Immunology 1979 38 509
Immunological properties of peptic fragments of bovine serum albumin
S. DOSA,* A. J. PESCE, D. J. FORD, ANNETTE MUCKERHEIDE & J. G. MICHAEL Departments of Medicine and Microbiology, University of Cincinnati College ofMedicine, Cincinnati, Ohio, U.S.A.
Acceptedfor publication 31 May 1979
INTRODUCTION
Summary. The effect of peptic degradation on the immunological and antigenic properties of bovine serum albumin (BSA) was investigated. Molecular fragments obtained after various times of digestion (3-360 min) were studied. Enzymatic digestion resulted in a rapid loss of serologically defined antigenic determinants. The immunogenicity of the fragments as measured by the level of reaginic and total anti-BSA antibody response in BDF1 mice was also diminished. Pre-treatment of mice with fragments exhibiting a low density of B-cell interacting determinants before immunization with BSA, resulted in significant suppression of both the primary and secondary antibody response. The most effective immunosuppressive fragments were obtained following removal of peptides which bound to anti-BSA antibodies. The suppression was shown to be antigen specific. It was concluded that separate determinants on the BSA molecule were responsible for the immunogenic and suppressive properties of the antigen.
The multideterminant nature of protein antigens is well established (Lapresle, Kaminski & Tanner, 1958; Feldhoff& Peters, 1975). Due to their structural complexity, information on the role of different determinants in the regulation of the immune response is still limited. Recently evidence has accumulated that alteration or destruction of serologically defined determinants by chemical modification (Bach & Brasher, 1975; Ishizaka, Okudaira & King, 1975; Takatsu & Ishizaka, 1975) or molecular fragmentation with proteolytic enzymes (Turkin & Sercarz, 1977; Muckerheide, Pesce & Michael, 1977) decreases the immunogenicity and enhances the immunosuppressive properties of protein antigens. The relationship between serologically defined determinants and the factors responsible for the immunosuppressive properties of these antigens has not been established. To examine this relationship in a systematic manner we investigated the properties of molecular fragments formed by peptic degradation of bovine serum albumin (BSA). BSA was selected because its antigenic structure is well characterized (Habeeb & Atassi, 1977; Peters, Feldhoff& Reed, 1977), its primary sequence is known (Brown 1977) and it is available in purified form in sufficient amounts. Results of our investigations show that certain peptides released during the course of degradation are highly immunosuppressive. These peptides are characterized by a low density of
* Present address: The George Washington University Medical Center, Department of Medicine, 2150 Pennsylvania Avenue, N.W., Washington, D.C. 20037. Correspondence: Professor J. Gabriel Michael, Department of Microbiology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267, U.S.A.
0019-2805/79/1 100-0509$02.00 © 1979 Blackwell Scientific Publications
509
S. Dosa et al.
510
serologically defined antigenic determinants and lack of immunogenicity. Our data provide evidence that in addition to determinants recognized by B cells, other determinants available on the antigenic structure have important immunoregulatory functions.
MATERIALS AND METHODS Preparation and characterization of antigens Limited peptic digestion of BSA was performed as described previously (Weber & Young, 1964a). Briefly, 2 g of BSA (Miles Research Products) were dissolved in distilled water to a concentration of 20 mg/ml. The pH was lowered to 2-9 with 2 M HCl. Digestion was initiated at 250 with pepsin at 1:650 pepsin (Worthington Biochemical Corp) to albumin ratio (w/w). During digestion, the pH of the solution was kept within + 0 05 pH units by titration with 0 1 M HCI. Aliquots (0 5 ml) were removed at 3,6, 12,20,40, 90, 180 and 360 min of digestion. The reaction was stopped by adjusting the pH to 7-5 with the addition of 1 M NaOH. The solution was left at room temperature for 20 min to allow denaturation of pepsin. The final pH was adjusted to 7T2 with 1 M HCl and the aliquots were stored at - 70°. Fluorescence-binding efficiency of various digests was determined as described by Weber & Young (1964b). The fluorescence of a BSA-I-anilino-naphthalene-8-sulphonic acid (ANS) complex before digestion (Fo) was defined as 1-0 and the relativefluorescence of samples (F) was expressed as the percentage of this
value (F/Fo x 100). The molecular weights offragments were determined by electrophoresis in 15% sodium dodecyl sulphate polyacrylamide gel (Laemli, 1970). The molecular weight standards were: immunoglobulin light chain dimers 44,000, light chain monomers 22,000, lysozyme 14,500, and insulin 5500. The presence of serologically defined antigenic determinants on the molecular fragments was examined by double immunodiffusion in agarose and passive haemagglutination inhibition studies. The antisera to BSA were raised in rabbits over a period of 6 weeks by weekly immunization with BSA in Freund's complete adjuvant. The antisera were pooled and decomplemented by heating at 560 for 30 min. The antiserum agglutinated BSA-coated tanned red cells at a dilution 1: 80,000. For the inhibition studies a 1: 5000 dilution of antiserum was used.
