Reduced Immunogenicity of Î²-Lactoglobulin by Conjugation with

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Bioconjugate Chem. 2000, 11, 84−93

Reduced Immunogenicity of β-Lactoglobulin by Conjugation with Carboxymethyl Dextran Makoto Hattori,*,† Koichi Nagasawa,† Koki Ohgata,† Noriko Sone,† Ayumu Fukuda,† Hiroshi Matsuda,‡ and Koji Takahashi† Department of Applied Biological Science and Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-City, Tokyo 183-8509, Japan. Received July 19, 1999; Revised Manuscript Received September 27, 1999

We prepared two β-lactoglobulin (β-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of β-LG. The molar ratios of β-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The β-LG-CMD conjugates maintained the retinol-binding activity of native β-LG. Intrinsic fluorescence study indicated that shielding of the surface of β-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around 125Thr-135Lys (R-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of β-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-β-LG antibody response was markedly reduced after immunization with the β-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/ He mice. We determined the B cell epitopes of β-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the β-LG-CMD conjugates were similar to those of β-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of β-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the β-LG in these conjugates.

INTRODUCTION

β-Lactoglobulin (β-LG),1 a major whey protein found in the milk of mammalian species (1), is a globular protein of Mr 18 400, with two disulfide bridges and one free cysteine residue, containing plenty of essential amino acids (2). The X-ray crystallography (3, 4) and protein sequence determination (5) have shown remarkable similarity between β-LG and plasma retinol-binding protein. Although the function of β-LG still remains unclear, β-LG can bind small hydrophobic ligands such as retinol, fatty acids, and so on and is categorized as a member of the lipocalin superfamily (6). β-LG has many useful functional properties such as emulsifying ability (7), foaming ability (8), and gelling ability (9). Although β-LG is considered to be a valuable protein in terms of food science, it is also known as a potent allergen associated with milk allergy; about 82% of milk allergy patients is sensitive to β-LG (10). Hence, it is very desirable to develop a new method to decrease the allergenicity of β-LG. Methods employed to decrease the allergenicity of proteins have involved their digestion and denaturation (11-15). However, these methods might destroy useful functions of the proteins and bring about * To whom correspondence should be addressed. Phone: +81-42-367-5879. Fax: +81-42-360-8830. E-mail: makoto@ cc.tuat.ac.jp. † Department of Applied Biological Science. ‡ Department of Veterinary Clinic. 1 Abbreviations: β-LG, β-lactoglobulin; CMD, carboxymethyl dextran; RCM-β-LG, reduced and carboxymethylated β-lactoglobulin; PBS, phosphate-buffered saline; PBS-Tween, PBS containing 0.05% Tween 20; SEC, size-exclusion chromatography; DSC, differential scanning calorimetry.

problems with their taste. In contrast, the conjugation of proteins with materials having low allergenicity can simultaneously decrease the allergenicity and improve the function of the proteins. To achieve low allergenicity of β-LG, we planned to prepare neoglycoconjugates of β-LG. Neoglycoconjugates have been widely investigated in the last 20 years, and various improvements to the functions of proteins have been reported. In fact, in our previous studies, we improved the emulsifying properties and thermal stability of β-LG while maintaining the retinol-binding activity by conjugating this protein with carboxymethyl dextran (CMD) (16-18). Many attempts to decrease the antigenicity and immunogenicity of proteins by conjugation have been reported. Since Lee and Sehon (19) found that conjugates of antigens (ovalbumin and ragweed pollen) and polyethylene glycol (PEG) derivatives were nonimmunogenic, immunological studies focusing on conjugates of protein antigens and PEGs were initiated. Some of PEG and polymer conjugated proteins have actually been used as pharmaceuticals for therapy (20, 21). Conjugation of proteins with dextrans also is reported to be effective to reduce immunogenicity. The conjugates of monoclonal and polyclonal antibodies with oxidized dextrans of low molecular weight have been reported to have optimal immunoreactivity, in vivo pharmacokinetics, and tumorlocalization properties, as well as low immunogenicity in vivo (22). Lipophilization is reported to be effective to suppress production of anti-β-LG IgG and IgE upon modification to a high degree (23). The shielding of epitopes by materials having low-antigenicity and immunogenicity is thought to be important to reduce the antigenicity and immunogenicity of the protein, and the

10.1021/bc990096q CCC: $19.00 © 2000 American Chemical Society Published on Web 12/21/1999

Immunogenicity of Protein−Polysaccharide Conjugate

use of a modifier with high molecular weight is desirable to achieve effective shielding of epitopes (24). Hence, we selected carboxymethyl dextran (CMD) as a modifier which is homogeneous in molecular weight and sequence, with a relatively high molecular weight (Mr 10 000) and low antigenicity and immunogenicity. Our purpose was to create a novel β-LG-CMD conjugate with high thermal stability and improved functional properties, while maintaining the retinol-binding activity, without allergenicity. In particular, we investigated the structure and function of novel protein conjugates in detail in an effort to develop an effective method to decrease the allergenicity of β-LG and to elucidate the mechanisms involved. Although many studies on protein conjugates have been carried out and functional improvement of proteins has been well described so far, the relationships between structural changes and these functional improvements have not been clearly elucidated and the precise mechanisms responsible for the decrease in antigenicity and immunogenicity of the proteins still remain unclear. Changes in epitope profiles of proteins after conjugation with saccharides have not been clarified. In this study, we prepared covalently bonded β-LGCMD conjugates. After exploring the structure of these conjugates, we investigated the immunogenicity profiles of β-LG and the conjugates in detail and demonstrated the reduced immunogenicity of β-LG when conjugated with CMD. The results of this study are essentially important to develop designing of novel foods with low allergenicity. MATERIALS AND METHODS

