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Evolution of low molecular weight immunoglobulins I. Relationship of 7S immunoglobulins of various vertebrates to chicken IgY
DEVELOPF~NTAL AND COF~ARATIVE IF~UNOLOGY, Vol. 4, pp. 501-514, 1980. 0145-305X/80/030501-15502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.
EVOLUTION OF LOW MOLECULAR WEIGHT IMMUNOGLOBULINS I. RELATIONSHIP OF 7S IMMUNOGLOBULINS OF VARIOUS VERTEBRATES TO CHICKEN IgY
Dietlind H~dge, Helmut Fiebig, snd Herwart Ambrosius Laboratory of Immunobiology, Section of Biosciences, Karl-Mmrx-University, Leipzig, G.D.R.
ABSTRACT. All of the birds (chicken, duck, goose), reptiles (sheltopusik, tortoise), end enuran amphibians (pond frog) included in these studies have 7S immunoglobulins with molecular weights between 160,000 and 180,5OO D, the H chains of which show molecular weights of 62,700 to 65,500 D. The molecular weights of these 7S immanoglobulins as well as those of their heavy chains ere markedly higher than the corresponding values for the IgG molecules of mammals, which are 150,000 end 50,000 D, respectively. The contents of carbokydrates (hexoses, hexosamines, eialic acid) of these low molecular weight immunoglobulins, which are between 4.6 and 6.4 %, are markedly higher than that of IgG which is of the order of 2.3 %. Immunochemical studies (double-antibody radioimmunosesay) made to determine similarities in the sntigenicity of low molecular weight immunoglobulins of the different species showed that there is s close antigenic relationship between the 7S immunoglobulins of birds, reptiles, end amphibians, but only s distsnt relationship to mammalian IgG. Therefore, we propose to include nonmammslian 7S immunoglobulins in one single class designsted as IgY sccording to the term used by Leslie and Clem for the 7S immunoglobulin of chicken.
INTRODUCTION Immunoglobulins (Ig'e) show an increase in the heterogeneity of isotypes in the course of evolution. All vertebrates hsve a high molecular weight type of immunoglobulin which is structurally and functionally related to mammalian IgM and which is the only class of immunoglobulins found in Agnatha (I), Chondrichthyes and Osteichthves (2-5). 501
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In amphibians (6-8) has been found s low molecular weight type of 7S immunoglobulin for the first time which differs in antigenicity from IgM and occurs also in reptiles (9-11) and in birds (12-14). These 7S immunoglobulins have often been termed IgG like the memm~lisn immunoglobulin. The structursl and functional characteristics of chicken 7S immunoglobulin have been studied most carefully (12,14) and designated as IgY by Leslie and Clem (12). In addition, s third 5.7S class sntigenically related to 7S immunoglobulin has been characterized in certain reptiles (9-11) and birds (14). In the current work, a number of physical and chemical data of various 7S immunoglobulins of representative vertebrates sre presented. Using the technique usually referred to as double-sntibo&y radioimmunoassay, immunoche~Lical studies on the antigenic relationship of these immunoglobulins were made to obtain information about the evolution of low molecular weight immunoglobulins, the major focus being on the relation between the 7S immunoglobulins of birds and lower vertebrates to mammalian IgG. MATERIALS AND METHODS Prepsrstion of immuno~lobulins. ~ material: Immunoglobulins were obtained from normal and immune sers of chickens (Gallus ~allus domesticus L.), ducks (Anser plstyrhynchos domestics L.), goose (Anser anser), tortoises (A~rlonemis horsfieldii GRAY), sheltopusiks ( P A h ~ saurus apodus /PALLAS/), and pond frogs (Rans e s c u l e n t a U . In addition, f~om human and guinea-pig sara were isolated IgG and IgG2, IgM(Go) from the serum of a Pstient with Wsldenstr~m's mscroglobulinemia, and IgM from the serum of carps (Cyprinus carpio L.). Gamm~ globulin obtained from cows (BGG) was made available by Serva Feinbiochemics, Heidelberg. The IgD psraprotein was 8 crude fraction precipitated with (NH4)2SO 4 st s finsl concentration of halfsaturation. Preparation of macro~lobulins: Macroglobulins were prepared Sy-r~Ya~gs ~ ~s~I~ of immunoglobulin by precipitation st 50 % saturation of (NH4)2S04, gel filtration on Sepharose 6B (Pharmscia) and Bio-Gel A - 1.5 m (Bio-Rsd Laboratories, Richmond), starch block electrophoresis, and rechromstography by gel filtration using s previously described method (4). PreRaration of 7S immuno~lobulins: The 7S immunoglobulins ~re ~ ~ - ~ ~e-~8~on of immunoglobulin (three precipitations with N82S04 at concentrations of 180, 140, and 140 mg/ml or three precipitations with (NHA)2SO A at 50 %, 33 %, and 33 % saturation, respectively). This-was ~ollowed by several steps of gel filtration on columns of Sephsdex G-200 (Pharmacia) or Bio-Gel A - 1.5 m. In the case of preparation and purification of 7S immunoglobulins obtained from chicken or sheltopusik sets as well 8s from human and
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guinea-pig sera, the steps of gel filtration were combined with chromstograpky on dietkylaminoetkv1 cellulose (DE-52, Whatman) by using a 0 . 0 1 M Tris HC1 buffer of pH 8.0 (conductivity: 5001uS) as starting buffer° Elution of the 7S immunoglobulin~ from the cellulose columns was carried out by means of a linear NaC1 gradient. The fractions containing the low molecular weight immunoglobulins were combined, concentrated by ultrsfiltration, and prepared for subsequent gel filtration. Confirmation of individual fractions for contents of 7S immunoglobulins was accomplished by immunoelectrophoretic analysis using antisera directed against the components of normal sera from the respective species. In all cases, the immunoglobulins thus obtained were immunoelectrophoretically pure preparations. Protein concentrations of samples were determined by extinction measurements at 280 nm assuming an extinction coefficient (1-cm cuvettes, 1 % solution) of 14.0 for the msmmslisn IgG preparations and of 12.0 for the other purified Ig materials. Determination of pkysical and chemical parameters. SDS - PAGE: Polyscrylamide gel electrophoresis performed in ~H~ presence of sodium dodecyl sulfate ~SDS) was used to estimate relative molecular weights of immunoglobulins and their polypeptide chains (15). Humsn IgG (150,000 D), its H and L chains (52,000 and 23,000 D, respectively) as well as H and L chains of carp IgM (76,000 and 23,000 D, respectively) served as standard substances for plotting a calibration curve. Preoaration of H and L chains of immuno~lobulins: H and L bulins with 2-mercaptoethanol (Ferak, Berlin-West) and by means of alkylation using iodoscetamide (Servs) (16). Determining the csrbokEdrate content: The contents of H~8~ ~S-H~8~n~s were S ~ L [ n e d using orcinol reagent (E. Merck AG, Darmstadt) and a modified version of the Elson-Morgsn reaction, respectively (4). To determine the proportions of sislic acid, we used the method of detection by thioberbituric acid (17). N-acetylneuraminic acid (17). N-acetylneuraminic acid (99 % synthetic) serving as a reference substance was made available by Serva. Double-antibody radioimmunoassa¥ Preoaration of sntisers: Carps weighing 1.5 kg snd kept at a-~m~era~-~-~-~ 2°C were immunized using 0.2 mg of chicken IgY emulsified in CFA (Difco, Detroit, U.S.A.). Two reimmunizations were mede at one-month intervals using I mg of IgY without an adjuvant; blood samples were taken by cardiac puncture two weeks after the second reimmunization. Rabbits were injected with 0.2 mg of chicken IgY emulsified in CFA into all four foot pads~ the second immunization was made after four months (o.4 mg). After ten ds~s, samples of blood were taken by cardiac puncture. These anti-IgY sets (sntisera I) were extensively absorbed with chicken IgM conjugated to CNBr-activated Sepharose 4B (18) rendering them H chain specific. Eor precipitatin antibodies (antisera II)
