Immunology 1990 70 281-283
Asymmetric Fab glycosylation in guinea-pig IgGi and IgG2 MALAN BOREL, T. GENTILE, J. ANGELUCCI, R. A. MARGNI & R. A. BINAGHI* Instituto de Estudios de la Inmunidad Humoral (CONICET-UBA), Departamento de Microbiologia, Inmunologia y Biotecnologia, Facultad de Farmacia y Bioquimica de la Universidad de Buenos Aires, Junin 956, Buenos Aires, Argentina and *Centre National de la Recherche Scientifique, Paris, France
I.
Acceptedfor publication 16 March 1990
SUMMARY The presence of asymmetric antibody molecules has been investigated in both IgGI and IgG2 subclasses of guinea-pig immunoglobulins. It was found that about 20% of the IgG I and 10% of the IgG2 were of asymmetric type. The proportion was essentially the same in the sera of normal animals, animals hyperimmunized with dinitrophenyl-bovine gamma globulin (DNP-BGG) and Freund's adjuvant, and animals infected with Trichinella spiralis. In the case of animals immunized with DNPBGG, no differences were observed in the proportion of asymmetric molecules between the specific antibodies and the IgG not specific for the immunizing antigen. It is concluded that the asymmetric glycosylation occurs to a different extent in each subclass and that it is not affected by the antigen specificity of the antibodies studied.
INTRODUCTION The existence of 'non-precipitating' or 'co-precipitating' antibodies has been well documented in many animal species (Margni & Binaghi, 1972; Margni & Hojos, 1973; Cordal & Margni, 1974; Margni & Binaghi, 1988), including man. In general, they represent about 10% of the IgG population and are present in all subclasses. Recently, it was demonstrated that these antibodies owe their non-precipitability to the fact that their molecules possess a monnose-rich oligosaccharide moiety attached to one of the Fab regions. The combination of the corresponding antibody site with a large antigen is sterically hindered by the carbohydrate group and, as a consequence, the molecule is functionally univalent (Labeta et al., 1986). When interaction takes place with a small ligand (hapten), the affinity constant of the hindered site is about 100-fold less than the affinity of the normal combining site. Since antigen/antibody aggregates are not formed, effector immune mechanisms are not triggered. The physicochemical and biological properties of non-precipitating antibodies, which have been designated asymmetric' antibodies, have been recently reviewed (Margni & Binaghi, 1988). It has been demonstrated that 10-15% of normal human IgG are asymmetrically glycosylated (Malan Borel et al., 1989). Also, a proportion of asymmetric antibodies is synthesized by the same cellular clones which produce precipitating antibodies (Morelli et al., 1989). Although the structural particularities of these asymmetric antibodies have been characterized, the same cannot be said Correspondence: Professor R. A. Margni, Facultad de Farmacia y Bioquimica, UBA, Junin 956, 1113-Buenos Aires, Argentina.
281
concerning their physiological role. Since they bind the antigen with high affinity but are unable to activate effector immune mechanisms, they may function as blocking antibodies which may be beneficial or harmful to the host according to the nature of the antigen and the particular situation in which they act. It has also been suggested that the asymmetric antibodies may participate in the regulation of the immune system itself, including induction of tolerance, stimulation or depression cells, modulation of the idiotypic network, etc. (Margni, 1989). In order to further study the biological function of the asymmetric antibodies, their quantitative distribution in both IgG subclasses of the guinea-pig, in normal, hyperimmunized and chronically infected animals, has been studied. MATERIALS AND METHODS
Animals, sera Random bred Hartley guinea-pigs were used. Pooled sera were obtained from normal animals, immunized with dinitrophenylbovine gamma globulin (DNP-BGG) or infected with Trichinella spiralis. Immunization with DNP-BGG was performed as previously reported (Binaghi, 1966), by injecting 1 mg protein in complete Freund's adjuvant into both hindlegs on Days I and 8. Animals were bled at Day 30. T. spiralis infection was induced by introducing 500 muscular larvae, orally, and sera were obtained 4 months later.