Materials. Dextran T10 was purchased from Pharmacia LKB (Uppsala, Sweden). Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), was purchased from Dojindo (Kumamoto, Japan). Carboxymethylation of Dextran. Carboxymethyl dextran was prepared by carboxymethylation with monochloroacetic acid according to the method described previously (16-18) on a larger experimental scale than that of the previous experiments. In brief, dextran T10 (10 g, Pharmacia LKB, Uppsala, Sweden) was dissolved in 47 mL of a 15% monochloroacetic acid solution containing 7 g of sodium hydroxide (pH 11.5), and then incubated at 40 °C for 48 h. The reaction mixture was neutralized to pH 6.5 with acetic acid after cooling to room temperature. After dialyzing against distilled water and lyophilizing, carboxymethyl dextran (CMD) was obtained. The degree of modification (DM) was determined by hydrochloride-methanol titration (25). The DM value for CMD was about 17 carboxyl group residues per dextran molecule, corresponding to 27 residues/100 glucose residues. Preparation of β-LG and RCM-β-LG. Crude β-LG (genotype AA) was prepared by the method of Armstrong et al. (26) and then purified by ion-exchange chromatography, using a DEAE-Sephacel column (2.5 ID x 50 cm; Pharmacia LKB, Uppsala, Sweden) as described previously (16-18). The purity of β-LG was confirmed by polyacrylamide gel electrophoresis (PAGE) according to the method of Davis (27). RCM-β-LG was prepared as described previously (31, 34, 35). The disulfide bonds in β-LG were reduced with 2-mercaptoethanol and then the free sulfhydryl groups were carboxymethylated with sodium iodoacetate. Preparation of the β-LG-CMD Conjugates. The β-LG-CMD conjugates were prepared by referring to the method described previously (16-18), on a larger experi-

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mental scale than in the previous experiments, by the following two methods. Method 1. β-LG (2.7 g) and CMD (2.7 g) were each dissolved in 324 mL of a 0.9% NaCl solution. An EDC solution (2.7 g/ 54 mL) was added to this CMD solution. After adjusting the solution to pH 5.0 with 1 N HCl, the β-LG solution was added. The reaction was stopped by adding acetic acid (22.7 mL), and the solution was dialyzed against distilled water. The coacervate, the phase separated during dialysis, was recovered by lyophilization as a crude β-LG-CMD conjugate (Conj. 10A). Method 2. β-LG (2.7 g) and 11.8 g of CMD were dissolved in 338 mL of distilled water, and the solution was adjusted to pH 4.75 with 1 N NaOH. An EDC solution (4.7 g/ 13.5 mL of distilled water) was gradually added over 30 min while maintaining the pH at 4.75. The reaction mixture was then incubated at room temperature for 3 h. The reaction was stopped by adding 22.5 mL of a 2 M acetate buffer (pH 5.5), and the solution was dialyzed against distilled water. The crude β-LGCMD conjugate (Conj. 10B) was recovered by lyophilization. Purification of these β-LG-CMD conjugates was then carried out as follows. Free CMD in each crude conjugate was removed by salting out with ammonium sulfate (final concentration, 5 M). After salting out, free β-LG and polymerized β-LG were removed by ion-exchange chromatography, using a DEAE-Sepharose Fast Flow column (2.5 ID x 40 cm, Pharmacia LKB, Uppsala, Sweden) as described previously (18). Size-Exclusion Chromatography (SEC). The molecular weight of each β-LG-CMD conjugate was measured by SEC. A TSKgel G3000SWXL column (7.8 ID × 300 mm, Tosoh, Tokyo, Japan) was equilibrated with a 0.067 M phosphate buffer containing 0.3 M NaCl at pH 7.0. Each β-LG-CMD conjugate (100 µg of protein/50 µL) was applied to the column and eluted at a flow rate of 1.0 mL/min. The absorbance was monitored at 280 nm. Conformational Analysis of the β-LG-CMD Conjugates by the Spectroscopic Methods. The CD spectrum for each β-LG-CMD conjugate was measured with a J-720 spectropolarimeter (Jasco, Tokyo, Japan), using a cell with a 1.0 mm path length. Samples were dissolved in PBS (phosphate-buffered saline; a 0.11 M phosphate buffer at pH 7.1 containing 0.04 M NaCl and 0.02% NaN3) at a protein concentration of 0.02%. The intrinsic fluorescence of each β-LG-CMD conjugate dissolved in PBS at 0.001% (as the protein concentration) was measured at an excitation wavelength of 283 nm by means of an RF-510 (Shimadzu, Kyoto, Japan) fluorescence spectrophotometer. Differential Scanning Calorimetry (DSC). Each sample was dissolved in PBS (pH 7.0) at a protein concentration of 5% (w/ v), and 50 µL of the sample solution was sealed in a silver DSC cell. Distilled water was used as a reference. DSC was performed with an SSC-5020 DSC 100 instrument (Seiko Instruments, Tokyo, Japan) at a heating rate of 2 K/min. Determination of the Carbohydrate Binding Site in the β-LG-CMD Conjugates. β-LG and the β-LGCMD conjugates (80 mg) were digested with Dextranase (0.8 mg, EC 3.2.1.11, Sigma, St. Louis, MO) in 0.1 M potassium phosphate buffer (pH 6.0) at 39 °C for 12 h. After digestion, the digested saccharides were removed by salting-out with ammonium sulfate (final concentration, 4 M). The precipitate was recovered by centrifugation (at 12 000 rpm for 10 min at 4 °C), washed with a 0.1 M potassium phosphate buffer (pH 6.0) containing 4