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we used anti-carp IgM serum obtained by immunization of rabbits with 0.5 mg of carp IgM in CFA and two reimmunizations with I mg each at four-week intervals and gost-snti-rsbbit gsmms globulin serum (SIFIN, Berlin-Wei6ensee), respectively. Radio-labelin~ of chicken IgY: Chicken IgY (0.5 mg) were I~I~-~-~H~-~I~=~ method (19), using 2 mBq of carrier-free iodine-125 (as Na125I, ROTOP, Nuclear Research Center at Rossendorf, G.D.R.). Free iodine was separated by gel filtration on Sephadex G-25. Radioimmunosssa~: All steps of dilution, including the dis~o-Y~-~-~ inhibitory proteins were made with PBS (phosphate-buffered saline, pH 7.4, containing 0.2 mg NaN3/ml). The following technique was used for the detection of crossreactivity: The specific antibodies (antisera I) were supplemented with normal sers in s I : 320 dilution. The exact amounts of double antibodies (sntisera II) to allow a complete precipitation of the whole Ig fractions were determined by a gel diffusion test. Amounts of specific antibodies were adjusted in such a manner that 50 % of labeled added chicken IgY wss bound by the antibodies. The following conditions were used: 0.05 ml of sntisers I (carp- or rabbit-snti-IgY sers in s I : 1,280 or s I : 10,000 dilution, respectively) was incubated with 0.05 ml of inhibition solution consisting of 7S Ig's of several species at 4oc for 2 hr. After this 125I-IgY (50,000 cpm) in 0.05 ml PBS was added, the contents mixed gently, and the polystyrene tubes agsin incubated at 4oc for 2 hr. Pinslly 0.05 mi of sntisers II (rabbit-anticarp IgM serum, diluted I : 32, and goat-snti-rabbit gamma globulin serum, 1 • 64, respectively) was added and the incubation continued for 24 hr st 4°C. Then two controls were included in which the anti-IgY sets were substituted for the corresponding normal sers and the inhibition solution by PBS. This wss done to determine the correction for coprecipitstion and cosdsorption. All tubes are set up in triplicate. Finally the tubes were centrifuged st 10,000 rpm for 10 min at 4°C and the radioactivity of supernatent (0.1-ml volume) which was carefully removed from the mixture was measured by s NaI(T1) crystal scintillator. RESULTS Estimation of relative molecular weights of immunoglobulins and of their H and L chains. To estimate the molecular weights, 5 % polyacrylamide gels c r o s s - l i n k e d w i t h N,N-methylene-bis-acrylamide with a ratio of polyscrylsmide to cross-linking agent (80 : I) were used. According to procedure there is a linear relation between the migration distances in gel and the logarithm of the molecular weights ranging from 20,000 to 200,000 D (15). Since the electrophoretic mobility of glycoproteins loaded with SDS anions depends upon both their carbokydrate contents, their conformation and the charge distributions on these molecules (20), the standard substsnces used for plotting of cslibration curves were, without exception, immunoglobulins
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with known molecular weights. In spite of that we are able to obtain only relative molecular weight values. Finally our standards differ also somewhat in its physical and chemical properties. The results of estimating the molecular weights of different 7S immunoglobulins are summarized in Table I. TABLE
1
Relative Molecular Weights of 7S Immunoglobulins of Different Vertebrates (Six Estimations) Species
Ig type
Molecular weights ± SD (in Daltons)
Man Cow Guinea-pig Chicken Duck Goose Tortoise Frog
IgG IgG IgG2 IgY 7S Ig 7S Ig 7S Ig 7S Ig
150,00 (assumed value) 148,500 ± 2,500 142,500 ± 3,010 169,500 ± 3,000 168,750 ± 3,400 165,000 ± 4,650 180,500 ± 2,500 160,000 ± 2,000
SD = standard deviation. Accordingly, chicken, duck, goose, tortoise, and frog may be regarded as having low molecular weight immunoglobulins with molecular weights between 160,000 and 180,500 D. As is Usually the case, the molecular weight of human IgG was assumed to be on the order of 150,000 D. Using SDS - PAGE on 5 % gels, s value of 142,500 D was calculated for guineapig IgG2, whereas a vslue of 148,500 D was obtained for BGG. The results of estimating the molecular weights of H and L chains of 7S immunoglobulins of different vertebrates are in Table 2 (see also Fig. 1). Clearly the 7S immunoglobulins of birds, reptiles, and amphibians, included in this investigation, possess an H chsln type with 62,700 to 65,500 D. These values are markedly higher than those of H chains with about 52,000 D for human IgG, guinea-pig IgG2, and BGG, but markedly lower than those for heavy chains of carp IgM (5) or IgM fractions (not shown in the Table),of the same species of mammals, birds, and lower vertebrates (70,000 to 76,000 D). In another series of experiments, the molecular weights of some polypep%ide chains were estimated by SDS PAGE using 10 % gels, since this tends to reduce the disturbing influence of carbokvdrate components of immunoglobulins upon their electrophoretic mobility. These measurements (four estimations) yielded only slightly lower vslue~ for the molecular weights of t~e H chains, namely, 61,300 z 470 D for chicken IgY, 61,800 z 430 D for duck 7S Ig, -
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62,000 ± 570 D for tortoise 7S Ig, and 74,500 ± 470 D for carp IgM. The values obtained for L chains remained unchanged and were about 24,000 D. Determination of the csrbokydrst e contents. The contents of carbokydrstes, i.e., hexoses, hexossmines, and sislic acid are compared with those values obtained for the IgM preparations of the respective species and for humsn and carp IgM, respectively (Table 3). Msnnose-galactose mixtures of I : I or, in the case of human IgM, of 2 : I served ss reference solutions. The hexosamine proportions are given as glucossmine bsse and the sialic acid residues as N-acetylenuraminic acid. TABLE
3
Csrbokydrste Composition of Immunoglobulins of Different Vertebrates (Two Determinations) Species
Ig type Hexoses
Man IgG Guinea-pig IgG2 Chicken IgY Duck 7S Ig Tortoise 7S I ~ Msn IgM(Go) Chicken IgM Duck IgM Tortoise IgM Sheltopusik IgM Carp . . . . I~M , n.d. -- not determined
1.3 1.3 3.2 4.5 3.3 7.1 4.1 4.1 2.8 5.3 4~.1 .