Purified anti-DNP antibodies Pooled antisera containing 2-3 mg/ml anti-DNP antibody were passed through a column containing DNP-bovine serum
282
2L
Malan Borel et al.
albumin (BSA) highly conjugated coupled to Sepharose 4B. The retained antibody was eluted with 01 M DNP-OH, passed through a column of IRA 400, dialysed against phosphate buffer, 0O01 M, pH 7-6, and chromatographed in DEAEcellulose. The peak obtained with this buffer was essentially IgG2; a elution of IgG I was subsequently obtained with 0 03 M, pH 7 6, buffer. IgGI and IgG2 These were isolated from normal serum, serum from animals infected with T. spiralis and from anti-DNP-BGG immune serum after immunoabsorption of the anti-DNP antibody. The gamma-globulin fraction was precipitated at 50% ammonium sulphate saturation, dissolved in buffer, 0 01 M phosphate, pH 7 6, and chromatographed on DEAE-cellulose. Using a gradient of phosphate buffer, IgG2 was eluted at 0 01 M and IgG I at 0-03
M.
Purification of non-immune IgGl was also performed by affinity chromatography employing, as immunoadsorbent, rat anti-IgGI guinea-pig immunoglobulin coupled to Sepharose 4B. Desorption was achieved with 0 1 M glycine-HCI buffer, pH 30. Immunoglobulin fragments F(ab')2 was obtained by pepsin digestion, according to Turner et al. (1970). Fab' was prepared by reduction of F(ab')2 fragment with 0 I M 2-mercaptoethanol and alkylation with 0 11 M iodoacetamide. The Fab' fragment was separated from the nonreduced IgG by filtration through a Sephadex G-200 column. Labelling with iodine Immunoglobulin fragments were labelled with 1251 by Greenwood, Hunter & Glover (1963).
as
described
Determination of symmetric and asymmetric IgG by the concanavalin A (Con A) test Con A-Sepharose was washed with elution buffer (Tris-HCI, 0-025 M; NaCl, 0-2 M; CaC12, MgCl2, MnCI2, 0-003 M each; Na Azide 002%, pH 7 2). The protein samples were dialysed against the same buffer. Equal volumes (about 1 ml) of 50% packed Con A-Sepharose and protein (about I mg/ml) were mixed in small tubes and gently agitated for 1 hr at room temperature, then overnight in the cold. After centrifugation the pellets were repeatedly washed with elution buffer and the protein in the collected supernates was estimated by measuring the optical density at 280 nm. This value corresponds to the symmetric IgG, not bound by the Con A. The asymmetric IgG was eluted from the pellets by repeatedly washing with 0-15 M alpha-methyl-mannoside in the elution buffer and estimated by measuring the optical density. All tests were made in triplicate. Recovery of protein (not retained plus retained by the Con A) was generally not less than 95% of the initial sample. RESULTS of immunoglobulins and their F(ab')2 Various preparations fragments were analysed by the Con A test in order to evaluate the proportion of molecules containing a carbohydrate group combining with the Con A. The results, reported in Table 1, show that about 8-10% of the protein was retained by Con A when IgG2 immunoglobulins or their F(ab')2 fragments were
Table 1. 0/)
binding in Con A
F(ab')2 from
Origin of the sample
IgG I
IgG2
IgG I
IgG2
Normal serum Purified anti-DNP antibody Supernate after adsorption of anti-DNP antibody
18 2
10-2
16-8
7-8
22-0
10 0
19-0
72
204
9-8
20 2
7-8
19 0
10-0
18-4
Anti-Trichinella serum
10
The results are the average of five determinations.