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M ammonium sulfate, and redissolved in the 0.1 M potassium phosphate buffer (pH 6.0). After dialysis against distilled water and lyophilization, samples with undigested small saccharide fragments were recovered. These samples were tagged with 2-aminopyridine by the method of Hase et al. (28). In brief, the samples were mixed with 0.7 mL of a reagent made by mixing 2-aminopyridine (1 g), acetic acid (0.47 mL), and methanol (0.6 mL). The solution was heated in a sealed tube at 90 °C for 15 min and dried at 60 °C to remove the nonreacted reagent. Acetic acid (1 mL) containing a borane-dimethylamine complex (59 mg) was added to the sample, and the solution was incubated at 90 °C for 30 min. The solution was mixed with toluene (1.5 mL) and dried at 40 °C to remove the nonreacted reagent. The dried sample was redissolved in 0.067 M phosphate buffer at pH 7.0. The 2-aminopyridine-tagged sample was obtained after dialysis and lyophilization. The 2-aminopyridine-tagged sample was reduced with 2-mercaptoethanol, carboxymethylated with sodium iodoacetate, and extensively digested with TPCK-trypsin (EC 3.4.21.4, Sigma, St. Louis, MO) at 37 °C for 24 h (S:E ) 100:1). TPCK-trypsin and the nondigested protein were removed by ultrafiltration with a USY-1 ultrafilter (Advantec, Tokyo, Japan). The digested fragments were separated by reversed-phase HPLC (ODS-120T column, 7.8 ID x 300 mm, Tosoh, Tokyo, Japan). Peptides were detected by their absorbance at 230 nm, and the 2-aminopyridine-tagged saccharides were detected by fluorescence (excitation at 310 nm and emission at 380 nm). The peaks containing peptide and 2-aminopyridine-tagged saccharide were further purified by reversed-phase HPLC, and the amino acid sequence was determined by means of a 470A peptide sequencer (Perkin-Elmer Applied Biosystems, Foster, CA). Measurement of the Retinol Binding Activity of the β-LG-CMD Conjugates. The retinol binding activity of the β-LG-CMD conjugates was measured by fluorescence titration as described previously (16, 2933). Preparation of Antisera. Female BALB/c, C57BL/ 6, and C3H/He mice were purchased from Charles River Japan (Yokohama, Japan). Five mice of each group at 6 weeks of age were immunized intraperitoneally with 100 µg of protein in Freund’s complete adjuvant (Difco Laboratories, Detroit, MI). Fourteen days after the primary immunization the mice were administered, a booster consisting of 100 µg of protein in Freund’s incomplete adjuvant (Difco Laboratories). The mice were bled 7 days after the primary and secondary immunization. Blood from five mice of each group was pooled and stored at 4 °C for 24 h to form a clot. Antisera were collected from each blood sample after clot formation. Enzyme-Linked Immunosorbent Assay (ELISA). A noncompetitive ELISA was carried out as follows. Antigen (β-LG, Conj. 10A, Conj. 10B, or RCM-β-LG) was dissolved in PBS at 0.01% (as protein). One hundred microliters of antigen solution was added to the wells of a polystyrene microtitration plate (Nunc, Roskilde, Denmark) and incubated at 25 °C for 2 h to coat the wells with each antigen. The plate was washed three times with 125 µL of PBS-Tween, and 125 µL of a 1% ovalbumin solution was then added. The plate was again incubated at 25 °C for 2 h and washed. One hundred microliters of antibody solution [the antiserum after the secondary immunization or monoclonal antibody (mAb)] was added. The plate was further incubated at 25 °C for 2 h. After washing, 100 µL of alkaline phosphataselabeled goat anti-mouse immunoglobulin (Dako A/S,

Hattori et al.

Glostrup, Denmark) diluted with PBS-Tween was added, and the plate was incubated again at 25 °C for 2 h. After washing, 100 µL of 0.1% p-nitrophenyl phosphate disodium salt dissolved in 1 M diethanolamine hydrochloride buffer (pH 9.8) was added as a substrate. The plate was incubated at 25 °C, the reaction was stopped by adding 20 µL of 6 M sodium hydroxide, and the absorbance at 405 nm was measured. A competitive ELISA was carried out to investigate the local conformational changes in β-LG after conjugation with CMD, using anti-β-LG mAbs (mAbs 21B3 and 61B4) as probes and to evaluate the antigenicity of the β-LGCMD conjugates as described previously (16-18, 34, 35). The equilibrium constant (KAS) of the mAb in binding to β-LG, RCM-β-LG, or β-LG-CMD conjugates was calculated according to the method of Hogg et al. (36), from the results of competitive and noncompetitive ELISA. Peptide Synthesis. Peptides were synthesized (37, 38) using a five-in-one B cell and T cell epitope scanning kit (Chiron Mimotopes, Clayton, Victoria, Australia). A series of overlapping 15-mer peptides moving one amino acid at a time in accordance with the amino acid sequence of β-LG was synthesized on activated polyethylene gears in each pin. Fmoc (9-fluorenylmethoxycarbonyl)-deprotection of the pins was performed by immersing in N,Ndimethylformamide (DMF) containing 20% piperidine at 25 °C for 20 min. The Fmoc-deprotected pins were successively washed with DMF for 2 min and methanol for 2 min. This washing procedure was conducted three times, before the washed pins were air-dried for 30 min in an acid-free fume. Each Fmoc-activated amino acid was dissolved in DMF containing 1-hydroxybenzotriazole (HOBt). The activated amino acid solutions were added to the wells of the reaction tray. Pins were placed in the wells and coupled with the activated amino acid at 25 °C overnight. After this coupling step, the pins were immersed in methanol for 5 min and air-dried for 2 min, before being immersed in DMF for 5 min. The Fmocdeprotection step and coupling step were repeated 14 times. After the last coupling step, the Fmoc-deprotection step was performed, and protective groups on amino acid residues were removed by treatment with trifluoroacetic acid (TFA) containing ethanedithiol and anisole. The pins were finally washed and air-dried. The success of the peptide synthesis was confirmed by ELISA using control peptide pins. Epitope Scanning. B cell epitopes were scanned by ELISA, using a series of overlapping 15-mer peptides on each pin as the antigen on a solid phase. A total of 200 µL of precoat buffer [0.11 M PBS at pH 7.0, containing 2% ovalbumin (w/v), 0.1% Tween 20 (v/v) and 0.1% NaN3 (w/v)] was added to the wells of the tray for blocking. Pins were put in the wells and incubated at 25 °C for 1 h to block with ovalbumin, before being washed in a bath of 0.01 M PBS (pH 7.2) for 10 min while shaking. Anti-βLG, anti-Conj. 10A, and anti-Conj. 10B antisera were each diluted with PBS (pH 7.0). Each antiserum (200 µL) prepared from blood after the secondary immunization was added to wells of a microtitration plate (Nunc Roskilde, Denmark). The pins were placed in the wells and incubated at 25 °C for 1.5 h before being removed from the plate and washed four times. A total of 200 µL of alkaline phosphatase-labeled goat anti-mouse immunoglobulin diluted with PBS-Tween was added to wells of a new plate. The pins were put in the wells again and incubated at 25 °C for 1 h. The pins were removed from the plate and washed four times. A total of 200 µL of 0.1% p-nitrophenyl phosphate disodium salt dissolved in a 1 M diethanolamine hydrochloride buffer (pH 9.8) was


Figure 1. Retinol binding activity of the β-LG-CMD conjugates. β-LG (b), Conj. 10A (2), Conj. 10B (4).

added to a new plate as a substrate. The pins were placed in the wells and incubated again at 25 °C. The enzyme reaction was stopped by removing the pins from the substrate solution, and the absorbance was determined at 405 nm. RESULTS AND DISCUSSION

Structural Features of the β-LG-CMD Conjugates. The covalent binding of CMD to β-LG was confirmed by the coincidence of the protein and saccharide stained bands observed upon analysis by SDSPAGE (data not shown). The molecular weights as determined by SEC were 282 000 for Conj. 10A and 251 000 for Conj. 10B; the respective molar ratios of β-LG to CMD were about 5:1 (Conj. 10A) and 2:1 (Conj. 10B). These results indicate that Conj. 10A and 10B were composed of 14 molecules of β-LG and 3 molecules of CMD and of 10 molecules of β-LG and 5 molecules of CMD, respectively. The thermal denaturation temperatures determined by DSC were 86.5 °C for Conj. 10A and 91.6 °C for Conj. 10B, which are much higher than that of β-LG (72.6 °C). The retinol-binding activity of the β-LG-CMD conjugates was found to be similar to that of β-LG (Figure 1).