Content (in %) HexosSialic Total amines acid 1.O O.1 2.4 0.9 O.1 2.3 1.8 0.2 5.2 1.7 0.2 6.4 1.~1 0.2 4.6 4.1 0.4 11.6 1.2 0.5 5.8 2.7 0.6 7.4 1.3 1.2 5.3 2.7 n.d. ,8.0 . 2,.7, . ~5 7.3
Clearly the carbokydrste contents of 7S immunoglobulins of birds (chicken, duck) end reptiles (tortoise), of which vslues were between 4.6 and 6.4 %, were higher than the proportions of about 2.3 % determined for the two IgG preparations (man, guinea-pig) (Tsble 3). Rather, the values of 7S immunoglobulins were nearby of those obtsined for IgM preparations without accounting for the extremely high value for human IgM(Go). Antigenic relstionship of chicken I~Y to the 7S immunoglobulins of different vertebrates. To detect sntigenic relationships, s radioimmunosssey system was used bssed upon the inhibition of bindin~ reaction between 125I-IgY snd csrp anti-IgY sntibodies ~Fig. 2) and rabbit snti-IgY sntiboaies (Pig. 3), respectively, by unlsbeled 7S Ig prepsrstions of goose, duck, sheltopusik, tortoise, frog, human (IgG and IgD), and cow.
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TABLE
507
2
Relative Molecular Weights of the H and L Chains of 7S Immunoglobulins of Different Vertebrates (Six Estimations) Species
Ig type
Molecular weights ± S D (in Daltons) H chains L chains
Man
IgG IgG IgG2 IgY 7S Ig 7S Ig 7S Ig 7S Ig 7S Ig
52,000 52,500 51,700 62,800 62,700 64,300 65,500 64,500 64,000
Cow
Guinea-pig Chicken Duck Goose Tortoise Sheltopusik Prog
(assumed value) f 800 23,000 23,000 ± 300 ± 2,170 22,000 ± 1,840 24,500 25,000 ± 2,450 ± 2,200 24,000 ± 2,200 25,500 ± 2,000 21,000
PIG.
± ± ± ± ± ± ± ±
1,000 1,000 1,700 1,800 2,000 1,750
1,850 2,500
I
SDS-PAGE of H add L Chains of Immunoglobulins of Different Vertebrates. s = Human IgG b = BGG c = Guinea-pig IgG2 d = Chicken IgY e = Duck 7S Ig f = Tortoise 7S Ig g = Human
IgM(Go)
h = Carp IgM ,5 % gels, 4.5 mA/gel, hrs.
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It is important to note that the 7S immunoglobulins of birds, reptiles, and mnuran amphibians appeared as an antigenically related group. As would be expected, immunoglobulins of the goose and duck were more closely related to chicken IgY than to those of the sheltopusik, tortoise, and frog. The proportion of species-specific determinants is responsible for the relatively large distance between the inhibition curves of IgY and those of other 7S immunoglobulins. Only a rather small degree of cross-reactivity wss observed for the IgG of men and cow. Likewise, the use of IgD paraprotein revealed only a slight degree of relationship to IgY add to the other 7S immunoglobulins of birds and lower vertebrates with respect to antigenicity. By using rmbbit anti-IgY sera, we were able to observe that 7S immunoglobulins of birds showed a somewhat closer relationship, whereas the immunoglobulins of the reptiles add the frog showed a less clearly evident cross-resctivity to chicken IgY. DISCUSSION The major focus of these studies dealt with relstions between the 7S immunoglobulins of different classes of vertebrstes. By comparing the pkvsical, chemical, and antigenic chsrscteristics of these immunoglobulins, we sttempted to classify them into previously known classes of immunoglobulins. Of particular interest in this connection was the structursl end sntigenic relstionship of the 7S immunoglobulins of lower vertebrstes to mammalian IgG. It wss already in the estimation of the molecular weights of intact immunoglobulin molecules that we observed s consistent variation in values of 7S immunoglobulins from those of IgG type immunoglobulins° All of the birds, reptiles, and amphibians included in these studies, for which vslues in the region of 170,000 D were obtained, showed higher molecular weights than immunoglobulins of mammals used for comparison, where the moleculmr weights were approximately 150,000 D. These vslues, obtained by SDS PAGE on 5 % gels, agreed with previous reports and measured 7S immunoglobulins of the most different vertebrate species by using other techniques such ms, for example, analytical gel filtration and sedimentation equilibrium analysis (8,11,12,13). Similar trends were observed for molecular weights of the H chains from 7S immunoglobulins. Thus the molecular weights of 7S immunoglobulins of birds and lower vertebrates which are higher than the corresponding values of the mammalian IgG type result from the presence of lodger H chains, also reported for s number of 7S immunoglobulins of different species (8,11,12,13). However, it must be considered that factors as carbokvdrate content or charge mentioned above may affect the migration of our immunoglobulins in SDS-gels. Nevertheless, the observed differences in the monmamm~lian heavy chain molecular weights in comparison to the mammalian 7 chain values indicate that they probably differ significantly in some structural features.