Preparation of immunoadsorption columns Rabbit anti-BSA antiserum was partially purified by precipitation with 35% ammonium sulphate. The antibody was conjugated to Sepharose 4B with cyanogen bromide according to the method of March, Parikh & Cuatrecasas (1974). Titration experiments showed that the conjugate adsorbed 0-6 mg of BSA/ml of packed gel. The amount of BSA or BSA digest applied to each column was 25-50% below the calculated column capacity. The non-adsorbed material was eluted with PBS, pH 7-2, and the adsorbed protein with 0-1 M glycine buffer, pH 2-8. The protein concentration in the eluted fractions was measured by absorbancy at 280 nm and by the biuret reaction. Immunization Female BDF1 mice, 8-12 weeks of age, were purchased from Jackson Laboratories, Bar Harbor, Maine. One hundred micrograms of BSA or BSA digest were absorbed to 1 mg of Al(OH)3 gel (Levine & Vaz, 1970) and injected intraperitoneally into groups of four to six mice. Animals were bled from the retro-orbital plexus and the bleedings (0-2 ml from each animal) were pooled. The reaginic anti-BSA antibody titre was estimated by passive cutaneous anaphylaxis (PCS) as described by Ovary (1964). This test was performed in duplicate or triplicate. The results were expressed in PCA units which are the reciprocal of the geometric means of the highest dilutions, resulting in PCA reactions 5 mm or greater in diameter. Variation for the same sample was two-fold or less; differences greater than this were taken to be significant. Heating sera at 560 for 4 h destroyed the 48 h PCA reactivity, confirming that the antibodies belong to the IgE class (Schwartz & Levine, 1973). A modification of the Farr technique (Muckerheide et al., 1977) was used to measure the total anti-BSA antibody response. Under the assay conditions, normal mouse serum consistently bound less than 2% of the antigen. Antigen-binding capacity (ABC) of the tested serum was calculated from the linear relationship between the binding capacity of the serum and the percentage of radioactivity precipitated. This linear relationship held between 0 and 35% of precipitation. The ABC was expressed as pg of BSA bound/ml of serum.
RESULTS Digestion of BSA with pepsin The rate of hydrogen ion uptake and the change in
Peptic fragments of BSA
ciency decreased to 63% of the initial value after 3 min
100 _
and to 2% after 40 min of digestion. Polyacrylamide gel electrophoresis (Fig. 2) revealed several peptides of various molecular sizes during the early stages of hydrolysis. After 3-12 min of digestion, the majority of peptides had a mol. wt of 16,000-44,000. With continuing degradation of the molecule, peptides of lower molecular weight were generated, forming three distinct bands in the 8000-16,000 region. In the late digest (360 min) a new distinct band appeared with a molecular weight around 5000.