The structure involved in retinol binding in the β-LGCMD conjugates is considered to have been maintained in the native form. The CD spectra of the β-LG-CMD conjugates are shown in Figure 2a. The CD spectrum of native β-LG showed a negative maximum at 216 nm, indicating that β-LG is rich in β-sheet structure, while that of each β-LG-CMD conjugate showed a broad negative maximum with a blue-shift. The β-sheet regions in β-LG are considered to have been changed by conjugation with CMD. The intrinsic fluorescence spectra of the β-LG-CMD conjugates were measured to evaluate the conformational changes around the Trp residues (19Trp and 61Trp) of β-LG (Figure 2b). The fluorescence emission maximum wavelength for β-LG was 337 nm, while those for Conj. 10A and 10B were 339 and 338 nm, respectively. It has previously been clarified that the fluorescence intensity increases with the red-shift of the wavelength for maximum emission as the conformation of β-LG changes (31, 34). Although a slight red-shift was observed in the case of each conjugate, the conformation around the Trp residues of the β-LG-CMD conjugates is considered to have retained a nativelike structure. The fluorescence intensity of each β-LG-CMD conjugate was lower than that of native β-LG, which indicates that the CMD polysaccharide chain shielded the area around the Trp residues (16). This effect of shielding of the Trp fluorescence was most marked in the case of Conj. 10B. Local conformational changes in β-LG after conjugation with CMD were evaluated by competitive ELISA with two anti-β-LG mAbs (21B3 and 61B4) as probes. These mAbs can detect the subtle conformational changes in local areas within the β-LG molecule during unfolding and refolding (31, 34, 35) and after conjugation with saccharides (16-18, 32, 33) by determining the affinity change. The epitope regions for mAbs 21B3 and 61B4 are 15Val-29Ile (β-sheet region) and 125Thr-135Lys (R-helix region), respectively. MAb 61B4 binds preferentially to native β-LG, while mAb 21B3 binds more strongly to RCM-β-LG (the denatured form of β-LG). The equilibrium constants (KAS) for binding of mAb 21B3 to the β-LG-CMD conjugates were similar to that for binding to native β-LG, while the KAS values for binding of mAb 61B4 to the β-LG-CMD conjugates were smaller than

Figure 2. Spectroscopic analysis of the β-LG-CMD conjugates. CD spectra (a), intrinsic fluorescence (b). β-LG (broken line), Conj. 10A (dotted line), Conj. 10B (solid line).


Hattori et al.

Figure 3. Equilibrium constants (KAS) of the β-LG-CMD conjugates in binding to anti-β-LG mAbs. β-LG (b), RCM-βLG (O), Conj. 10A (2), Conj. 10B (4). KAS values were calculated from the results of competitive and noncompetitive ELISA according to the procedure of Hogg et al. (36).

that for binding to native β-LG (Figure 3). Hence, the conformation around 15Val-29Ile (β-sheet region) is considered to have maintained its native form, while the conformation around 125Thr-135Lys (R-helix region) is considered to have been changed by conjugation with CMD. These results also raise the possibility that steric hindrance by CMD in the conjugates brought about the weak affinity of mAb 61B4 in binding to the β-LG-CMD conjugates. Carbohydrate-binding sites in the β-LG-CMD conjugates were partially determined to be 47Lys, 60Lys, and 138Lys for Conj. 10A and 101Lys for Conj. 10B, which are located on the surface of the β-LG molecule (3, 4). The low fluorescence intensity of the β-LG-CMD conjugates can be ascribed to the carbohydrate binding at 60Lys which is adjacent to 61Trp. Antigenicity of the β-LG-CMD Conjugates. The antigenicity of each β-LG-CMD conjugate in three strains of mice was evaluated by competitive ELISA (Figure 4). β-LG was adsorbed to the solid phase, and β-LG, Conj. 10A, Conj. 10B, and RCM-β-LG were used as competitive antigens. Although anti-β-LG antisera were elicited by immunization with native β-LG, they showed higher affinity to the denatured form of β-LG (RCM-β-LG) than to the native material in the case of all strains. These results are consistent with findings obtained by Takahashi et al. (39) that the binding of a trypsin-generated fragment of RCM-β-LG to anti-β-LG antisera of BALB/c mice was as strong as that of native β-LG. In our previous study (31) on the unfolding/ refolding of β-LG, using mAbs as probes, we found that a region of β-LG within the molecule was easily exposed during the denaturation process and was hard to refold. Such a structural feature of β-LG may be responsible for triggering the production of antibodies that preferentially react with the unfolded molecule although the antibodies were elicited by immunization with native material. CMD showed no reactivity with the anti-β-LG antiserum and was proven to have low antigenicity. Since the reactivity of the anti-β-LG antiserum with each β-LG-CMD conjugate was similar to that with native β-LG, the antigenicity of the β-LG-CMD conjugates is considered to have been similar to that of native β-LG. This nativelike antigenicity is believed to result from the total of an increase in reactivity with anti-β-LG antiserum due to conformational changes in β-LG that occurred as a result of conjugation with CMD and of a decrease in reactivity of the β-LG molecule due to the shielding effect of the CMD. Immunogenicity of the β-LG-CMD Conjugates. The immunogenicity of each β-LG-CMD conjugate in BALB/c, C57BL/6, and C3H/He mice was evaluated by

Figure 4. Antigenicity of the β-LG-CMD conjugates. The reactivity of anti-β-LG antisera after the secondary immunization from BALB/c (a), C57BL/6 (b), and C3H/He mice (c) with β-LG (b), RCM-β-LG (O), Conj. 10A (2), Conj. 10B (4), and CMD (0) was evaluated by competitive ELISA. B/B0 is the ratio of the absorbance at the last step of ELISA in the presence of various concentrations of a competitive antigen to the absorbance in the absence of the competitive antigen.