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The degree of immunological cross-reactivity, representing the presence of equal and similar determinants, was inferred from our immunochemical studies on antigenic relationship, undertaken as part of a rsdioimmunoassay. EvidentLy, the 7S immunoglobulins of birds, reptiles, and amphibians are characterized by a high degree of similarity in antigenicity. Moreover, these studies showed that using csrp-anti-IgY sera allowed a far better agreement between the antigenic properties of IgY-like immunoglobulins than when rabbit anti-IgY sera were used. Unlike rabbits, carps, which produce only IgM antibodies, also showed an intense immune response, because of the pkylogenetic distance of the antiserum donor from the chicken IgY antigen, to those determinants which are relatively conservetive in the course of evolution. Because of the consistently higher values of the molecular weights of 7S immunoglobulins of birds, reptiles, and amphibians, the markedly higher carbokydrate contents (5 - 7 % as compared with 2 - 3 % for mammalian IgG), and the agreement between antigenic properties, we propose that low molecular weight immunoglobulins of birds and lower vertebrates be delimited from IgG type immunoglobulins by the designation Ig's of IgY type or IgY like Ig's, which was chosen by Leslie and Clem (12) for the 7S Ig of chicken. Considering the genetic background for the evolution of Ig's it is assumed that at the pkylogenetic level of anuran amphibians there must have evolved an H chain gene differing from the mu-chain gene, which can also be found in the Sauropsida. Hill et al. (21) suggested that for all Ig's there is an ancestral primordial gene capable of coding for a poLTpeptide chain of about 110 amino acid residues (with a molecular weight of about 11,500 D). All of the genes capable of coding for the variable and constant regions of the Ig's are assumed to have originated therefrom through successive tandem duplication and diversification. The development of the heavy chain of IgY is believed to have its origin in a primordial gene which, in a similar way to the mu chain gene, has evolved through point mutation and tandem duplication. The occurrence of this primordial gene is considered to coincide with the transition from life in water to life on land of ancestral Amphibia. It appears probable that this has taken place during the process of evolution of the Amphibia, since until now only IgM could be detected in Urodela (9). The development of H chains having less than five domains might be explained by what is usually referred to as defect mutation. It is reasonable to assume that in the case of IgG, for example, the hinge region has developed from the CH2 domain adjacent to the mu chain. Domain loss is a widespread phenomenon in the evolution of immunoglobulins. For example, the presence of 5,7S Ig's in reptiles and birds antigenicalLv deficient the IgY like Ig's may be readily explained by the fact that their H chains of 36,000 D originated from those of IgY by the loss of two C-terminal C H domains. What remains to be answered is the question as to whether mammals possess an equivalent of the IgY type Ig's. A possible candidate would be IgD. The reasons given for
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this assumption are the similarity of the molecular weight of the H chains which is in the neighbourhood of 69,000 D, a carbohydrate content of 1 1 % (25), its first ontogenetic occurrence, and its function as an antigen specific l~mphocyte receptor (26). The results of our preliminary experiments conducted using an IgD paraprotein do not, for the present, allow a positive answer. Further, human subclass IgG3 which, compared to the other three subclasses, has a molecular weight that is some 8,000 D higher for the H chain and also has a carbohydrate content of 5 %, is discussed as possible candidate for an IgY equivslent (27), though a great number of interheav¥ chain half-cystine residues is charscteristic for IgG3 (25), but not for IgY-like immunoglobulins (13,26). Further studies are in progress to investigate the antigenic relationship of Ig's of the IgD class and the IgG3 subclass to IgY. A CKNOWLED GMENT The authors are indebted to Mrs. Christa Fritzsche for her most conscientious technical assistance. REFERENCES I. MARCHALONIS, J.J. and EDELMAN, G.M. Phylogenetic origins of antibo&¥ structure. III. Antibodies in the primary immune response of the sea lympre¥ Petromyzon msrinus. 2. CLEM, L.W. and SMALL, P.A. Phylogeny of immunoglobulin structure and function. I. Immunoglobulins of the lemon shark. J.Exp.Med. 125, 893, 1967. 3. ~RCHALONIS, J.J. Isolation and partial characterization of immunoglobulins of goldfish (Carsssius auratus) and carp (Cyprinus carpio). Immunology 20, Ibl, 1971. 4. RICHTER, R., FRENZEL, E.-M., HADGE, D., KOPPERSCHLAGER, G., and AMBROSIUS, H. Strukturelle und immunchemische Untersuchungen am Immunglobulin des Karpfens (Cyprinus carpio L.). I. Analyse am GessmtmolekG1. Acts Biol.Med. Germ. 30, V35, 1973. 5. ANDREAS, E.-M., RICHTER, R.F., HADGE, D., and AMBROSIUS, H. Strukturelle und immunchemische Untersuchungen am I ~ u n globulin des Karpfens (C¥orinus carpio L.) II. Analyse der Untereinheiten. Acta ~iol.Med. Germ. 34~ 1407, 1975. 6. ACTON, R.T., EVANS, E.E., WEINHEIMER, P.F., NIEDERMEIER, W., and BENNETT, J.C. Purification and characterization of two classes of immunoglobulins from the marine toad (Bufo marinus). Biochemistry 11, 2751, 1972. 7. STEINER, L.A., MAKORYAK, C.A., LOPES, A.D., and GREEN, C. Immunoglobulins in ranid frogs and tadpoles in: Immunologic Phylogeny. W.H. HILDEMANN snd A.A. BENEDICT (Eds.) New York and London, Plenum Press, 1975, p. 173. 8. ATWELL, J.L. and MARCHALONIS, J.J. Phylogenetic emergence of immunoglobulin classes distinct from IgM. J.Immunogenetics I, 367, 1975.
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9. AMBROSIUS, H., HEMMERLING, J., RICHTER, R., and SCHIMKE, R. Immunoglobulins and the ~vnamics of antibody formation in poikilothermic vertebrates (Pisces t Urodela I Reptilia) in: Developmental aspects of antibo&y formation and structure. J. STERZL and I. RIHA (Eds.) New York and London, Academic Press. 1970, p. 727. 10. AMBROSIUS, H. Immunoglobulins and antibody production in eptiles in: Comparative immunolo@v. J.J. MARCHALONIS Ed.) Oxford, London, Edinburgh, and Melbourne. Blackwell Scientific Publications. 1976, p. 298. 11. LESLIE, G.A. and CLEM, L.W. Pkvlogeny of immumoglobulin structure and function. VI. 17S, 7,5S and 5,7S anti-DNP of the turtle, Pseudamys scripts. J.Immunol. 108, 1656, 1972. 12. LESLIE, G.A. and CLEM, L.W. Pkylogeny of immunoglobulin structure and function. III. Immunoglobulins of the chicken. J.Exp.Med. 130, 1337, 1969. 13. ZIMME~RMAN, B., SHALATIN, N., and GREY, H.M. Structural studies on the duck 5.7S and 7.8S immunoglobulins. Biochemistry 10, 482, 1971. 14. GALLAGHER, J.S. and VOSS, E.W. Conformational state of chicken 7S immunoglobulin. Immunochemistr~ 11, 461, 1974. 15. WEBER, K. and 0SBORN, M. The reliability of molecular weight determination by dodecyl sulfste-polyacr~lsmide gel electrophoresis. J.Biol.Chem. 244, 4406, 1969. 16. EDELMAN, G.M. and POULIK, M.D. Studies on structural units of the~-globulin. J.Exp.Med. 113, 861, 1961. 17. WARREN, L. The thiobsrbituric acid assay of sialic acids. J.Biol.Chem. 234, 1971, 1959. 18. CUATRECASAS, P. Protein purification by affinity chromatogrspk¥. Derivatization of agarose and polyacr~lamide beads. J.Biol.Chem. 245, 3059, 1970. 19. HUNTER, W.M. and GREENWOOD, F.C. Preparation of iodine131 labelled human growth hormone of high specific activity. Nature 194, 495, 1962. 20. REYNOLDS, J.A. and TANFORD, C. The gross conformation of protein-sodium dodecyl sulfate complexes. J.Biol.Chem. 245, 5161, 1970. 21. HILL, R.L., DELANEY, R., FELLOWS, R.E., and LEBOVITZ, H.E. The evolutionary origins of the im~noglobulins. Proc. Nat.Acad. Sci. U.S. 56, 1762, 1966. 22. LESLIE, G.A. and ROWE, D.S. The molecular weight of hum~nIgD heavy chains. Immunochemistr~ 8, 565, 1971. 23. ROWE, D.A., HUGH, S., FAULK, W., PAGE, W., McCORMICK, J.N., and GERBER, H. IgD on the surface of peripheral blood lymphocytes on the human newborn. Nature New Biol. 242, 155, 1973.
~
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24. SALUK, P.H., and CLEM, L.W. The unique moleculsr weight of the hesvy chsin from human IgG3. J.Immunol. 107, 298, 1970. 25. MICHAELSEN, T.E. Evidence of 15 S-S bridges in the hinge region of human IgG3. Scsnd.J.Immunol. 2, 523, 1973. 26. TENENHOUSE, H.S. end DEUTSCH, H.F. Some pkysical-chemicsl properties of chicken ~-globulins end their pepsin end pepsin digestion products. Immunochemistry 3, 11, 1966.