tested. In the case of IgGl the proportion retained was much higher, practically twice as much. No significant differences were observed between the samples obtained from normal immunoglobulins, or from immunoglobulins of anti-DNP hyperimmune sera (purified antibody or IgG non-specific for DNP) or from sera of animals infected with T. spiralis. In another experiment the F(ab')2 fragment from purified anti-DNP IgGI antibody was passed through a Con ASepharose column and the fraction retained (19%) was thereafter eluted with alpha-methyl-mannoside and reduced, to obtain the Fab' fragment. When these fragments were examined by the Con A test, it was found that 55% was retained, indicating that only half of the molecules contained the carbohydrate group. Similar results were obtained with the Fab' fragment from F(ab')2 of IgG2. These results confirm previously reported observations demonstrating that only one Fab region of the F(ab')2 or the IgG molecule possesses the carbohydrate group attaching to the Con A. DISCUSSION It is clear that the proportion of asymmetric molecules in the two subclasses of guinea-pig IgG is significantly different, at least twice as much within IgGI than IgG2. This difference has been found in all samples studied, whether obtained from normal serum or from sera of animals immunized with a DNPconjugated protein and complete Freund's adjuvant or from animals infected with T. spiralis. For the anti-DNP immunized animals, the proportion of asymmetric molecules was shown to be the same for each subclass for the specific antibodies and the non-specific IgG. It must be remarked that the method employed to immunize the animals with DNP-BGG induces a very important increase in the level of immunoglobulins (Binaghi, 1966), which in the case of IgGl may become 50-100 times higher than that of the normal animals before immunization, although only a minor proportion of this increase is specific antibody. Incidentally, it is to be noted that when the immunizing antigen is highly conjugated DNP-BGG, with 30 or more DNP groups per molecule, practically no antibodies are formed against the BGG carrier. The same observation, to a lesser degree, applies in the case of the animals infected by T. spiralis (Binaghi & Perrudet-Badoux, 1978). It appears, then, that the relative proportion of asymmetric molecular in newly
IgG asymmetric molecules synthesized IgG I and IgG2 has not been modified in the course of the antigenic stimulation. It has been shown previously, in a number of animal species, that asymmetric antibody is present in all IgG subclasses. The observations now reported demonstrate, for the first time, that the proportion present in each subclass may be different. The carbohydrate group which characterizes the asymmetric molecule is in the Fd region of the H chain. This has been demonstrated by studies of binding of Fd fragments to Con A and also by inhibition of binding to guinea-pig erythrocytes Con A induced by Fd fragments. In general, asymmetric antibodies represent 10-15% of the total serum antibodies. This proportion can increase in some cases, as when the antigen is given in particulate form for long periods of time or in some chronic infections. In view of the results reported in this paper, the variations in the proportion of asymmetric antibodies frequently observed during immunization may represent variations in the relative proportion of the various subclasses. It cannot be excluded, however, that modifications in the mechanisms of synthesis of the asymmetric molecule, related to the nature and the form of presentation of the antigen, could take place in some cases. For example, when rabbits were immunized with repeated injections of polymerized egg albumin, up to 60% of total anti-egg albumin antibodies was the asymmetric type; such a proportion is hardly conceivable without assuming a modified glycosylation of the molecule. The quantification of the asymmetric molecules has been made by determining the quantity of immunoglobulins binding to Con A and assuming that the carbohydrate group responsible for binding is present in only one Fab region, as previously shown in rabbit and sheep. This assumption was confirmed by studying the fragments of F(ab')2 obtained from purified IgGl and IgG2 anti-DNP antibody. The protein bound by the Con A was eluted with alpha-methyl-mannoside, reduced to obtain the Fab' fragment and analysed by the Con A test. It was found that half of the molecules (55%) bound to Con A, thus confirming that the carbohydrate group was present in only one Fab region of each F(ab')2 molecule. The reason for using F(ab')2 instead of the whole IgG molecule is to avoid binding to Con A by carbohydrate groups present in the Fc region, which are not relevant to the asymmetric or symmetric nature of the molecule. In conclusion, the results reported indicate that asymmetric molecules are not present at the same ratio in the two subclasses of guinea-pig IgG. The reasons for these differences, as well as the biological implications, are unknown for the moment, but given the quite different properties of this two subclasses, it can
283
be predicted that the presence of asymmetric molecules may be in some way related to their physiological role.
ACKNOWLEDGMENTS This work was supported in part by a scientific cooperation agreement between CONICET (Argentina) and INSERM (France) and by a grant from CONICET.