measuring the reactivity of 10000-fold diluted antisera with antigen (β-LG, RCM-β-LG, Conj. 10A, and Conj. 10B) adsorbed to the solid phase of a microtitration plate by noncompetitive ELISA (Figure 5). As already described, the anti-β-LG antisera reacted strongly with the denatured form of β-LG, so evaluation of the reactivity of each antiserum with the denatured material (RCMβ-LG) is considered to be required for examining immunogenicity. The anti-β-LG antibody response was markedly low in those three strains of mice immunized with each β-LG-CMD conjugate (Figure 5, panels a, e, and i). Effective reduction in immunogenicity of β-LG was observed with Conj. 10B. The antibody response to denatured β-LG (RCM-β-LG) was also only slightly elicited by immunization with each β-LG-CMD conjugate (Figure 5, panels b, f, and j). When each β-LG-CMD conjugate was used as the coating antigen on the solid



Figure 5. Immunogenicity of the β-LG-CMD conjugates in three strains of mice. The anti-β-LG response, anti-RCM-β-LG response, anti-Conj. 10A response, and anti-Conj. 10B response after the secondary immunization in BALB/c (a-d), C57BL/6 (e-h), and C3H/ He mice (i-l) were evaluated by noncompetitive ELISA.

phase to evaluate the productivity of the specific antibody for each conjugate (Figure 5c, d, g, h, k, and l), both antiConj. 10A antisera and anti-Conj. 10B antisera showed a higher antibody titer than anti-β-LG antisera. These results indicate the production of antibodies specific not for β-LG but for each conjugate and the emergence of novel immunogenicity in β-LG as a result of conjugation with CMD. The conformational changes in β-LG due to conjugation with CMD may have brought about new epitopes. Berzofsky (40) has reported that the hydrophobic region tended to become an epitope. New epitopes may have emerged as a result of exposure of the hydrophobic region, buried in the native state, upon conjugation with CMD. However, the decrease in immunogenicity of β-LG observed upon conjugation with CMD surpassed the emergence of novel immunogenicity. Thus, the conjugation of CMD to β-LG is considered to be effective for decreasing the immunogenicity of β-LG. In particular, immunization with Conj. 10B was more effective than that with Conj. 10A to lower anti-β-LG antibody production. Since shielding of the β-LG molecule by CMD was more marked with Conj. 10B than with Conj. 10A, as described in the section on the structural features of the β-LG-CMD conjugates, it is considered that masking of the epitopes by CMD was more effectively achieved in Conj. 10B than in Conj. 10A. Scanning of Epitopes in the β-LG-CMD Conjugates. Since the anti-β-LG antiserum reacted more strongly with the denatured form of β-LG (RCM-β-LG) than with native β-LG, determination of the linear epitopes (41) is important to clarify the B cell epitope profiles of β-LG and to clarify how conjugation with CMD leads to reduced immunogenicity of β-LG. In this study, the B cell epitopes were scanned by ELISA using overlapping 15-mer multipin peptides.

The results obtained with BALB/c mice are shown in Figure 6, panels a-c. The horizontal axis indicates the number of the N-terminal amino acid residue in each peptide, and the vertical axis shows the reactivity of each 15-mer peptide with each antiserum. In determining the immunogenicity profiles, an A405 value above 0.1 was adopted as positive. The epitopes were identified by determining the common amino acid sequence among the immunoreactive peptides by referring to the method of Gammon et al. (42) as summarized in Figure 7, in which the horizontal axis indicates the sequence number in β-LG, and the line thickness indicates the response to each epitope. Anti-β-LG-antiserum reacted to the peptides 1-3, 6-12, 21-31, 46-54, 61-69, 73-79, 92-93, 127-129, 133-137, and 141-143 (the number indicates residue number of N-terminus of the peptides). The linear B cell epitopes of β-LG recognized in BALB/c mice were determined to be 3Val-15Val, 12Ile-20Tyr, 31Leu-35Gln, 54Leu60Lys, 69Lys-75Lys, 79Pro-87Leu, 93Leu-106Cys, 129Asp141Lys, 137Asp-147Ile, and 143Leu-155Gln, the dominant epitope being 69Lys-75Lys, which is located in a β-sheet region. Although other B cell epitopes recognized in BALB/c mice were found in various regions, they tended to exist in regions with R-helix or β-sheet secondary structure (Figure 7). As for the anti-conjugates response in BALB/c mice (Figure 6, panels b and c), the epitope distribution did not change, while the decrease in response was marked through the entire amino acid sequence as compared with the anti-β-LG antiserum, which was eminent with Conj. 10B. The B cell epitopes of conjugates recognized in BALB/c mice were determined to be 3Val-15Val, 11Asp-20Tyr, 28Asp-42Tyr, and 69Lys80 Ala for Conj. 10A and 3Val-15Val and 11Asp-20Tyr for Conj. 10B (Figure 7). A decrease in the immunogenicity


Hattori et al.

Figure 6. Reactivity of anti-β-LG, anti-Conj. 10A and anti-Conj. 10B antisera with overlapping 15-mer peptides covering the amino acid sequence of β-LG. The reactivity of anti-β-LG (a, d, g), anti-conjugate 10A (b, e, h), and anti-conjugate 10B (c, f, i) antisera after the secondary immunization from BALB/c (a-c), C57BL/6 (d-f), and C3H/He mice (g-i) with multipin peptides was evaluated by ELISA.

of β-LG by conjugation with CMD was achieved in each conjugate as far as the linear B cell epitopes were concerned. In particular, the immunogenicity of β-LG was almost completely nullified in Conj. 10B. However, 3Val15 Val showed a stronger signal with the anti-Conj. 10A antiserum than with the anti-β-LG serum, so this region might be involved in the emergence of novel immunogenicity in Conj. 10A. The results obtained with C57BL/6 and C3H/He mice are shown in Figure 6, panels d-i, and B cell epitope profiles of β-LG and the conjugates were determined as shown in Figure 7. The B cell epitopes of β-LG recognized in C57BL/6 mice were determined to be 12Ile-20Tyr, 19 Trp-33Asp, 36Ser-49Thr, 47Lys-61Trp, 54Leu-67Ala, 61Trp-74Glu, 70Lys-82Phe, 93Leu-106Cys and 138Lys147 Ile, the dominant epitopes being 12Ile-20Tyr and 138Lys-147Ile (Figure 7), most of which contained loop regions of the β-LG molecule. With respect to the anticonjugates response in C57BL/6 mice, the epitope distribution was similar to that of β-LG, and the response was less throughout the entire amino acid sequence than the anti-β-LG response (Figure 6, panels e and f). In particular, an effective reduction of the immunogenicity of β-LG was achieved by conjugation with CMD in the case of Conj. 10B so far as the linear B cell epitopes were concerned. The B cell epitopes of the conjugates in C57BL/6 mice were determined to be 11Asp-19Trp, 19Trp33 Asp, 28Asp-42Tyr, 69Lys-82Phe, 99Tyr-106Cys, 136Phe-