EVOLUTION OF LOW MOLECULAR WEIGHT IMMUNOGLOBULINS I. RELATIONSHIP OF 7S IMMUNOGLOBULINS OF VARIOUS VERTEBRATES TO CHICKEN IgY
Dietlind H~dge, Helmut Fiebig, snd Herwart Ambrosius Laboratory of Immunobiology, Section of Biosciences, Karl-Mmrx-University, Leipzig, G.D.R.
ABSTRACT. All of the birds (chicken, duck, goose), reptiles (sheltopusik, tortoise), end enuran amphibians (pond frog) included in these studies have 7S immunoglobulins with molecular weights between 160,000 and 180,5OO D, the H chains of which show molecular weights of 62,700 to 65,500 D. The molecular weights of these 7S immanoglobulins as well as those of their heavy chains ere markedly higher than the corresponding values for the IgG molecules of mammals, which are 150,000 end 50,000 D, respectively. The contents of carbokydrates (hexoses, hexosamines, eialic acid) of these low molecular weight immunoglobulins, which are between 4.6 and 6.4 %, are markedly higher than that of IgG which is of the order of 2.3 %. Immunochemical studies (double-antibody radioimmunosesay) made to determine similarities in the sntigenicity of low molecular weight immunoglobulins of the different species showed that there is s close antigenic relationship between the 7S immunoglobulins of birds, reptiles, end amphibians, but only s distsnt relationship to mammalian IgG. Therefore, we propose to include nonmammslian 7S immunoglobulins in one single class designsted as IgY sccording to the term used by Leslie and Clem for the 7S immunoglobulin of chicken.
INTRODUCTION Immunoglobulins (Ig'e) show an increase in the heterogeneity of isotypes in the course of evolution. All vertebrates hsve a high molecular weight type of immunoglobulin which is structurally and functionally related to mammalian IgM and which is the only class of immunoglobulins found in Agnatha (I), Chondrichthyes and Osteichthves (2-5). 501
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In amphibians (6-8) has been found s low molecular weight type of 7S immunoglobulin for the first time which differs in antigenicity from IgM and occurs also in reptiles (9-11) and in birds (12-14). These 7S immunoglobulins have often been termed IgG like the memm~lisn immunoglobulin. The structursl and functional characteristics of chicken 7S immunoglobulin have been studied most carefully (12,14) and designated as IgY by Leslie and Clem (12). In addition, s third 5.7S class sntigenically related to 7S immunoglobulin has been characterized in certain reptiles (9-11) and birds (14). In the current work, a number of physical and chemical data of various 7S immunoglobulins of representative vertebrates sre presented. Using the technique usually referred to as double-sntibo&y radioimmunoassay, immunoche~Lical studies on the antigenic relationship of these immunoglobulins were made to obtain information about the evolution of low molecular weight immunoglobulins, the major focus being on the relation between the 7S immunoglobulins of birds and lower vertebrates to mammalian IgG. MATERIALS AND METHODS Prepsrstion of immuno~lobulins. ~ material: Immunoglobulins were obtained from normal and immune sers of chickens (Gallus ~allus domesticus L.), ducks (Anser plstyrhynchos domestics L.), goose (Anser anser), tortoises (A~rlonemis horsfieldii GRAY), sheltopusiks ( P A h ~ saurus apodus /PALLAS/), and pond frogs (Rans e s c u l e n t a U . In addition, f~om human and guinea-pig sara were isolated IgG and IgG2, IgM(Go) from the serum of a Pstient with Wsldenstr~m's mscroglobulinemia, and IgM from the serum of carps (Cyprinus carpio L.). Gamm~ globulin obtained from cows (BGG) was made available by Serva Feinbiochemics, Heidelberg. The IgD psraprotein was 8 crude fraction precipitated with (NH4)2SO 4 st s finsl concentration of halfsaturation. Preparation of macro~lobulins: Macroglobulins were prepared Sy-r~Ya~gs ~ ~s~I~ of immunoglobulin by precipitation st 50 % saturation of (NH4)2S04, gel filtration on Sepharose 6B (Pharmscia) and Bio-Gel A - 1.5 m (Bio-Rsd Laboratories, Richmond), starch block electrophoresis, and rechromstography by gel filtration using s previously described method (4). PreRaration of 7S immuno~lobulins: The 7S immunoglobulins ~re ~ ~ - ~ ~e-~8~on of immunoglobulin (three precipitations with N82S04 at concentrations of 180, 140, and 140 mg/ml or three precipitations with (NHA)2SO A at 50 %, 33 %, and 33 % saturation, respectively). This-was ~ollowed by several steps of gel filtration on columns of Sephsdex G-200 (Pharmacia) or Bio-Gel A - 1.5 m. In the case of preparation and purification of 7S immunoglobulins obtained from chicken or sheltopusik sets as well 8s from human and
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guinea-pig sera, the steps of gel filtration were combined with chromstograpky on dietkylaminoetkv1 cellulose (DE-52, Whatman) by using a 0 . 0 1 M Tris HC1 buffer of pH 8.0 (conductivity: 5001uS) as starting buffer° Elution of the 7S immunoglobulin~ from the cellulose columns was carried out by means of a linear NaC1 gradient. The fractions containing the low molecular weight immunoglobulins were combined, concentrated by ultrsfiltration, and prepared for subsequent gel filtration. Confirmation of individual fractions for contents of 7S immunoglobulins was accomplished by immunoelectrophoretic analysis using antisera directed against the components of normal sera from the respective species. In all cases, the immunoglobulins thus obtained were immunoelectrophoretically pure preparations. Protein concentrations of samples were determined by extinction measurements at 280 nm assuming an extinction coefficient (1-cm cuvettes, 1 % solution) of 14.0 for the msmmslisn IgG preparations and of 12.0 for the other purified Ig materials. Determination of pkysical and chemical parameters. SDS - PAGE: Polyscrylamide gel electrophoresis performed in ~H~ presence of sodium dodecyl sulfate ~SDS) was used to estimate relative molecular weights of immunoglobulins and their polypeptide chains (15). Humsn IgG (150,000 D), its H and L chains (52,000 and 23,000 D, respectively) as well as H and L chains of carp IgM (76,000 and 23,000 D, respectively) served as standard substances for plotting a calibration curve. Preoaration of H and L chains of immuno~lobulins: H and L bulins with 2-mercaptoethanol (Ferak, Berlin-West) and by means of alkylation using iodoscetamide (Servs) (16). Determining the csrbokEdrate content: The contents of H~8~ ~S-H~8~n~s were S ~ L [ n e d using orcinol reagent (E. Merck AG, Darmstadt) and a modified version of the Elson-Morgsn reaction, respectively (4). To determine the proportions of sislic acid, we used the method of detection by thioberbituric acid (17). N-acetylneuraminic acid (17). N-acetylneuraminic acid (99 % synthetic) serving as a reference substance was made available by Serva. Double-antibody radioimmunoassa¥ Preoaration of sntisers: Carps weighing 1.5 kg snd kept at a-~m~era~-~-~-~ 2°C were immunized using 0.2 mg of chicken IgY emulsified in CFA (Difco, Detroit, U.S.A.). Two reimmunizations were mede at one-month intervals using I mg of IgY without an adjuvant; blood samples were taken by cardiac puncture two weeks after the second reimmunization. Rabbits were injected with 0.2 mg of chicken IgY emulsified in CFA into all four foot pads~ the second immunization was made after four months (o.4 mg). After ten ds~s, samples of blood were taken by cardiac puncture. These anti-IgY sets (sntisera I) were extensively absorbed with chicken IgM conjugated to CNBr-activated Sepharose 4B (18) rendering them H chain specific. Eor precipitatin antibodies (antisera II)