80 _
'j60 401J20
0QQ 0
20 40 60 90180 360 MINUTES OF DIGESTION Figure 1. Hydrogen ion uptake and changes in fluorescencebinding efficiency of BSA during the course of pepsin digestion at pH 2 9. > Q
511
*0o
fluorescence-binding efficiency of BSA after various periods of pepsin digestion are shown in Fig. 1. The initial fast phase of hydrolysis was followed by a slower degradation of the molecule. An average of ten to eleven peptide bonds per molecule were cleaved at 6 min, and thirty-four to thirty-six peptide bonds per molecule at 40 min. The fluorescence-binding effi-
Immunochemical characterization of various digests To determine the effect of degradation on the serologically defined determinants, digests were immunoadsorbed on anti-BSA-Sepharose 4B columns. The quantification of adsorption is shown in Table 1. Fragments produced by a digestion period greater than 20 min were not retained by the column. On 15% polyacrylamide gel electrophoresis (Fig. 3) the unadsorbed peptides migrated into the low mol. wt region (I 2,000-30,000), whereas absorbed peptides generally had higher molecular weights (20,000-50,000).
m.w4 44000
22000 14500
3
6 12 20 40 90 180 360 DURATION OF DIGESTION (min)
Figure 2. SDS-polyacrylamide gel electrophoretic analysis of peptic digests of BSA.
S. Dosa et al.
512
CD
mw.
CD AF UF
AF UF -,mono ',, I-,M", .
............
-v'I;.'-;
-
UF UF UF
:. '-:* -h
f:: :."; so
69000 44000 22000 14500
LSIbrag
_
3 20 90 360 12 DURATION OF DIGESTION (min) Figure 3. SDS-polyacrylamide gel electrophoretic analysis of peptic digests of BSA after fractionation by anti-BSA-Sepharose 4B columns. CD, crude digest; AF, adsorbed fraction; UF, unadsorbed fraction.
When examined in an Ouchterlony double diffusion test in agarose, peptides obtained by digestion for longer than 12 min as well as those not retained by the immunoadsorption columns did not precipitate. Since the formation of precipitating BSA-anti-BSA immune complexes depends on the presence of two or more antigenic sites on the molecule (Peters et al., 1977), non-precipitating peptides may also bear antigenic determinants present on the native molecule. To determine the presence of such determinants, haemagglutination inhibition studies were done. As shown in Table 2, no inhibition of haemagglutination of BSA-
coated erythrocytes with anti-BSA serum occurred with peptides digested for 20 min or longer, nor with fractions unadsorbed by the columns. ld
,lt Isa
3520 w a-
BSA in Alum
BSA in Alum
20
A
I
I
-0
80 _
*
*
-
5
Immunological properties of peptic fragments of bovine serum albumin
S. DOSA,* A. J. PESCE, D. J. FORD, ANNETTE MUCKERHEIDE & J. G. MICHAEL Departments of Medicine and Microbiology, University of Cincinnati College ofMedicine, Cincinnati, Ohio, U.S.A.
Acceptedfor publication 31 May 1979
INTRODUCTION
Summary. The effect of peptic degradation on the immunological and antigenic properties of bovine serum albumin (BSA) was investigated. Molecular fragments obtained after various times of digestion (3-360 min) were studied. Enzymatic digestion resulted in a rapid loss of serologically defined antigenic determinants. The immunogenicity of the fragments as measured by the level of reaginic and total anti-BSA antibody response in BDF1 mice was also diminished. Pre-treatment of mice with fragments exhibiting a low density of B-cell interacting determinants before immunization with BSA, resulted in significant suppression of both the primary and secondary antibody response. The most effective immunosuppressive fragments were obtained following removal of peptides which bound to anti-BSA antibodies. The suppression was shown to be antigen specific. It was concluded that separate determinants on the BSA molecule were responsible for the immunogenic and suppressive properties of the antigen.
The multideterminant nature of protein antigens is well established (Lapresle, Kaminski & Tanner, 1958; Feldhoff& Peters, 1975). Due to their structural complexity, information on the role of different determinants in the regulation of the immune response is still limited. Recently evidence has accumulated that alteration or destruction of serologically defined determinants by chemical modification (Bach & Brasher, 1975; Ishizaka, Okudaira & King, 1975; Takatsu & Ishizaka, 1975) or molecular fragmentation with proteolytic enzymes (Turkin & Sercarz, 1977; Muckerheide, Pesce & Michael, 1977) decreases the immunogenicity and enhances the immunosuppressive properties of protein antigens. The relationship between serologically defined determinants and the factors responsible for the immunosuppressive properties of these antigens has not been established. To examine this relationship in a systematic manner we investigated the properties of molecular fragments formed by peptic degradation of bovine serum albumin (BSA). BSA was selected because its antigenic structure is well characterized (Habeeb & Atassi, 1977; Peters, Feldhoff& Reed, 1977), its primary sequence is known (Brown 1977) and it is available in purified form in sufficient amounts. Results of our investigations show that certain peptides released during the course of degradation are highly immunosuppressive. These peptides are characterized by a low density of
* Present address: The George Washington University Medical Center, Department of Medicine, 2150 Pennsylvania Avenue, N.W., Washington, D.C. 20037. Correspondence: Professor J. Gabriel Michael, Department of Microbiology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267, U.S.A.