REFERENCES BINAGHI R.A. (1966) Production of 7S immunoglobulins in immunized guinea-pig. J. Immunol. 97, 159. BINAGHI R.A. & PERRUDET-BADoux A. (1978) Stimulation of immunoglobulin synthesis during infestation with Trichinella spiralis. In: Trichinelosis (ed. C. Kim), p. 175. University Press of New England. CORDAL M.E. & MARGNI R.A. (1974) Isolation, purification and biological properties of horse precipitating and non-precipitating antibodies. Immunochemistry, 11, 765. GREENWOOD F., HUNTER W.M. & GLOVER J. (1963) The preparation of '25I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114. LABETA M., MARGNI R.A., LEONI J. & BINAGHI R.A. (1986) Structure of asymmetric nonprecipitating antibodies: presence of a carbohydrate residue in only one Fab region of the molecule. Immunology, 57, 311. MALAN BOREL I., GENTILE T., ANGELUCCI J., MARGNI R.A. & BINAGHI R.A. (1989) Asymmetrically glycosylated IgG isolated from nonimmune human sera. Biochim. Biophys. Acta, 990, 162. MARGNI R.A. (1989) Are the nonprecipitating asymmetric antibodies autoprotective and regulatory antibodies? One opinion. Res. Immunol. 140, 725. MARGNI R.A. & BINAGHI R.A. (1972) Purification and properties of non-precipitating rabbit antibodies. Immunology, 22, 557. MARGNI R.A. & BINAGHI R.A. (1988) Nonprecipitating asymmetric antibodies. Ann. Rev. Immunol. 6, 535. MARGNI R.A. & HAJOS S.E. (1973) Biological and physicochemical properties of purified anti-DNP guinea-pig non-precipitating antibodies. Immunology, 24, 435. MORELLI L., LEONI J., FOSSATI C.A. & MARGNI R.A. (1989) Symmetric and asymmetric IgG antibodies are synthesized by the same cellular clone. Mol. Immunol. 26, 789. PORTER R.R. (1959) The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochem. J. 73, 119. TURNER M.W., BENNICH H. & NATVIG J.B. (1970) Pepsin digestion of human G myeloma proteins of different subclasses. I. The characteristic features of pepsin cleavage as a function of time. Clin. exp. Immunol. 7, 603.
Asymmetric Fab glycosylation in guinea-pig IgGi and IgG2 MALAN BOREL, T. GENTILE, J. ANGELUCCI, R. A. MARGNI & R. A. BINAGHI* Instituto de Estudios de la Inmunidad Humoral (CONICET-UBA), Departamento de Microbiologia, Inmunologia y Biotecnologia, Facultad de Farmacia y Bioquimica de la Universidad de Buenos Aires, Junin 956, Buenos Aires, Argentina and *Centre National de la Recherche Scientifique, Paris, France
I.
Acceptedfor publication 16 March 1990
SUMMARY The presence of asymmetric antibody molecules has been investigated in both IgGI and IgG2 subclasses of guinea-pig immunoglobulins. It was found that about 20% of the IgG I and 10% of the IgG2 were of asymmetric type. The proportion was essentially the same in the sera of normal animals, animals hyperimmunized with dinitrophenyl-bovine gamma globulin (DNP-BGG) and Freund's adjuvant, and animals infected with Trichinella spiralis. In the case of animals immunized with DNPBGG, no differences were observed in the proportion of asymmetric molecules between the specific antibodies and the IgG not specific for the immunizing antigen. It is concluded that the asymmetric glycosylation occurs to a different extent in each subclass and that it is not affected by the antigen specificity of the antibodies studied.
