggesting that this protein contained a mannose side chain. After endo-3-glycosidase H treatment, the mobility of pho-

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resis as described in the text. The amount of radioactivity applied was 1,800 cpm in both experiments. Numbers on the right indicate the positions of molecular weight markers (10~). Lanes: 1, STh200B; 2, STR-200A.

FIG. 6. a-Mannosidase treatment of STR-200A. STR-200A labeled with 1"I-ANB-STh[5-19] was treated with a-mannosidase as described in the text for 1 or 5 h at 30°C. Samples were then subjected to SDS-polyacrylamide gel electrophoresis as described in the text. Numbers at the left are molecular weights (10') of markers. Lanes 1, 3, and 5; samples without enzyme treatment; lanes 2, 4, and 6; samples with enzyme treatment. Lanes 1 and 2, controls without incubation; lanes 3 and 4, samples after 1 h of incubation; lanes 5 and 6, samples after 5 h of incubation.

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45-

FIG. 7. Endo-p-glycosidase H treatment of STR-200A. Labeled STR-200A was treated with endo-,-glycosidase H as described in the text for various times at 30'C. Samples were then subjected to SDS-polyacrylamide gel electrophoresis. Numbers at the left show molecular weights (103) of markers. Lanes 1, 3, 5, 7, and 9, samples without enzyme treatment; lanes 2, 4, 6, 8, and 10, samples with enzyme treatment. Lanes 1 and 2, controls without incubation; lanes 3 and 4, samples after 15 min of incubation; lanes 5 and 6, samples after 1 h of incubation; lanes 9 and 10, samples after 20 h of incubation.

toaffinity-labeled STR-200A on an SDS-polyacrylamide gel also increased from a position corresponding to a 70-kDa protein to a position corresponding to a 53-kDa protein (Fig. 7, lanes 8 and 10), with two intermediate cleavage products (lower bands of lanes 4 and 6 in Fig. 7). The 53-kDa-protein band was observed after incubation for 5 to 20 h and seemed to be the final product of the 70-kDa protein treatment with endo-,-glycosidase H. Since endo-pglycosidase H specifically released N-linked high-mannosetype and hybrid-type oligosaccharides from glycoprotein (39), these results indicate that STR-200A has at least three N-linked oligosaccharide side chains. Similar treatment of affinity-labeled STR-200A with neuraminidase had no effect on the migration of the 70-kDa protein on two-dimensional electrophoresis (data not shown). Thus, we concluded that the N-linked oligosaccharides on the 70-kDa protein of STR-200A are high-mannose-type and/or hybrid-type oligosaccharide chains. Figure 8 shows an autoradiogram of photoaffinity-labeled STR-200B treated with various glycosidases, which cleave a glycosylamine linkage of complex-type, high-mannose-type, and hybrid-type oligosaccharides linked to asparagine (44). Treatment of STR-200B with N-glycosidase F slightly increased the electrophoretic mobility of the 53-kDa protein on SDS-polyacrylamide gel electrophoresis (Fig. 8, lane 4). However, other glycosidases, such as endo-,B-glycosidase H and O-glycanase, which release 0-linked oligosaccharides linked to serine or threonine residues (22), and neuraminidase, did not affect the migration of either the 53- or 77-kDa protein. These results suggest that the 53-kDa protein in STR-200B contains N-linked complex-type oligosaccharides. The 53-kDa protein from STR-200B was demonstrated to be different from the 53-kDa protein from STR-200A by the difference in mobilities of the two proteins on twodimensional electrophoresis (data not shown). Moreover, treatment of the deglycosylated 53-kDa protein derived from the 70-kDa protein of STR-200A did not further increase the electrophoretic mobility as treatment of the 53-kDa protein from STR-200B did. Binding of STh to deglycosylated 70-kDa protein of STR200A. The results in Fig. 9 show that the N-linked highmannose-type oligosaccharide chain of the 70-kDa protein of STR-200A does not contribute to the binding of STh to the 70-kDa protein. In this experiment, STR-200A was first

was

36-

FIG. 8. Treatment of STR-200B with various glycosidases. STR200B labeled with '25I-ANB-STh[5-19] was treated with various enzymes for 20 h at 30°C as described in the text. Samples (980 cpm) were then subjected to SDS-polyacrylamide gel electrophoresis as described in the text. Numbers on the left are molecular weights (103) of markers. Lane 1, control after 20 h of incubation at 4°C without enzyme; lane 2, control after 20 h of incubation at 30°C without enzyme; lane 3, with endo-,B-glycosidase H; lane 4, with N-glycosidase F; lane 5; with O-glycanase; lane 6, with neuraminidase; lane 7; with O-glycanase plus neuraminidase.

treated with endo-3-glycosidase H for 1 or 5 h, and then the STh-binding activity of each digestion product of the 70-kDa protein was assayed by photoaffinity labeling with l25I_ANB1 STh[5-19]. As shown in Fig. 9, lanes 2 and 4, protein bands that incorporated radioactivity were detected after incuba-

1

2

3

4

116

.X~~~2 _3

l#W

-=W. 3

g

8:

-36 -30

FIG. 9. Effect of endo-3-glycosidase H treatment of STR-200A labeling with 251I-ANB-STh[5-19]. STR-200A was treated with 0.004 U of endo-3-glycosidase H for 1 h (lane 2) or 5 h (lane 4) at 30°C and then labeled with 125I-ANB-STh[5-19]. Radiolabeled materials were then separated by SDS-polyacrylamide gel electrophoresis as described in the text. Lanes 1 and 3, controls incubated for 1 h (lane 1) or 5 h (lane 3) without enzyme. Numbers on the right show molecular weights (103) of markers. on its

