------~-- "'~. Developmental Aspects of Immunoglobulins and

Herwart Ambrosius

Institute of Zoology, University, D-Leipzig

Summary: Zusammenfassung: Aspekte der Entwicklung von Immunglo- This short review discusses the evolulution of the immu- bulinen und Antikorpern. noglobulins and the development of the diversity of antibod- Die vorliegende Ubersicht diskutiert die Evolution der ies. and represent two lines of development Intmunglobuline und die Ausbildung del' Vielfalt del' having divided more than 300 million years ago. In the Antikorper. Vogel und Siiugetiere sind heutige Typen von immunoglobulins and -systems there are many Entwicklungslinien, die sicti vor mehr als 300 Millionen similarities which, however, are based on analogies. In this Jahren getrennt haben. In den Immunglobulinen und sense the dominant humoral type of immunoglobulin is Ig Y Aruikorpersystemen gibt es viele Ahnlichkeiten, die jedoch in birds as opposed to IgG in the mammals. Also, the oft auf Analogien beruhen. So ist der dominierende humora- mechanisms differ which lead to the diversity of antibodies. le Immunglobulintyp der Vogel das IgY im Gegensat: zum In mammals predominantly recombination of gen-segments IgG der Siiugetiere. Auch die Mechanismen, die zum and somatic mutations lead to this diversity, in birds gen- Antikorperrepertoire fuhren, sind unterschiedlich. Bei den conversion shows a greater significance. In this way Siiugetieren sind es VOl'allem Rekombination der Genseg- comparable results are produced through different struc- mente und somatische Mutationen, die das groj3e Antikor- tures or mechanisms. perrepertoire liefern, wdhrend bei Yogeln der Prozej3 der Genkonversion die grofste Bedeutung hat. So werden im Keywords.immunoglobulins, Ig Y, IgG, evolution, diversity, Immunsystem der Vogel und Siiugetiere vergleichbare birds, mammals Leistungen durch unterschiedliche Strukturen bzw. Mecha- nismen erreicht.

1 Evolution of immunoglobulins mune reaction of the hagfish again, but weight immunoglobulins of non-mam- he adapted the animals to a higher malian vertebrates, they were called IgG In the fifties and the sixties the group water temperature. After injec- in analogy to the dominant humoral of Robert Good in Minneapolis studied tion he found with binding immunoglobulin class of mammals. But the ontogeny and phylogeny of the activity to the antigen and believed to in the paper of Leslie and Clem (1969) . One of the main have induced antibodies (Raison et aI., about the low molecular weight immu- results was the finding that the thymus 1978). But in 1994 he questioned his noglobulin of the chicken, the chicken plays an essential role in the immune previous results after new experiments, immunoglobulin was called IgY and system. The first data about the dichot- assuming the induced proteins to be showed well defined differences be- omy of the immune system with the complement components. tween that immunoglobulin and the IgG humoral and the cell-mediated parts Because published data about the of mammals. Unfortunately, the conclu- were found using the chicken as exper- antibodies of other groups of cyclos- sions of Leslie and Clem were not noted imental animal. It could be shown that tomes, especially the lampreys, are by the majority of later authors. Only the thymus is a primary immune organ. very controversial, hard facts about the during recent years the name IgY has It is the origin of the thymus-dependent humoral begin with been accepted for the dominant humoral and a regulatory organ cartilaginous and bony fishes and in- low molecular weight immunoglobulin also in the humoral immune response. clude all other groups of vertebrates. of amphibians, , and birds, as in One question, already dealt with by The only immunoglobulin class, oc- the review of WaIT et al. (1995). Robert Good, is about the most primi- curing in all vertebrate groups from Already in 1977 we published data tive species with a functioning immune fish to mammals, is the IgM. In some about the low molecular weight immu- system. Good and his coworkers stud- species, especially in bony fishes, it noglobulins of amphibians, reptiles, ied the hagfish Eptatretus stouti, a can be found in different types, and for and birds in comparison to the immu- cyclostome, and could not find a thy- special functions. noglobulins of mammals (Hadge et al., mus or induce antibodies. So they One question, discussed for many 1977). These investigations about the called the hagfish the "negative hero" years, concemes the first appearance of immunoglobulins of non-mammalian of immunology. A few years later low molecular weight antibody class in vertebrates have been extended in the Robert Raison in the lab of Bill Hilde- phylogeny. Throughout many years, in following years and produced the fol- mann in Los Angeles studied the im- nearly all papers about low molecular lowing picture:

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2 Structure of IgY-immuno- Table 1: Relative Molar Mass of the Heavy and Light Chains of Low Molecular globulins Weight Immunoglobulins of Different Vertebrates (Six Estimations)

Molar Mass (in KD) There are well defined structural diffe- rences of IgY-type immunoglobulins Species Ig Type H Chains L Chains and the 19G. That includes the molar mass of the heavy chains (table 1) and Man IgG 52 (Ref. Value) the carbohydrate content of the immun- Cow IgG 52.5 23,0 oglobulins (table 2). Guinea-Pig IgG 2 51,7 23.0 On the other hand, the IgY-type Chicken IgY 62.8 22.0 immunoglobulins are much less flexi- Duck IgY 62.7 24.5 ble than IgG as shown by fluorescence Goose IgY 64.3 25.0 polarisation technique (table 3). But Tortoise Agrion. horsf. IgY 65.5 24.0 the comparison of the data of the IgY Lizard Ophis. apodus IgY 64.5 25.5. of frog, tortoise, and chicken show a Frog Rana esculenta IgY 64.0 21.0 significant higher flexibility of the Data from Hadqe et aI., 1980 chicken IgY than the other IgY types. Also, the structures of the Fe part of the immunoglobulin isotypes IgY and Table 2: Carbohydrate Composition of Immunoglobulins of Different Vertebrates IgG are different. The Fe part of IgY is thinner than the Fe part of IgG and Species Ig Type Hexos. Content (in %) Sialic acid does not contain any hole in its middle Hexosamines Total part, as was revealed for the IgG Fe fragment (Cser et al., 1982). The ab- Man IgG 1.3 1.0 0.1 2.4 sence of the hole agrees with the Guinea-Pig IgG 2 1.3 0.9 0.1 2.3 supposition that the excess mass in the Chicken IgY 3.2 1.8 0.2 5.2 6.4 H-chain is distributed in the Fe region Duck IgY 4.5 1.7 0.2 near the centre of mass of the whole mo- Tortoise IgY 3.2 1.1 0.2 4.6 lecule, and it also supports the observa- Data from Hadqe et aI., 1980 tion that chicken IgY is less flexible in comparison with mammalian IgG. Unfortunately, the comparison of Table 3: Rotational Correlation Time of DNS-Conjugates of Immunoglobu- amino acid sequences is hardly suitable lins of Different Vertebrates for the elucidation of the relationships between the different immunoglobulin DNS-Conjugate Oocalc (nsec) OhexP (nsec) Oh/Oo classes occuring in the different verte- brate groups because the differences Frog IgY 54 67 1.24 are too great. It only points to IgY as Tortoise IgY 54 68 1.26 the ancestor of IgG and IgE (Warr et Chicken IgY 61 43 0.69 al., 1995). On the other hand, the Human IgG 59 20 0.35 phenomenon of immunological cross- Data from Zagyansky, 1975 reactivity could be used very success- fully for the identification and classifi- cation of immunoglobulins in species other than man which also corresponds as quantitative value for the antigenic points to a strong relationship to the with the nomeclature rules (WHO re- cross-reactivity which is nearly identi- chicken IgY. On the other hand, the port, 1969; Ambrosius et al., 1978). cal with the structural relationship. IgG-type immunoglobulins do not in- The technique mostly used for the Extensive experiments of Dietlind hibit the antigen-antibody system measurement of antigenic cross-reac- Hadge and others in our laboratory measurably. Therefore, no clear rela- tivity of proteins, is the inhibition of with alltogether 43 different antisera of tionship is detectable. IgG antibodies binding of specific antibodies to the different experimental animal species are only found in mammals. The struc- radio-labelled antigen by the proteins showed that the dominant humoral low tural difference between IgY and IgG to be compared. Fig. 1 shows the molecular weight antibodies of the are not surprising in light of the fact results of such an experiment, using non-mammalian vertebrates, excluding that the evolutionary lines of reptiles carp anti-chicken IgY antibodies of H- fishes, are all of the IgY type. Table 4 and birds separated from the one of the chain specificity and different immu- shows the data of experiments with mammals in the Permian or even earli- noglobulin preparations as inhibitors IgY fragment-specific carp antibodies. er, which means 300 million years ago. (Ambrosius and midge, 1987). Be- The 50 % inhibition values are, in the This is independent of the possibility cause in that system the point of 50 % cases of IgY-type immunoglobulins, that IgY may be the ancestor of IgG inhibition is measurable, it can be used always in the range of 1-2000 which (Warr et al., 1995).