INTRODUCTION The existence of 'non-precipitating' or 'co-precipitating' antibodies has been well documented in many animal species (Margni & Binaghi, 1972; Margni & Hojos, 1973; Cordal & Margni, 1974; Margni & Binaghi, 1988), including man. In general, they represent about 10% of the IgG population and are present in all subclasses. Recently, it was demonstrated that these antibodies owe their non-precipitability to the fact that their molecules possess a monnose-rich oligosaccharide moiety attached to one of the Fab regions. The combination of the corresponding antibody site with a large antigen is sterically hindered by the carbohydrate group and, as a consequence, the molecule is functionally univalent (Labeta et al., 1986). When interaction takes place with a small ligand (hapten), the affinity constant of the hindered site is about 100-fold less than the affinity of the normal combining site. Since antigen/antibody aggregates are not formed, effector immune mechanisms are not triggered. The physicochemical and biological properties of non-precipitating antibodies, which have been designated asymmetric' antibodies, have been recently reviewed (Margni & Binaghi, 1988). It has been demonstrated that 10-15% of normal human IgG are asymmetrically glycosylated (Malan Borel et al., 1989). Also, a proportion of asymmetric antibodies is synthesized by the same cellular clones which produce precipitating antibodies (Morelli et al., 1989). Although the structural particularities of these asymmetric antibodies have been characterized, the same cannot be said Correspondence: Professor R. A. Margni, Facultad de Farmacia y Bioquimica, UBA, Junin 956, 1113-Buenos Aires, Argentina.
281
concerning their physiological role. Since they bind the antigen with high affinity but are unable to activate effector immune mechanisms, they may function as blocking antibodies which may be beneficial or harmful to the host according to the nature of the antigen and the particular situation in which they act. It has also been suggested that the asymmetric antibodies may participate in the regulation of the immune system itself, including induction of tolerance, stimulation or depression cells, modulation of the idiotypic network, etc. (Margni, 1989). In order to further study the biological function of the asymmetric antibodies, their quantitative distribution in both IgG subclasses of the guinea-pig, in normal, hyperimmunized and chronically infected animals, has been studied. MATERIALS AND METHODS
Animals, sera Random bred Hartley guinea-pigs were used. Pooled sera were obtained from normal animals, immunized with dinitrophenylbovine gamma globulin (DNP-BGG) or infected with Trichinella spiralis. Immunization with DNP-BGG was performed as previously reported (Binaghi, 1966), by injecting 1 mg protein in complete Freund's adjuvant into both hindlegs on Days I and 8. Animals were bled at Day 30. T. spiralis infection was induced by introducing 500 muscular larvae, orally, and sera were obtained 4 months later.
Purified anti-DNP antibodies Pooled antisera containing 2-3 mg/ml anti-DNP antibody were passed through a column containing DNP-bovine serum
282
2L
Malan Borel et al.
albumin (BSA) highly conjugated coupled to Sepharose 4B. The retained antibody was eluted with 01 M DNP-OH, passed through a column of IRA 400, dialysed against phosphate buffer, 0O01 M, pH 7-6, and chromatographed in DEAEcellulose. The peak obtained with this buffer was essentially IgG2; a elution of IgG I was subsequently obtained with 0 03 M, pH 7 6, buffer. IgGI and IgG2 These were isolated from normal serum, serum from animals infected with T. spiralis and from anti-DNP-BGG immune serum after immunoabsorption of the anti-DNP antibody. The gamma-globulin fraction was precipitated at 50% ammonium sulphate saturation, dissolved in buffer, 0 01 M phosphate, pH 7 6, and chromatographed on DEAE-cellulose. Using a gradient of phosphate buffer, IgG2 was eluted at 0 01 M and IgG I at 0-03
M.
Purification of non-immune IgGl was also performed by affinity chromatography employing, as immunoadsorbent, rat anti-IgGI guinea-pig immunoglobulin coupled to Sepharose 4B. Desorption was achieved with 0 1 M glycine-HCI buffer, pH 30. Immunoglobulin fragments F(ab')2 was obtained by pepsin digestion, according to Turner et al. (1970). Fab' was prepared by reduction of F(ab')2 fragment with 0 I M 2-mercaptoethanol and alkylation with 0 11 M iodoacetamide. The Fab' fragment was separated from the nonreduced IgG by filtration through a Sephadex G-200 column. Labelling with iodine Immunoglobulin fragments were labelled with 1251 by Greenwood, Hunter & Glover (1963).