146His,

and 148Arg-155Gln for Conj. 10A and 12Ile-20Tyr, Trp- Asp, 61Trp-74Glu, 70Lys-80Ala, 93Leu-106Cys, and 138Lys-148Arg for Conj. 10B (Figure 7). 28Asp-42Tyr and 148Arg-155Gln showed slightly stronger signals with the anti-Conj. 10A antiserum than with the anti-β-LG serum. This region might be involved in the emergence of novel immunogenicity in Conj. 10A. As for the anti-β-LG antiserum response in C3H/He mice, the linear B cell epitopes of β-LG recognized in C3H/He mice were determined to be 12Ile-20Tyr, 38Pro42Tyr, 54Leu-61Trp, 67Ala-77Lys, 73Ala-82Phe, 79Pro90 Asn, 90Asn-99Tyr, 93Leu-106Cys, and 138Lys-146His, the dominant epitope being 138Lys-146His (Figure 7), most of which also contained loop regions. Regarding the anticonjugates response in C3H/He mice, the epitope distribution was also similar to that of β-LG, and for each peptide, the signal obtained with the anti-conjugates sera was weaker than that with the anti-β-LG antiserum (Figure 6, panels h and i). A decrease in the immunogenicity of β-LG in C3H/He mice was achieved in the case of each conjugate as far as the linear B cell epitopes were concerned. In particular, the response of anti-Conj. 10B antisera was almost completely lacking, except for the weak signals obtained with peptides corresponding to the C-terminal region. The B cell epitopes of conjugates recognized in C3H/He mice were determined to be 11Asp20Tyr, 33Asp-42Tyr, 77Lys-80Ala, 93Leu-106Cys, and 138 Lys-146His for Conj. 10A and 11Asp-22Leu, 69Lys-8019

33



Figure 7. B cell epitope profiles of β-LG and the β-LG-CMD conjugates. The common regions which gave high ELISA values are shown. Hatched lines represent the antigenic peptides obtained from β-LG by tryptic digestion that were previously found to be immunogenic in BALB/c mice by Takahashi et al. (39).

Ala, and 138Lys-147Ile for Conj. 10B. (Figure 7). However, the reactivity of 11Asp-20Tyr and 33Asp-42Tyr with the anti-Conj. 10A antiserum was greater than that with the anti-β-LG antiserum, so these regions might be involved in the emergence of novel immunogenicity in Conj. 10A. The results of the precise analysis with multipin peptides enabled the profiles of the B cell epitopes of β-LG recognized in three strains of mice to be clarified, and reduction of the immunogenicity of β-LG by conjugation with CMD was demonstrated. A reduction of the immunogenicity of β-LG as a result of conjugation with CMD was achieved with each conjugate. Although the precise mechanism responsible for the reduction of the immunogenicity of proteins through conjugation with polysaccharides is still unclear, a plausible one is that shielding of the epitopes in β-LG by CMD brought about its escape from recognition by the immune system. In fact, Conj. 10B, in which the surface of β-LG was effectively shielded by CMD, showed very low immunogenicity. Moreover, after conjugation with CMD, the response to the epitope regions around the carbohydrate-binding sites in the β-LG-CMD conjugates (47Lys, 60Lys, and 138Lys for Conj. 10A and 101Lys for Conj. 10B) was lowered in each strain of mice. For instance, responses to 54Leu-60Lys, 129Asp141Lys, and 137Asp-147Ile in BALB/c mice, 47Lys-61Trp in C57BL/6 mice, and 54Leu-61Trp in C3H/He mice were dramatically reduced. CMD-conjugation was especially effective in reduction of the immunogenicity of β-LG in the case of BALB/c mice, for which the epitopes recognized were found in various regions. Several studies on the mechanisms responsible for the reduction of immunogenicity of proteins as a result of conjugation with macromolecules have been carried out so far. Among them, the induction of regulatory T cells upon immunization with conjugates has been reported. In the pioneering

work of Lee et al. (43), they investigated the mechanism of suppression of IgE antibody production upon immunization with ovalbumin-polyethylene glycol conjugate and suggested that the induction of regulatory T cells would bring about the suppressive effect. Kishimoto et al. (44) also have reported that regulatory T cells were induced by injection of dinitrophenol (DNP)-tubercle bacillus conjugates and that the production of anti-DNP IgE antibody was suppressed. Takata et al. (45) reported that regulatory T cells and a soluble factor derived from these cells suppressed the growth of spleen cells from mice sensitized by treatment with antigen. One of the other reasons for the low immunogenicity of modified proteins is the increased resistance of modified proteins to proteolysis during antigen processing. So et al. (46, 47) have reported that PEG-modified lysozyme showed a level of incorporation by spleen cells similar to that of lysozyme, but it showed low susceptibility to digestion by various proteases. In the case of the β-LG-CMD conjugates prepared in the present work, there is a possibility that the induction of regulatory T cells upon immunization of the mice with each conjugate and low susceptibility to digestion by endosomal/lysosomal enzymes, as well as masking of epitopes by CMD, may contribute to the diminished antiβ-LG antibody response. Further studies focusing on these aspects should be carried out. Totsuka et al. (48) have recently determined the T cell determinants of β-LG recognized in BALB/c, C57BL/6, and C3H/He mice. We intend to clarify the change in T cell response to β-LG after conjugation with CMD. Concluding Remarks. Although many protein conjugates have been prepared and studied to decrease the antigenicity and immunogenicity so far, changes in the epitope profiles have not been clarified. Our work is the


first study to clarify the changes in the epitope profiles of a protein after conjugation with saccharides. We could show that the immunogenicity of β-LG is dramatically reduced by conjugation with CMD. However, the possibility of the emergence of novel immunogenicity was also indicated. Since the reduction of the immunogenicity of β-LG was most marked in the case of Conj. 10B, which contains a greater amount of polysaccharide than Conj. 10A and in which shielding of the surface of β-LG was effective, it is evident that the problem of the emergence of novel immunogenicity can be overcome by glycoconjugation with a high shielding effect. It would be of interest to evaluate the effects of different amounts of CMD of differing molecular weights conjugated with β-LG in terms of the reduction of immunogenicity. We conclude that the conjugation with CMD performed in this study is an effective method for diminishing the immunogenicity of β-LG. Further studies on the mechanism responsible for the reduction of immunogenicity by this method should be carried out, and it is strongly hoped that such conjugates will contribute to the development of new foods with low allergenicity. ACKNOWLEDGMENT