ALTEX 13, SUPPLEMENT 96 11 AMBROSIUS ~r;") ------h~~-- ~0~,,~.

IN HI BiTtON ['f,] 31gB in birds

In 1972 several authors found a third Ig class in the serum and in the secretions of chickens with H chains clearly different in structure and anti- genicity from H chains of IgM and IgY (Bienenstock et aI., 1972; Lebacq- Verheyden et aI., 1972; Orlans and Rose, 1972). Owing to its preferential occurrence in secretory fluids like sali- va, bronchial, intestinal, or oviduct secretions, and since that third chicken Ig is nearly the only immunoglobulin component in the bile, it has been presumed to be homologous to mam- malian IgA. Since we were interested 16 64 250 1000 4000 16000 64000 25GOOO in the phylogenetic status of that "so- INHIBITOR [ng] called chicken IgA", we studied the

Figure 1: Inhibition of the binding of 125 I-chicken IgY to carp anti-chicken IgY antigenic structure similarity of that antibodies by different immunoglobulins. S = chicken IgY, 1 = goose IgY, 2 = duck immunoglobulin to mammalian IgA, IgY, 3 = tortoise IgY, 4 = sheltopusik IgY, 5 = frog IgY, 6 = human IgG, 7 = bovine IgG, IgM, and non-mammalian IgYs IgG, 8 = human IgD. From Ambrosius and Hadqe (1987). using a series of double-antibody RIA systems and antisera against the "so- called chicken IgA" as well as against Table 4: Antigenic Structure Comparison with Carp anti-Chicken human IgA (Ambrosius and midge, 1982; Hudge and Ambrosius, 1983; IgY (Fc) IgY (Fab) Ambrosius and midge, 1987; midge and Ambrosius, 1988). In not one of Immunoglobulins 50 % Inhibition the systems a measurable antigenic cross-reactivity of the "so-called chik- Chicken IgY 1 1 ken IgA" with one of the other immun- Goose IgY 36 133 Duck IgY 53 767 oglobulin types could be found. This is Tortoise IgY 300 2.200 in agreement with the fact, that this Frog IgY 66 1.495 type of the "so-called chicken IgA" is Human IgG > 30.000 > 100.000 to be found only in gallinaceous birds. Bovine IgG > 30.000 > 100.000 Probably it is a special immunoglobu- lin type which evolved within that Personal Data from D. Hadqe group of birds. It should not any longer be called IgA because it is not a homologue to mammalian IgA. We Contrary to IgG, the mammalian IgA propose to use the term 19B in conside- has a high antigenic cross-reactivity with IgY. This could be shown by Table 5: Antigenic Structure Compari- experiments with many antisera against son with anti-Chicken IgY chicken IgY and its fragments as well Table 6: Antigenic Structure Compari- as against turkey IgY in our laboratory. Immunoglobulins 50 % Inhibition son with Carp anti-Chicken IgY (Fe) Tables 5 and 6 show some results with H chain-specific rabbit anti-chicken Chicken IgY 1 Immunoglobulins 50% (Inhibition) IgY antibodies and carp anti-chicken Turkey IgY 33 IgY (Fc) antibodies, respectively. In all Human IgAFr 250 Chicken IgY 1 experiments the antigenic cross-reac- Human IgAHec 450 Porcine colostrum IgA 100 tivity of mammalian IgA with chicken Goose IgY 1.350 Goose IgY 200 or turkey IgY was in the same range as Porcine IgA 1.500 Tortoise IgY 200 that of the IgYs of other amphibian, Tortoise IgY 1.750 Duck IgY 300 Duck IgY 1.900 reptilian, or avian species. Therefore, Human IgAHec 450 Carp IgM Human colostrum IgA 500 we conclude, that the mammalian IgA Chicken 19B Chicken 19B is the direct descendant of the IgY, the Human IgG Human IgG dominant humoral antibody type of non-mammalian vertebrates. Data from Hadqe and Ambrosius, 1984 Data from Hadqe and Ambrosius, 1984

12 ALTEX 13, SUPPLEMENT 96 :;:~ AMBROSIUS --h~~------¥ct ration of the fuct that it is a biliary Murine kappa chain locus immunoglobulin of some birds.