as
described
Determination of symmetric and asymmetric IgG by the concanavalin A (Con A) test Con A-Sepharose was washed with elution buffer (Tris-HCI, 0-025 M; NaCl, 0-2 M; CaC12, MgCl2, MnCI2, 0-003 M each; Na Azide 002%, pH 7 2). The protein samples were dialysed against the same buffer. Equal volumes (about 1 ml) of 50% packed Con A-Sepharose and protein (about I mg/ml) were mixed in small tubes and gently agitated for 1 hr at room temperature, then overnight in the cold. After centrifugation the pellets were repeatedly washed with elution buffer and the protein in the collected supernates was estimated by measuring the optical density at 280 nm. This value corresponds to the symmetric IgG, not bound by the Con A. The asymmetric IgG was eluted from the pellets by repeatedly washing with 0-15 M alpha-methyl-mannoside in the elution buffer and estimated by measuring the optical density. All tests were made in triplicate. Recovery of protein (not retained plus retained by the Con A) was generally not less than 95% of the initial sample. RESULTS of immunoglobulins and their F(ab')2 Various preparations fragments were analysed by the Con A test in order to evaluate the proportion of molecules containing a carbohydrate group combining with the Con A. The results, reported in Table 1, show that about 8-10% of the protein was retained by Con A when IgG2 immunoglobulins or their F(ab')2 fragments were
Table 1. 0/)
binding in Con A
F(ab')2 from
Origin of the sample
IgG I
IgG2
IgG I
IgG2
Normal serum Purified anti-DNP antibody Supernate after adsorption of anti-DNP antibody
18 2
10-2
16-8
7-8
22-0
10 0
19-0
72
204
9-8
20 2
7-8
19 0
10-0
18-4
Anti-Trichinella serum
10
The results are the average of five determinations.
tested. In the case of IgGl the proportion retained was much higher, practically twice as much. No significant differences were observed between the samples obtained from normal immunoglobulins, or from immunoglobulins of anti-DNP hyperimmune sera (purified antibody or IgG non-specific for DNP) or from sera of animals infected with T. spiralis. In another experiment the F(ab')2 fragment from purified anti-DNP IgGI antibody was passed through a Con ASepharose column and the fraction retained (19%) was thereafter eluted with alpha-methyl-mannoside and reduced, to obtain the Fab' fragment. When these fragments were examined by the Con A test, it was found that 55% was retained, indicating that only half of the molecules contained the carbohydrate group. Similar results were obtained with the Fab' fragment from F(ab')2 of IgG2. These results confirm previously reported observations demonstrating that only one Fab region of the F(ab')2 or the IgG molecule possesses the carbohydrate group attaching to the Con A. DISCUSSION It is clear that the proportion of asymmetric molecules in the two subclasses of guinea-pig IgG is significantly different, at least twice as much within IgGI than IgG2. This difference has been found in all samples studied, whether obtained from normal serum or from sera of animals immunized with a DNPconjugated protein and complete Freund's adjuvant or from animals infected with T. spiralis. For the anti-DNP immunized animals, the proportion of asymmetric molecules was shown to be the same for each subclass for the specific antibodies and the non-specific IgG. It must be remarked that the method employed to immunize the animals with DNP-BGG induces a very important increase in the level of immunoglobulins (Binaghi, 1966), which in the case of IgGl may become 50-100 times higher than that of the normal animals before immunization, although only a minor proportion of this increase is specific antibody. Incidentally, it is to be noted that when the immunizing antigen is highly conjugated DNP-BGG, with 30 or more DNP groups per molecule, practically no antibodies are formed against the BGG carrier. The same observation, to a lesser degree, applies in the case of the animals infected by T. spiralis (Binaghi & Perrudet-Badoux, 1978). It appears, then, that the relative proportion of asymmetric molecular in newly