0019-2805/79/1 100-0509$02.00 © 1979 Blackwell Scientific Publications
509
S. Dosa et al.
510
serologically defined antigenic determinants and lack of immunogenicity. Our data provide evidence that in addition to determinants recognized by B cells, other determinants available on the antigenic structure have important immunoregulatory functions.
MATERIALS AND METHODS Preparation and characterization of antigens Limited peptic digestion of BSA was performed as described previously (Weber & Young, 1964a). Briefly, 2 g of BSA (Miles Research Products) were dissolved in distilled water to a concentration of 20 mg/ml. The pH was lowered to 2-9 with 2 M HCl. Digestion was initiated at 250 with pepsin at 1:650 pepsin (Worthington Biochemical Corp) to albumin ratio (w/w). During digestion, the pH of the solution was kept within + 0 05 pH units by titration with 0 1 M HCI. Aliquots (0 5 ml) were removed at 3,6, 12,20,40, 90, 180 and 360 min of digestion. The reaction was stopped by adjusting the pH to 7-5 with the addition of 1 M NaOH. The solution was left at room temperature for 20 min to allow denaturation of pepsin. The final pH was adjusted to 7T2 with 1 M HCl and the aliquots were stored at - 70°. Fluorescence-binding efficiency of various digests was determined as described by Weber & Young (1964b). The fluorescence of a BSA-I-anilino-naphthalene-8-sulphonic acid (ANS) complex before digestion (Fo) was defined as 1-0 and the relativefluorescence of samples (F) was expressed as the percentage of this
value (F/Fo x 100). The molecular weights offragments were determined by electrophoresis in 15% sodium dodecyl sulphate polyacrylamide gel (Laemli, 1970). The molecular weight standards were: immunoglobulin light chain dimers 44,000, light chain monomers 22,000, lysozyme 14,500, and insulin 5500. The presence of serologically defined antigenic determinants on the molecular fragments was examined by double immunodiffusion in agarose and passive haemagglutination inhibition studies. The antisera to BSA were raised in rabbits over a period of 6 weeks by weekly immunization with BSA in Freund's complete adjuvant. The antisera were pooled and decomplemented by heating at 560 for 30 min. The antiserum agglutinated BSA-coated tanned red cells at a dilution 1: 80,000. For the inhibition studies a 1: 5000 dilution of antiserum was used.
Preparation of immunoadsorption columns Rabbit anti-BSA antiserum was partially purified by precipitation with 35% ammonium sulphate. The antibody was conjugated to Sepharose 4B with cyanogen bromide according to the method of March, Parikh & Cuatrecasas (1974). Titration experiments showed that the conjugate adsorbed 0-6 mg of BSA/ml of packed gel. The amount of BSA or BSA digest applied to each column was 25-50% below the calculated column capacity. The non-adsorbed material was eluted with PBS, pH 7-2, and the adsorbed protein with 0-1 M glycine buffer, pH 2-8. The protein concentration in the eluted fractions was measured by absorbancy at 280 nm and by the biuret reaction. Immunization Female BDF1 mice, 8-12 weeks of age, were purchased from Jackson Laboratories, Bar Harbor, Maine. One hundred micrograms of BSA or BSA digest were absorbed to 1 mg of Al(OH)3 gel (Levine & Vaz, 1970) and injected intraperitoneally into groups of four to six mice. Animals were bled from the retro-orbital plexus and the bleedings (0-2 ml from each animal) were pooled. The reaginic anti-BSA antibody titre was estimated by passive cutaneous anaphylaxis (PCS) as described by Ovary (1964). This test was performed in duplicate or triplicate. The results were expressed in PCA units which are the reciprocal of the geometric means of the highest dilutions, resulting in PCA reactions 5 mm or greater in diameter. Variation for the same sample was two-fold or less; differences greater than this were taken to be significant. Heating sera at 560 for 4 h destroyed the 48 h PCA reactivity, confirming that the antibodies belong to the IgE class (Schwartz & Levine, 1973). A modification of the Farr technique (Muckerheide et al., 1977) was used to measure the total anti-BSA antibody response. Under the assay conditions, normal mouse serum consistently bound less than 2% of the antigen. Antigen-binding capacity (ABC) of the tested serum was calculated from the linear relationship between the binding capacity of the serum and the percentage of radioactivity precipitated. This linear relationship held between 0 and 35% of precipitation. The ABC was expressed as pg of BSA bound/ml of serum.