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we used anti-carp IgM serum obtained by immunization of rabbits with 0.5 mg of carp IgM in CFA and two reimmunizations with I mg each at four-week intervals and gost-snti-rsbbit gsmms globulin serum (SIFIN, Berlin-Wei6ensee), respectively. Radio-labelin~ of chicken IgY: Chicken IgY (0.5 mg) were I~I~-~-~H~-~I~=~ method (19), using 2 mBq of carrier-free iodine-125 (as Na125I, ROTOP, Nuclear Research Center at Rossendorf, G.D.R.). Free iodine was separated by gel filtration on Sephadex G-25. Radioimmunosssa~: All steps of dilution, including the dis~o-Y~-~-~ inhibitory proteins were made with PBS (phosphate-buffered saline, pH 7.4, containing 0.2 mg NaN3/ml). The following technique was used for the detection of crossreactivity: The specific antibodies (antisera I) were supplemented with normal sers in s I : 320 dilution. The exact amounts of double antibodies (sntisera II) to allow a complete precipitation of the whole Ig fractions were determined by a gel diffusion test. Amounts of specific antibodies were adjusted in such a manner that 50 % of labeled added chicken IgY wss bound by the antibodies. The following conditions were used: 0.05 ml of sntisers I (carp- or rabbit-snti-IgY sers in s I : 1,280 or s I : 10,000 dilution, respectively) was incubated with 0.05 ml of inhibition solution consisting of 7S Ig's of several species at 4oc for 2 hr. After this 125I-IgY (50,000 cpm) in 0.05 ml PBS was added, the contents mixed gently, and the polystyrene tubes agsin incubated at 4oc for 2 hr. Pinslly 0.05 mi of sntisers II (rabbit-anticarp IgM serum, diluted I : 32, and goat-snti-rabbit gamma globulin serum, 1 • 64, respectively) was added and the incubation continued for 24 hr st 4°C. Then two controls were included in which the anti-IgY sets were substituted for the corresponding normal sers and the inhibition solution by PBS. This wss done to determine the correction for coprecipitstion and cosdsorption. All tubes are set up in triplicate. Finally the tubes were centrifuged st 10,000 rpm for 10 min at 4°C and the radioactivity of supernatent (0.1-ml volume) which was carefully removed from the mixture was measured by s NaI(T1) crystal scintillator. RESULTS Estimation of relative molecular weights of immunoglobulins and of their H and L chains. To estimate the molecular weights, 5 % polyacrylamide gels c r o s s - l i n k e d w i t h N,N-methylene-bis-acrylamide with a ratio of polyscrylsmide to cross-linking agent (80 : I) were used. According to procedure there is a linear relation between the migration distances in gel and the logarithm of the molecular weights ranging from 20,000 to 200,000 D (15). Since the electrophoretic mobility of glycoproteins loaded with SDS anions depends upon both their carbokydrate contents, their conformation and the charge distributions on these molecules (20), the standard substsnces used for plotting of cslibration curves were, without exception, immunoglobulins
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with known molecular weights. In spite of that we are able to obtain only relative molecular weight values. Finally our standards differ also somewhat in its physical and chemical properties. The results of estimating the molecular weights of different 7S immunoglobulins are summarized in Table I. TABLE
1
Relative Molecular Weights of 7S Immunoglobulins of Different Vertebrates (Six Estimations) Species
Ig type
Molecular weights ± SD (in Daltons)
Man Cow Guinea-pig Chicken Duck Goose Tortoise Frog
IgG IgG IgG2 IgY 7S Ig 7S Ig 7S Ig 7S Ig
150,00 (assumed value) 148,500 ± 2,500 142,500 ± 3,010 169,500 ± 3,000 168,750 ± 3,400 165,000 ± 4,650 180,500 ± 2,500 160,000 ± 2,000
SD = standard deviation. Accordingly, chicken, duck, goose, tortoise, and frog may be regarded as having low molecular weight immunoglobulins with molecular weights between 160,000 and 180,500 D. As is Usually the case, the molecular weight of human IgG was assumed to be on the order of 150,000 D. Using SDS - PAGE on 5 % gels, s value of 142,500 D was calculated for guineapig IgG2, whereas a vslue of 148,500 D was obtained for BGG. The results of estimating the molecular weights of H and L chains of 7S immunoglobulins of different vertebrates are in Table 2 (see also Fig. 1). Clearly the 7S immunoglobulins of birds, reptiles, and amphibians, included in this investigation, possess an H chsln type with 62,700 to 65,500 D. These values are markedly higher than those of H chains with about 52,000 D for human IgG, guinea-pig IgG2, and BGG, but markedly lower than those for heavy chains of carp IgM (5) or IgM fractions (not shown in the Table),of the same species of mammals, birds, and lower vertebrates (70,000 to 76,000 D). In another series of experiments, the molecular weights of some polypep%ide chains were estimated by SDS PAGE using 10 % gels, since this tends to reduce the disturbing influence of carbokvdrate components of immunoglobulins upon their electrophoretic mobility. These measurements (four estimations) yielded only slightly lower vslue~ for the molecular weights of t~e H chains, namely, 61,300 z 470 D for chicken IgY, 61,800 z 430 D for duck 7S Ig, -
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62,000 ± 570 D for tortoise 7S Ig, and 74,500 ± 470 D for carp IgM. The values obtained for L chains remained unchanged and were about 24,000 D. Determination of the csrbokydrst e contents. The contents of carbokydrstes, i.e., hexoses, hexossmines, and sislic acid are compared with those values obtained for the IgM preparations of the respective species and for humsn and carp IgM, respectively (Table 3). Msnnose-galactose mixtures of I : I or, in the case of human IgM, of 2 : I served ss reference solutions. The hexosamine proportions are given as glucossmine bsse and the sialic acid residues as N-acetylenuraminic acid. TABLE
3
Csrbokydrste Composition of Immunoglobulins of Different Vertebrates (Two Determinations) Species
Ig type Hexoses
Man IgG Guinea-pig IgG2 Chicken IgY Duck 7S Ig Tortoise 7S I ~ Msn IgM(Go) Chicken IgM Duck IgM Tortoise IgM Sheltopusik IgM Carp . . . . I~M , n.d. -- not determined
1.3 1.3 3.2 4.5 3.3 7.1 4.1 4.1 2.8 5.3 4~.1 .