This work was supported in part by the grant of The Japan Food Industry Center (JAFIC). We are grateful to Professor Shuichi Kaminogawa of The University of Tokyo for his help in the ELISA experiments. We are grateful to Dr. Akio Ametani of La Jolla Institute for Allergy and Immunology for critical review of the manuscript. We gratefully acknowledge helpful discussions with Drs. Jun-ich Kurisaki and Mamoru Totsuka on several points in this paper. We are very grateful to Takashi Takakuwa of Jasco, Tokyo, Japan for his help in measuring the CD spectra. LITERATURE CITED (1) Jennes, R. (1979) Comparative aspects of milk proteins. J. Dairy Res. 46, 197-210. (2) McKenzie, H. A. (1971) β-Lactoglobulins. In Milk Proteins: Chemistry and Molecular Biology (H. A. McKenzie, Ed.) pp 257-330, Academic Press, New York. (3) Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadrao, R., Jones, T. A., Newcomer, M. E., and Kraulis, P. J. (1986) The Structure of β-Lactoglobulin and Its Similarity to Plasma Retinol-Binding Protein. Nature 324, 383-385. (4) Brownlow, S., Morais, C. J., Cooper, R., Flower, D. R., Yewdall, S. J., Polikarpov, I., North, A. C., and Sawyer, L. (1997) Bovine β-Lactoglobulin at 1.8 A ResolutionsStill an Enigmatic Lipocalin. Structure 5, 481-495. (5) Pervaiz, S., and Brew, K. (1985) Homology of β-Lactoglobulin, Serum Retinol Binding Protein, and Protein HC. Science 228, 335-337. (6) A° kerstrom, B., and Lo¨gdberg, L. (1990) An Intriguing Member of the Lipocalin Protein Family: R1-Microglobulin. Trends Biochem. Sci. 15, 240-243. (7) Shimizu, M., Saito, M., and Yamauchi, K. (1985) Emulsifying and Structural Properties of β-Lactoglobulin at Different pHs. Agric. Biol. Chem. 49, 189-194. (8) Waniska, R. D., and Kinsella, J. E. (1988) Foaming and Emulsifying Properties of Glycosylated β-Lactoglobulin. Food Hydrocolloids 2, 439-449. (9) Mulvihill, D. M., and Kinsella, J. E. (1987) Gelation Characteristics of Whey Proteins and β-Lactoglobulin. Food Technol. 41 (9), 102-111. (10) Spies, J. (1973) Milk Allergy. J. Milk Food Technol. 36, 225-231. (11) Kurisaki, J., Nakamura, S., Kaminogawa, S., and Yamauchi, K. (1982) The Antigenic Properties of β-Lactoglobulin Examined with Mouse IgE Antibody. Agric. Biol. Chem. 46, 2069-2075.

Hattori et al. (12) Watanabe, M., Suzuki, T., Ikezawa, Z., and Arai, S. (1994) Controlled Enzymatic Treatment of Wheat Proteins for Production of Hypoallergenic Flour. Biosci. Biotech. Biochem. 58, 288-390. (13) Patterson, R., Suszko, I. M., Pruzansky, J. J., and Zeiss, R. (1973) Polymerized Ragweed Antigen E. ii. In vivo Elimination Studies and Reactivity with IgE Antibody Systems. J. Immunol. 110, 1413-1418. (14) Takatsu, K., and Ishizaka, K. (1976) Reaginic Antibody Formation in the Mouse. VII. Induction of Suppressor T cells for IgE and IgE Antibody Response. J. Immunol. 116, 12571264. (15) Ishizaka, K., Okudaira, H., and King, T. P. (1975) Immunogenic Properties of Modified Antigen E II. Ability of Urea-Denatured Antigen and a-Polypeptide Chain to Prime T cells Specific For Antigen C. J. Immunol. 114, 110-115. (16) Hattori, M., Nagasawa, K., Ametani, A., Kaminogawa, S., and Takahashi, K. (1994) Functional Changes in β-Lactoglobulin by Conjugation with Carboxymethyl Dextran. J. Agric. Food Chem. 42, 2120-2125. (17) Nagasawa, K., Takahashi, K., and Hattori, M. (1996) Improved Emulsifying Properties of β-Lactoglobulin by Conjugating with Carboxymethyl Dextran. Food Hydrocolloids 10, 63-67. (18) Nagasawa, K., Ohgata, K., Takahashi, K., and Hattori, M. (1996) Role of the Polysaccharide Content and Net Charge on the Emulsifying Properties of β-Lactoglobulin-Carboxymethyl Dextran Conjugates. J. Agric. Food Chem. 44, 25382543. (19) Lee, W. Y., and Sehon, A. H. (1977) Abrogation of reaginic antibodies with modified allergens. Nature 267, 618-619. (20) Kimoto, A., Konno, T., Kawaguchi, T., Miyauchi, Y., and Maeda, H. (1992) Antitumor effects of SMANCS on Rat Mammary Tumor Induced by 7,12-Dimethylbenz[a]anthracene. Cancer Res. 52, 1013-1017. (21) Holle, L. M. (1997) Pegaspargase: An Alternative? Ann. Pharmacother. 31, 616-624. (22) Fagnani, R., Hagan, M. S., and Bartholomew, R. (1990) Reduction of Immunogenicity by Covalent Modification of Murine and Rabbit Immunoglobulins with Oxidized Dextrans of Low Molecular Weight. Cancer Res. 50, 3638-3645. (23) Akita, E. M., and Nakai, S. (1990) Lipophilization of β-lactoglobulin: Effect on Allergenicity and Digestibility. J. Food Sci. 55, 718-723. (24) Sehon, A. H. (1982) Suppression of IgE Antibody Responses with Tolerogenic Conjugates of Allergens and Haptens. Prog. Allergy 32, 161-202. (25) Smith, R. J. (1967) Characterization and Analysis of Starches. In Starch, Chemistry and Technology (R. L. Whistler and F. F. Paschall, Eds.) pp 569-635, Academic Press, New York. (26) Armstrong, J. M., McKenzie, H. A., and Sawyer, W. H. (1967) On the Fractionation of β-Lactoglobulin and R-Lactalbumin. Biochim. Biophys. Acta 147, 60-72. (27) Davis, B. J. (1964) DISC ELECTROPHORESIS - II: Method and Application to Human Serum Proteins. Ann. N. Y. Acad. Sci. 121, 404-427. (28) Hase, S., Ikenaka, T., and Matsushima, Y. (1978) Structure Analyses of Oligosaccharides by Tagging of the Reduced End Sugars with a Fluorescent Compound. Biochem. Biophys. Res. Commun. 85, 257-263. (29) Cogan, U., Kopelman, M., Mokady, S., and Shintzky, M. (1976) Binding Affinities of Retinol and Related Compounds to Retinol Binding Proteins. Eur. J. Biochem. 65, 71-78. (30) Futterman, S., and Heller, J. (1972) The Enhancement of Fluorescence and Decreased Susceptibility to Enzyme Oxidation of Retinol Complexed with Bovine Serum Albumin, β-Lactoglobulin, and the Retinol-Binding Protein of Human Plasma. J. Biol. Chem. 247, 5168-5172. (31) Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., and Kaminogawa, S. (1993) Unfolding/Refolding Studies on Bovine β-Lactoglobulin with Monoclonal Antibodies as Probes: Does a Renatured Protein Completely Refold? J. Biol. Chem. 268, 22414-22419.