IgG asymmetric molecules synthesized IgG I and IgG2 has not been modified in the course of the antigenic stimulation. It has been shown previously, in a number of animal species, that asymmetric antibody is present in all IgG subclasses. The observations now reported demonstrate, for the first time, that the proportion present in each subclass may be different. The carbohydrate group which characterizes the asymmetric molecule is in the Fd region of the H chain. This has been demonstrated by studies of binding of Fd fragments to Con A and also by inhibition of binding to guinea-pig erythrocytes Con A induced by Fd fragments. In general, asymmetric antibodies represent 10-15% of the total serum antibodies. This proportion can increase in some cases, as when the antigen is given in particulate form for long periods of time or in some chronic infections. In view of the results reported in this paper, the variations in the proportion of asymmetric antibodies frequently observed during immunization may represent variations in the relative proportion of the various subclasses. It cannot be excluded, however, that modifications in the mechanisms of synthesis of the asymmetric molecule, related to the nature and the form of presentation of the antigen, could take place in some cases. For example, when rabbits were immunized with repeated injections of polymerized egg albumin, up to 60% of total anti-egg albumin antibodies was the asymmetric type; such a proportion is hardly conceivable without assuming a modified glycosylation of the molecule. The quantification of the asymmetric molecules has been made by determining the quantity of immunoglobulins binding to Con A and assuming that the carbohydrate group responsible for binding is present in only one Fab region, as previously shown in rabbit and sheep. This assumption was confirmed by studying the fragments of F(ab')2 obtained from purified IgGl and IgG2 anti-DNP antibody. The protein bound by the Con A was eluted with alpha-methyl-mannoside, reduced to obtain the Fab' fragment and analysed by the Con A test. It was found that half of the molecules (55%) bound to Con A, thus confirming that the carbohydrate group was present in only one Fab region of each F(ab')2 molecule. The reason for using F(ab')2 instead of the whole IgG molecule is to avoid binding to Con A by carbohydrate groups present in the Fc region, which are not relevant to the asymmetric or symmetric nature of the molecule. In conclusion, the results reported indicate that asymmetric molecules are not present at the same ratio in the two subclasses of guinea-pig IgG. The reasons for these differences, as well as the biological implications, are unknown for the moment, but given the quite different properties of this two subclasses, it can
283
be predicted that the presence of asymmetric molecules may be in some way related to their physiological role.
ACKNOWLEDGMENTS This work was supported in part by a scientific cooperation agreement between CONICET (Argentina) and INSERM (France) and by a grant from CONICET.
REFERENCES BINAGHI R.A. (1966) Production of 7S immunoglobulins in immunized guinea-pig. J. Immunol. 97, 159. BINAGHI R.A. & PERRUDET-BADoux A. (1978) Stimulation of immunoglobulin synthesis during infestation with Trichinella spiralis. In: Trichinelosis (ed. C. Kim), p. 175. University Press of New England. CORDAL M.E. & MARGNI R.A. (1974) Isolation, purification and biological properties of horse precipitating and non-precipitating antibodies. Immunochemistry, 11, 765. GREENWOOD F., HUNTER W.M. & GLOVER J. (1963) The preparation of '25I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114. LABETA M., MARGNI R.A., LEONI J. & BINAGHI R.A. (1986) Structure of asymmetric nonprecipitating antibodies: presence of a carbohydrate residue in only one Fab region of the molecule. Immunology, 57, 311. MALAN BOREL I., GENTILE T., ANGELUCCI J., MARGNI R.A. & BINAGHI R.A. (1989) Asymmetrically glycosylated IgG isolated from nonimmune human sera. Biochim. Biophys. Acta, 990, 162. MARGNI R.A. (1989) Are the nonprecipitating asymmetric antibodies autoprotective and regulatory antibodies? One opinion. Res. Immunol. 140, 725. MARGNI R.A. & BINAGHI R.A. (1972) Purification and properties of non-precipitating rabbit antibodies. Immunology, 22, 557. MARGNI R.A. & BINAGHI R.A. (1988) Nonprecipitating asymmetric antibodies. Ann. Rev. Immunol. 6, 535. MARGNI R.A. & HAJOS S.E. (1973) Biological and physicochemical properties of purified anti-DNP guinea-pig non-precipitating antibodies. Immunology, 24, 435. MORELLI L., LEONI J., FOSSATI C.A. & MARGNI R.A. (1989) Symmetric and asymmetric IgG antibodies are synthesized by the same cellular clone. Mol. Immunol. 26, 789. PORTER R.R. (1959) The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochem. J. 73, 119. TURNER M.W., BENNICH H. & NATVIG J.B. (1970) Pepsin digestion of human G myeloma proteins of different subclasses. I. The characteristic features of pepsin cleavage as a function of time. Clin. exp. Immunol. 7, 603.