Content (in %) HexosSialic Total amines acid 1.O O.1 2.4 0.9 O.1 2.3 1.8 0.2 5.2 1.7 0.2 6.4 1.~1 0.2 4.6 4.1 0.4 11.6 1.2 0.5 5.8 2.7 0.6 7.4 1.3 1.2 5.3 2.7 n.d. ,8.0 . 2,.7, . ~5 7.3
Clearly the carbokydrste contents of 7S immunoglobulins of birds (chicken, duck) end reptiles (tortoise), of which vslues were between 4.6 and 6.4 %, were higher than the proportions of about 2.3 % determined for the two IgG preparations (man, guinea-pig) (Tsble 3). Rather, the values of 7S immunoglobulins were nearby of those obtsined for IgM preparations without accounting for the extremely high value for human IgM(Go). Antigenic relstionship of chicken I~Y to the 7S immunoglobulins of different vertebrates. To detect sntigenic relationships, s radioimmunosssey system was used bssed upon the inhibition of bindin~ reaction between 125I-IgY snd csrp anti-IgY sntibodies ~Fig. 2) and rabbit snti-IgY sntiboaies (Pig. 3), respectively, by unlsbeled 7S Ig prepsrstions of goose, duck, sheltopusik, tortoise, frog, human (IgG and IgD), and cow.
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TABLE
507
2
Relative Molecular Weights of the H and L Chains of 7S Immunoglobulins of Different Vertebrates (Six Estimations) Species
Ig type
Molecular weights ± S D (in Daltons) H chains L chains
Man
IgG IgG IgG2 IgY 7S Ig 7S Ig 7S Ig 7S Ig 7S Ig
52,000 52,500 51,700 62,800 62,700 64,300 65,500 64,500 64,000
Cow
Guinea-pig Chicken Duck Goose Tortoise Sheltopusik Prog
(assumed value) f 800 23,000 23,000 ± 300 ± 2,170 22,000 ± 1,840 24,500 25,000 ± 2,450 ± 2,200 24,000 ± 2,200 25,500 ± 2,000 21,000
PIG.
± ± ± ± ± ± ± ±
1,000 1,000 1,700 1,800 2,000 1,750
1,850 2,500
I
SDS-PAGE of H add L Chains of Immunoglobulins of Different Vertebrates. s = Human IgG b = BGG c = Guinea-pig IgG2 d = Chicken IgY e = Duck 7S Ig f = Tortoise 7S Ig g = Human
IgM(Go)
h = Carp IgM ,5 % gels, 4.5 mA/gel, hrs.
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CHICKEN IgY
It is important to note that the 7S immunoglobulins of birds, reptiles, and mnuran amphibians appeared as an antigenically related group. As would be expected, immunoglobulins of the goose and duck were more closely related to chicken IgY than to those of the sheltopusik, tortoise, and frog. The proportion of species-specific determinants is responsible for the relatively large distance between the inhibition curves of IgY and those of other 7S immunoglobulins. Only a rather small degree of cross-reactivity wss observed for the IgG of men and cow. Likewise, the use of IgD paraprotein revealed only a slight degree of relationship to IgY add to the other 7S immunoglobulins of birds and lower vertebrates with respect to antigenicity. By using rmbbit anti-IgY sera, we were able to observe that 7S immunoglobulins of birds showed a somewhat closer relationship, whereas the immunoglobulins of the reptiles add the frog showed a less clearly evident cross-resctivity to chicken IgY. DISCUSSION The major focus of these studies dealt with relstions between the 7S immunoglobulins of different classes of vertebrstes. By comparing the pkvsical, chemical, and antigenic chsrscteristics of these immunoglobulins, we sttempted to classify them into previously known classes of immunoglobulins. Of particular interest in this connection was the structursl end sntigenic relstionship of the 7S immunoglobulins of lower vertebrstes to mammalian IgG. It wss already in the estimation of the molecular weights of intact immunoglobulin molecules that we observed s consistent variation in values of 7S immunoglobulins from those of IgG type immunoglobulins° All of the birds, reptiles, and amphibians included in these studies, for which vslues in the region of 170,000 D were obtained, showed higher molecular weights than immunoglobulins of mammals used for comparison, where the moleculmr weights were approximately 150,000 D. These vslues, obtained by SDS PAGE on 5 % gels, agreed with previous reports and measured 7S immunoglobulins of the most different vertebrate species by using other techniques such ms, for example, analytical gel filtration and sedimentation equilibrium analysis (8,11,12,13). Similar trends were observed for molecular weights of the H chains from 7S immunoglobulins. Thus the molecular weights of 7S immunoglobulins of birds and lower vertebrates which are higher than the corresponding values of the mammalian IgG type result from the presence of lodger H chains, also reported for s number of 7S immunoglobulins of different species (8,11,12,13). However, it must be considered that factors as carbokvdrate content or charge mentioned above may affect the migration of our immunoglobulins in SDS-gels. Nevertheless, the observed differences in the monmamm~lian heavy chain molecular weights in comparison to the mammalian 7 chain values indicate that they probably differ significantly in some structural features.
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The degree of immunological cross-reactivity, representing the presence of equal and similar determinants, was inferred from our immunochemical studies on antigenic relationship, undertaken as part of a rsdioimmunoassay. EvidentLy, the 7S immunoglobulins of birds, reptiles, and amphibians are characterized by a high degree of similarity in antigenicity. Moreover, these studies showed that using csrp-anti-IgY sera allowed a far better agreement between the antigenic properties of IgY-like immunoglobulins than when rabbit anti-IgY sera were used. Unlike rabbits, carps, which produce only IgM antibodies, also showed an intense immune response, because of the pkylogenetic distance of the antiserum donor from the chicken IgY antigen, to those determinants which are relatively conservetive in the course of evolution. Because of the consistently higher values of the molecular weights of 7S immunoglobulins of birds, reptiles, and amphibians, the markedly higher carbokydrate contents (5 - 7 % as compared with 2 - 3 % for mammalian IgG), and the agreement between antigenic properties, we propose that low molecular weight immunoglobulins of birds and lower vertebrates be delimited from IgG type immunoglobulins by the designation Ig's of IgY type or IgY like Ig's, which was chosen by Leslie and Clem (12) for the 7S Ig of chicken. Considering the genetic background for the evolution of Ig's it is assumed that at the pkylogenetic level of anuran amphibians there must have evolved an H chain gene differing from the mu-chain gene, which can also be found in the Sauropsida. Hill et al. (21) suggested that for all Ig's there is an ancestral primordial gene capable of coding for a poLTpeptide chain of about 110 amino acid residues (with a molecular weight of about 11,500 D). All of the genes capable of coding for the variable and constant regions of the Ig's are assumed