Immunogenicity of Protein−Polysaccharide Conjugate (32) Hattori, M., Aiba, Y., Nagasawa, K., and Takahashi, K. (1996) Functional Improvements in Alginic Acid by Conjugating with β-Lactoglobulin. J. Food Sci. 61, 1171-1176. (33) Hattori, M., Ogino, A., Nakai, H., and Takahashi, K. (1997) Functional Improvement of β-Lactoglobulin by Conjugating with Alginate Lyase-Lysate. J. Agric. Food Chem. 45, 703708. (34) Kaminogawa, S., Hattori, M., Ando, O., Kurisaki, J., and Yamauchi, K. (1987) Preparation of Monoclonal Antibody against Bovine β-Lactoglobulin and Its Unique Affinity. Agric. Biol. Chem. 51, 797-802. (35) Kaminogawa, S., Shimizu, M., Ametani, A., Hattori, M., Ando, O., Hachimura, S., Nakamura, Y., Totsuka, M., and Yamauchi, K. (1989) Monoclonal antibodies as probes for monitoring the denaturation process of bovine β-lactoglobulin. Biochim. Biophys Acta 998, 50-56. (36) Hogg, P. J., Johnson, S. C., Bowles, M. R., Pond, S. M., and Winzor, D. J. (1987) Evaluation of Equilibrium Constants for Antigen-Antibody Interaction by Solid-phase immunoassay: the Binding of Paraquant to Its Elicited Mouse Monoclonal Antibody. Mol. Immunol. 24, 797-801. (37) Maeji, N. J., Bray, A. M., and Geysen, H. M. (1990) Multipin peptide synthesis strategy for T cell determinant analysis. J. Immunol. Methods 134, 23-33. (38) Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) Strategies for epitope analysis using peptide synthesis. J Immunol. Methods 102, 259-274. (39) Takahashi, T., Yamauchi, K., and Kaminogawa, S. (1990) Comparison between the antigenicity of native and unfolded β-lactoglobulin. Agric. Biol. Chem. 54, 691-697. (40) Berzofsky, J. A. (1985) Intrinsic and extrinsic factors in protein antigenic structure. Science 229, 932-940. (41) Atassi, M. Z. (1984) Antigenic Structures of Proteins. Their Determination Has Revealed Important Aspects of Immune Recognition and Generated Strategies for Synthetic Mimicking of Protein Binding Sites. Eur. J. Biochem. 145, 1-20.

Bioconjugate Chem., Vol. 11, No. 1, 2000 93 (42) Gammon, G., Geysen, H. M., Apple, R. J., Pickett, E., Palmer, M., Ametani, A., and Sercarz, E. E. (1991) T Cell Determinant Structure: Cores and Determinant Envelopes in Three Mouse Major Histocompatibility Complex Haplotypes. J. Exp. Med. 173, 609-617. (43) Lee, W. Y., Sehon, A. H., and A° kerblom, E. (1981) Suppression of Reaginic Antibodies with Modified Allergens. IV. Induction of Suppressor T cells by Conjugates of Polyethylene glycol (PEG) and Monomethoxy PEG with Ovalbumin. Int. Arch. Allergy. Appl. Immunol. 64, 100-114. (44) Kishimoto, T., Hirai, Y., Suemura, M., and Yamamura, Y. (1976) Regulation of Antibody Response in Different Immunoglobulin Classes. I. Selective Suppression of Anti-DNP IgE Antibody Response by Preadministration of DNP-coupled Mycobacterium. J. Immunol. 117, 396-404. (45) Takata, M., Maiti, P. K., Kubo, R. T., Chen, Y. H., Holford, S. V., Rector, E. S., and Sehon, A. H. (1990) Cloned Suppressor T Cells Derived from Mice Tolerized with Conjugates of Antigen and Monomethoxypolyethylene glycol. Relationship between Monoclonal T Suppressor Factor and the T Cell Receptor. J. Immunol. 145, 2846-2853. (46) So, T., Ito, H. O., Koga, T., Ueda, T., and Imoto, T. (1996) Reduced Immunogenicity of Monomethoxypolyethylene glycolModified Lysozyme for Activation of T Cells. Immunol. Lett. 49, 91-97. (47) So, T., Ito, H. O., Koga, T., Watanabe, S., Ueda, T., and Imoto, T. (1997) Depression of T-cell Epitope Generation by Stabilizing Hen Lysozyme. J. Biol. Chem. 272, 32136-32140. (48) Totsuka, M., Ametani, A., and Kaminogawa, S. (1997) Fine Mapping of T-cell Determinants of Bovine β-Lactoglobulin. Cytotechnology 25, 101-113.

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