V200 (------~ V, J, ~---) Js C • It----BJ~ • I~~r_ f- 4 Origin of antlhody-diversity Germ-lino ~ The efficiency of an antibody system VJ-Recombinatlon depends not only on the quantity of antibody production. Very important is J, also the quality of the produced anti- L c body types. Here, the affinity plays a --I1I1-IIII"-1{ ~~!!8888I---I'''''-~-I---.. }-~_II-- leading part. As we could demonstrate in studies with tortoises, chickens, and the produced antibodies of IgY as well as IgO class show the same J, increase in affinity following the first and second immunisation which is well L V J c known from the fully matured immune --I.HIII •• ~{,~,~~~~~m---.~~~m~I~r-~_~ response (Ambrosius et al., 1972; Fie- big, 1972; Fiebig and Balcerova, Figure 2: Diversification of the mouse kappa chain locus. The locus consists of 1975). A condition for that process is a approximately 200 V segments, 5 J segments, and a C gene segment. VJ genetic mechanism which delivers a recombination joins one of the V gene segments to one of the J gene segments. high number of differing antibodies Following antigen stimulation somatic mutations are introduced into the rearran- within a short time. In mammals that ged V gene. mechanism is well known for some years (fig. 2). Chicken light chain locus One of a number of 200-1000 V gene segments recombine with one of .V25 ~------~ .V1 L V J C mostly 5 J segments and in the case of ,..~----, 11- -a--_4_---I'I-- __ 1------{~ •.III!!Im Ii H-chains one of 20-50 D segments. • Germ-line Therefore, only the process of recom- V bination produces a diversity of about VJ-Recomblnatlon 1000 (L-chains) or 100.000 (H-chains) J. immunoglobulin chains. The associati- on of L- and H-chains can yield 108 L C different antibodies. That number can • increase by a factor of about 100 in consequence of imprecise DNA recom- Gene Conversion bination which leads to changes in the J. amino acids at the junction sites of the v J C V, D, and J gene segments, and also by point mutations. These somatic mutati- ons, which primarily occur after stimu- lation by antigen, lead to a selection for mutations of antibodies with higher Figure 3: Diversification of the chicken light chain locus. The locus contains only affinity for the antigen. a single V, J, and C gene segment downstream of a pool of 25 pseudogenes. VJ rearrangement joins the V and J gene segment. Afterwards, gene conversion Chicken, the only analysed species within the bursa of Fabricius introduces sequence substitutions in the functionally with dominant IgY production, devel- rearranged V gene. ops its antibody repertoire by a differ- ent strategy (fig. 3) (Bezzubova and Buerstedde,1994). junction of the V and J gene segments. Presently, we can not accurately The chicken light chain locus con- Afterwards, gene conversion introduc- calculate the antibody repertoire of tains only a single functional V and J es sequence substitutions in the func- chicken. But it may be in the same gene segment. 25 pseudo- V gene seg- tionally rearranged V gene. Blocks of range as that of mammals. There is no ments are homologous to the V gene pseudo gene sequenzes appear in the V sign that the specificity of chicken but lack the common transcription reg- gene. The conversion tracts comprise antibodies of IgY class differ from the ulatory and signal recognition sequenc- from 10 to more than 120 bp, and a specifity of mammalian antibodies of es. The single V and J gene segments single V gene can receive segmental IgO class. Chickens can recognise rearrange by VJ recombination and exchange from up to six different mammalian proteins or pep tides as create only a limited diversity at the pseudogenes. foreign and produce antibodies. Mam-