VOL. 60, 1992

tions of STR-200A with endo-3-glycosidase H for 1 and 5 h, respectively, indicating that the deglycosylated products of the 70-kDa protein still bound 1"I-ANB-STh[5-19]. Lane 4 shows that the final product of digestion (Mr = 53,000) of the 70-kDa protein with endo-13-glycosidase H could bind to STh, although the amount of radioactivity was less than that with the 70-kDa protein. DISCUSSION Biochemical characterization of the ST receptor on the intestinal cell membrane has been a focus of interest of many investigators, including those working on bacterial enterotoxins, signal transduction, and membrane structures. Kuno et al. (23) and Gariepy and Schoolnik (12) demonstrated a variety of binding proteins of 57, 60, 68, 75, and 80 kDa by affinity labeling. The discrepancies in their results may have been because they used whole STh, and so may not have labeled the binding site of STh, and also because they used different techniques. Studies on the structure-activity relationships of STh have shown that the carboxy-terminal peptide region possesses full biological activity (4, 6, 7). Therefore, in this work we used a short STh probe, 125I_ ANB-STh[5-19], in which the site of affinity labeling is much closer to the receptor-binding region than it is in a longer probe. Our results showed that the main protein labeled with 125I-ANB-STh[5-19] was a 70-kDa protein, suggesting that this is the main receptor protein of STh. Robertson and Jaso-Friedmann (31) attempted to purify the STh receptor by lectin affinity chromatography and reported that the affinity of the STh receptor for a ConASepharose column is effective for its purification. By ConASepharose column chromatography, we demonstrated the presence of two glycoprotein receptors (STR-200A and STR-200B). Previously, Kuno et al. (23) reported that the ST receptor was a 200-kDa protein consisting of 60-, 68-, and 80-kDa subunits. The sizes of these three receptor proteins are similar to those found in this study (53, 70, and 77 kDa), but unlike Kuno et al. (23) we found two distinct glycoprotein receptors. Recently, Hugues et al. (18) demonstrated the existence of a high-affinity ST receptor without guanylate cyclase activity by kinetic analysis of 1"I-STp binding to its receptor. Although STR-200A and STR-200B, reported in this paper, did not show guanylate cyclase activity, the relationship of their receptor to ours remains to be studied. Using specific glycosidases, we characterized the carbohydrate moieties of the STh receptors, STR-200A and STR200B. Digestion of the affinity-labeled 70-kDa protein of STR-200A with a-mannosidase or endo-o-glycosidase H increased the electrophoretic mobility of the 70-kDa band on SDS-polyacrylamide gel electrophoresis. Complete digestion of the 70-kDa protein with endo-3-glycosidase H yielded a 53-kDa protein, but limited digestion demonstrated two additional proteins of intermediate sizes. Endo-,-glycosidase H releases N-linked high-mannose-type and hybridtype oligosaccharides, so our results suggest that the 70-kDa protein contains one or both types of oligosaccharides at three sites. The 53-kDa protein of STR-200B was digested by N-glycosidase F but was resistant to endo-,-glycosidase H (Fig. 8). This finding suggests that the 53-kDa protein contains complex-type oligosaccharides but not high-mannose-type or hybrid-type oligosaccharides. Complex-type oligosaccharides are usually terminated by sialic acid. However, neuraminidase digestion did not increase the electrophoretic

mobility of the 53-kDa protein of STR-200B, suggesting that

GLYCOPROTEIN RECEPTOR FOR E. COLI ST

4219

it is devoid of sialic acid. This finding is consistent with previous reports that brush border membranes of rat and human small intestines have low contents of sialic acid (8, 26). Treatment of the photoaffinity-labeled 53-kDa protein of STR-200B with O-glycanase caused no change in its mobility on SDS-polyacrylamide gel electrophoresis. The 0-linked group may terminate in sialic acid, which interferes with the action of O-glycanase, so we also examined the effect of neuraminidase on STR-200B but found that it did not change the electrophoretic mobility of the labeled band. This result supports a previous finding that N-linked oligosaccharides are the major forms of brush border membrane glycoproteins (16). To determine the role of the carbohydrate in the function of STh receptors, we examined the effect of the carbohydrate of the 70-kDa protein of STR-200A on its binding with STh. As shown in Fig. 9, the product of digestion of the 70-kDa protein with endo-,B-glycosidase H was still capable of binding STh and was labeled with 15I-ANB-STh[5-19], suggesting that the N-linked oligosaccharides on the 70-kDa protein of STR-200A are not essential for ST binding. Schulz et al. (33) recently determined the sequence of a gene encoding a new form of guanylate cyclase (GC-C), to which STp binds, from cDNA of rat intestinal cells. In the present study, since the guanylate cyclase activity in the membrane preparation was eluted in different fractions from those containing 200-kDa proteins in Sephacryl S-300 HR column chromatography (Fig. 2A) and STR-200A and STR200B did not show guanylate cyclase activity, it was assumed that STR-200A and STR-200B were different ST receptor molecules from the GC-C reported by Schulz et al. (33). However, further studies to examine the relationships of STR-200A and STR-200B to GC-C are necessary to conclude the existence of multiple ST receptors in the membrane. ACKNOWLEDGMENTS We thank K. Matsumoto for excellent technical assistance. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and by a research grant from the Japanese Panel of the U.S.-Japan Cooperative Medical Science Program, Cholera and Related Diarrheal Diseases.

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