RESULTS Digestion of BSA with pepsin The rate of hydrogen ion uptake and the change in
Peptic fragments of BSA
ciency decreased to 63% of the initial value after 3 min
100 _
and to 2% after 40 min of digestion. Polyacrylamide gel electrophoresis (Fig. 2) revealed several peptides of various molecular sizes during the early stages of hydrolysis. After 3-12 min of digestion, the majority of peptides had a mol. wt of 16,000-44,000. With continuing degradation of the molecule, peptides of lower molecular weight were generated, forming three distinct bands in the 8000-16,000 region. In the late digest (360 min) a new distinct band appeared with a molecular weight around 5000.
80 _
'j60 401J20
0QQ 0
20 40 60 90180 360 MINUTES OF DIGESTION Figure 1. Hydrogen ion uptake and changes in fluorescencebinding efficiency of BSA during the course of pepsin digestion at pH 2 9. > Q
511
*0o
fluorescence-binding efficiency of BSA after various periods of pepsin digestion are shown in Fig. 1. The initial fast phase of hydrolysis was followed by a slower degradation of the molecule. An average of ten to eleven peptide bonds per molecule were cleaved at 6 min, and thirty-four to thirty-six peptide bonds per molecule at 40 min. The fluorescence-binding effi-
Immunochemical characterization of various digests To determine the effect of degradation on the serologically defined determinants, digests were immunoadsorbed on anti-BSA-Sepharose 4B columns. The quantification of adsorption is shown in Table 1. Fragments produced by a digestion period greater than 20 min were not retained by the column. On 15% polyacrylamide gel electrophoresis (Fig. 3) the unadsorbed peptides migrated into the low mol. wt region (I 2,000-30,000), whereas absorbed peptides generally had higher molecular weights (20,000-50,000).
m.w4 44000
22000 14500
3
6 12 20 40 90 180 360 DURATION OF DIGESTION (min)
Figure 2. SDS-polyacrylamide gel electrophoretic analysis of peptic digests of BSA.
S. Dosa et al.
512
CD
mw.
CD AF UF
AF UF -,mono ',, I-,M", .
............
-v'I;.'-;
-
UF UF UF
:. '-:* -h
f:: :."; so
69000 44000 22000 14500
LSIbrag
_
3 20 90 360 12 DURATION OF DIGESTION (min) Figure 3. SDS-polyacrylamide gel electrophoretic analysis of peptic digests of BSA after fractionation by anti-BSA-Sepharose 4B columns. CD, crude digest; AF, adsorbed fraction; UF, unadsorbed fraction.
When examined in an Ouchterlony double diffusion test in agarose, peptides obtained by digestion for longer than 12 min as well as those not retained by the immunoadsorption columns did not precipitate. Since the formation of precipitating BSA-anti-BSA immune complexes depends on the presence of two or more antigenic sites on the molecule (Peters et al., 1977), non-precipitating peptides may also bear antigenic determinants present on the native molecule. To determine the presence of such determinants, haemagglutination inhibition studies were done. As shown in Table 2, no inhibition of haemagglutination of BSA-
coated erythrocytes with anti-BSA serum occurred with peptides digested for 20 min or longer, nor with fractions unadsorbed by the columns. ld
,lt Isa
3520 w a-
BSA in Alum
BSA in Alum
20
A
I
I
-0
80 _
*
*
-
5