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------AMBROSIUS

mals sometimes cannot recognise Hadge, D. and Ambrosius, H. (1986). Correspondence address mammalian material as foreign anti- Evolution of low molecular weight im- Prof. Herwart Ambrosius gens and produce no antibodies or only munoglobulins. V. Degree of antigenic Universitat Leipzig in very low quantities. Therefore, relationship between the 7S immunoglo- Fakultat fur Biowissenschaften, bulins of mammals, birds, and lower chickens can be excellent experimental Pharmazie und Psychologie vertebrates to the turkey IgY. Developm. animals for the production of antibod- Institut fur Zoologie/Immunbiologie Compo Immunol. 10,377-385. ies of diagnostic value in human and Hadge, D. and Ambrosius, H. (1988). TalstraBe 33 veterinary medicine. Comparative studies on the structure of D-04103 Leipzig biliary immunoglobulins of some avian species. II. Antigenic properties of bilia- References ry immunoglobulins of chicken, turkey, duck and goose. Developm. Compo Im- Ambrosius, H., Asofsky, R., Binaghi, R. A. munol. 12,319-329. et al. (1978). Proposed rules for the Hadge, D., Fiebig, H. and Ambrosius, H. designation of immunoglobulins of ani- (1977). Strukturelle Untersuchungen an mal origin. Bull. WId. Hlth. Org. 56, lmmunglobulinen niederer Wirbeltiere. 815-817. 1. Uber die Evolution der niedermoleku- Ambrosius, H., Frenzel, E.-M. und Fiebig, laren Immunglobuline. Wiss. Z. KMU H. (1972). Untersuchungen zur Struktur Leipzig 26, 67-80. und Affinitat der Antikorper von Schild- Hadge, D., Fiebig, H. und Ambrosius, H. kroten. Ann. Immunol. Hung. 16, 15-36. (1980a). Evolution of low molecular Ambrosius, H. and Hadge, D. (I 982). A weight immunoglobulins. 1. Relationship phylogenetic view of avian immunology. of 7S immunoglobulins of various verte- Folia BioI. (Praha) 28, 1-21. brates to chicken IgY. Developm. Compo Ambrosius, H.and Hadge, D. (1987). Chik- Immunol. 4, 501-513. ken Immunoglobulins. Vet. Immunol. Im- Hadge, D., Fiebig, H., Puskas, E. and munopathol. 17,57-67. Ambrosius, H. (1980b). Evolution of Bezzubova, O. Y. and Buerstedde, J. M. low molecular weight immunoglobulins. (1994). Gene conversion in the chicken II. No antigenic cross-reactivity of hu- immunoglobulin locus: A paradigm of man IgD, human IgG and IgG3 to homologous recombination in higher eu- chicken IgY. Developm. Compo Immun- karyotes. Experientia 50, 270-276. 01. 4, 725-736. Bienenstock, J., Perey, D. Y. E., Gauldie, J. Lebacq-Verheyden, A. M., Vaerman, J. P. and Underdown, B. J. (1972). Chicken and Herernans, J. F. (1972). A possible immunoglobulin resembling IgA. 1. lm- homologue of mammalian IgA in chik- munol. 109,403-406. ken serum and secretions. Immunology Cser, L., Gladkih, I. A., Hadge, D. and 22, 165-175. Ambrosius, H. (1982). X-ray small-angle Leslie, G. A. and Clem, L. W. (1969). scattering study of general structure of Phylogeny of immunoglobulin structure chicken immunoglobulin Y. Immunology and function. III. Immunoglobulins of Leu. 4, 15-19. the chicken. 1. Exp. Med. 130, 1337- Fiebig, H. (1972). Vergleichende Untersu- 1352. chung der Affinitiit von Anti-- Orlans, E. and Rose, M. E. (1972). An IgA- Antikiirpern von Vertretern verschiede- like immunoglobulin in the fowl. Im- ner Wirbeltierklassen. Univ. Leipzig: munochemistry 9, 833-838. Dissertation. Raison, R. L., Hull, C. J. and Hildemann, Fiebig, H. (1973). Die Entwicklung der W. H. (1978). Characterization of immu- Antikorperaffinitat in der Phylogenese. noglobulin from the Pacific hagfish, a Allerg.1mmunol. 19,248-255. primitive vertebrate. Proc. Natl. Acad. Fiebig, H. and Balcerova, J. (1975). Studies Sci. U.S.A. 75, 5679-5682. on the affinity of anti-DNP antibodies of Vainoi, O. and Imhof, B. A. (1995). The the chicken inbred lines. Folia Biol. 21, immunology and developmental biology 365-366. of the chicken. Immunol. Today 16, 365- Hadge, D. and Ambrosius, H. (1983). 370. Evolution of low molecular weight im- Warr, G. W., Magor, K. E. and Higgins, D. munoglobulins - III. The immunoglobu- A. (1995). IgY: clues to the origins of lin of chicken bile - not an IgA. Mol. modern antibodies. lmmunol. Today 16, lmmunol. 20, 597-606. 392-398. Hadge, D. and Ambrosius, H. (1984). WHO (1969). An extension of the nomen- Evolution of low molecular weight im- clature for immunoglobulins. Bull. WId. munoglobulins - IV. IgY-like immuno- Hlth. Org. 41,975-978. globulins of birds, reptiles and amphibi- Zagyansky, Y. A. (1975). Phylogenesis of ans, precursors of mammalian IgA. Mol. the general structure of immunoglobulins. lmmunol. 21,699-707. Arch. Biochem. Biophys. 166,371-381.

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