to have originated therefrom through successive tandem duplication and diversification. The development of the heavy chain of IgY is believed to have its origin in a primordial gene which, in a similar way to the mu chain gene, has evolved through point mutation and tandem duplication. The occurrence of this primordial gene is considered to coincide with the transition from life in water to life on land of ancestral Amphibia. It appears probable that this has taken place during the process of evolution of the Amphibia, since until now only IgM could be detected in Urodela (9). The development of H chains having less than five domains might be explained by what is usually referred to as defect mutation. It is reasonable to assume that in the case of IgG, for example, the hinge region has developed from the CH2 domain adjacent to the mu chain. Domain loss is a widespread phenomenon in the evolution of immunoglobulins. For example, the presence of 5,7S Ig's in reptiles and birds antigenicalLv deficient the IgY like Ig's may be readily explained by the fact that their H chains of 36,000 D originated from those of IgY by the loss of two C-terminal C H domains. What remains to be answered is the question as to whether mammals possess an equivalent of the IgY type Ig's. A possible candidate would be IgD. The reasons given for
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this assumption are the similarity of the molecular weight of the H chains which is in the neighbourhood of 69,000 D, a carbohydrate content of 1 1 % (25), its first ontogenetic occurrence, and its function as an antigen specific l~mphocyte receptor (26). The results of our preliminary experiments conducted using an IgD paraprotein do not, for the present, allow a positive answer. Further, human subclass IgG3 which, compared to the other three subclasses, has a molecular weight that is some 8,000 D higher for the H chain and also has a carbohydrate content of 5 %, is discussed as possible candidate for an IgY equivslent (27), though a great number of interheav¥ chain half-cystine residues is charscteristic for IgG3 (25), but not for IgY-like immunoglobulins (13,26). Further studies are in progress to investigate the antigenic relationship of Ig's of the IgD class and the IgG3 subclass to IgY. A CKNOWLED GMENT The authors are indebted to Mrs. Christa Fritzsche for her most conscientious technical assistance. REFERENCES I. MARCHALONIS, J.J. and EDELMAN, G.M. Phylogenetic origins of antibo&¥ structure. III. Antibodies in the primary immune response of the sea lympre¥ Petromyzon msrinus. 2. CLEM, L.W. and SMALL, P.A. Phylogeny of immunoglobulin structure and function. I. Immunoglobulins of the lemon shark. J.Exp.Med. 125, 893, 1967. 3. ~RCHALONIS, J.J. Isolation and partial characterization of immunoglobulins of goldfish (Carsssius auratus) and carp (Cyprinus carpio). Immunology 20, Ibl, 1971. 4. RICHTER, R., FRENZEL, E.-M., HADGE, D., KOPPERSCHLAGER, G., and AMBROSIUS, H. Strukturelle und immunchemische Untersuchungen am Immunglobulin des Karpfens (Cyprinus carpio L.). I. Analyse am GessmtmolekG1. Acts Biol.Med. Germ. 30, V35, 1973. 5. ANDREAS, E.-M., RICHTER, R.F., HADGE, D., and AMBROSIUS, H. Strukturelle und immunchemische Untersuchungen am I ~ u n globulin des Karpfens (C¥orinus carpio L.) II. Analyse der Untereinheiten. Acta ~iol.Med. Germ. 34~ 1407, 1975. 6. ACTON, R.T., EVANS, E.E., WEINHEIMER, P.F., NIEDERMEIER, W., and BENNETT, J.C. Purification and characterization of two classes of immunoglobulins from the marine toad (Bufo marinus). Biochemistry 11, 2751, 1972. 7. STEINER, L.A., MAKORYAK, C.A., LOPES, A.D., and GREEN, C. Immunoglobulins in ranid frogs and tadpoles in: Immunologic Phylogeny. W.H. HILDEMANN snd A.A. BENEDICT (Eds.) New York and London, Plenum Press, 1975, p. 173. 8. ATWELL, J.L. and MARCHALONIS, J.J. Phylogenetic emergence of immunoglobulin classes distinct from IgM. J.Immunogenetics I, 367, 1975.
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9. AMBROSIUS, H., HEMMERLING, J., RICHTER, R., and SCHIMKE, R. Immunoglobulins and the ~vnamics of antibody formation in poikilothermic vertebrates (Pisces t Urodela I Reptilia) in: Developmental aspects of antibo&y formation and structure. J. STERZL and I. RIHA (Eds.) New York and London, Academic Press. 1970, p. 727. 10. AMBROSIUS, H. Immunoglobulins and antibody production in eptiles in: Comparative immunolo@v. J.J. MARCHALONIS Ed.) Oxford, London, Edinburgh, and Melbourne. Blackwell Scientific Publications. 1976, p. 298. 11. LESLIE, G.A. and CLEM, L.W. Pkvlogeny of immumoglobulin structure and function. VI. 17S, 7,5S and 5,7S anti-DNP of the turtle, Pseudamys scripts. J.Immunol. 108, 1656, 1972. 12. LESLIE, G.A. and CLEM, L.W. Pkylogeny of immunoglobulin structure and function. III. Immunoglobulins of the chicken. J.Exp.Med. 130, 1337, 1969. 13. ZIMME~RMAN, B., SHALATIN, N., and GREY, H.M. Structural studies on the duck 5.7S and 7.8S immunoglobulins. Biochemistry 10, 482, 1971. 14. GALLAGHER, J.S. and VOSS, E.W. Conformational state of chicken 7S immunoglobulin. Immunochemistr~ 11, 461, 1974. 15. WEBER, K. and 0SBORN, M. The reliability of molecular weight determination by dodecyl sulfste-polyacr~lsmide gel electrophoresis. J.Biol.Chem. 244, 4406, 1969. 16. EDELMAN, G.M. and POULIK, M.D. Studies on structural units of the~-globulin. J.Exp.Med. 113, 861, 1961. 17. WARREN, L. The thiobsrbituric acid assay of sialic acids. J.Biol.Chem. 234, 1971, 1959. 18. CUATRECASAS, P. Protein purification by affinity chromatogrspk¥. Derivatization of agarose and polyacr~lamide beads. J.Biol.Chem. 245, 3059, 1970. 19. HUNTER, W.M. and GREENWOOD, F.C. Preparation of iodine131 labelled human growth hormone of high specific activity. Nature 194, 495, 1962. 20. REYNOLDS, J.A. and TANFORD, C. The gross conformation of protein-sodium dodecyl sulfate complexes. J.Biol.Chem. 245, 5161, 1970. 21. HILL, R.L., DELANEY, R., FELLOWS, R.E., and LEBOVITZ, H.E. The evolutionary origins of the im~noglobulins. Proc. Nat.Acad. Sci. U.S. 56, 1762, 1966. 22. LESLIE, G.A. and ROWE, D.S. The molecular weight of hum~nIgD heavy chains. Immunochemistr~ 8, 565, 1971. 23. ROWE, D.A., HUGH, S., FAULK, W., PAGE, W., McCORMICK, J.N., and GERBER, H. IgD on the surface of peripheral blood lymphocytes on the human newborn. Nature New Biol. 242, 155, 1973.
~
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24. SALUK, P.H., and CLEM, L.W. The unique moleculsr weight of the hesvy chsin from human IgG3. J.Immunol. 107, 298, 1970. 25. MICHAELSEN, T.E. Evidence of 15 S-S bridges in the hinge region of human IgG3. Scsnd.J.Immunol. 2, 523, 1973. 26. TENENHOUSE, H.S. end DEUTSCH, H.F. Some pkysical-chemicsl properties of chicken ~-globulins end their pepsin end pepsin digestion products. Immunochemistry 3, 11, 1966.