---------------------------~--"'~.

Developmental Aspects of Immunoglobulins andAntibodies

Herwart AmbrosiusInstitute of Zoology, University, D-Leipzig

Summary:This short review discusses the evolulution of the immu-noglobulins and the development of the diversity of antibod-ies. Birds and mammals represent two lines of developmenthaving divided more than 300 million years ago. In theimmunoglobulins and antibody-systems there are manysimilarities which, however, are based on analogies. In thissense the dominant humoral type of immunoglobulin is Ig Yin birds as opposed to IgG in the mammals. Also, themechanisms differ which lead to the diversity of antibodies.In mammals predominantly recombination of gen-segmentsand somatic mutations lead to this diversity, in birds gen-conversion shows a greater significance. In this waycomparable results are produced through different struc-tures or mechanisms.

Keywords.immunoglobulins, Ig Y, IgG, evolution, diversity,birds, mammals

1 Evolution of immunoglobulins

In the fifties and the sixties the groupof Robert Good in Minneapolis studiedthe ontogeny and phylogeny of theimmune system. One of the mainresults was the finding that the thymusplays an essential role in the immunesystem. The first data about the dichot-omy of the immune system with thehumoral and the cell-mediated partswere found using the chicken as exper-imental animal. It could be shown thatthe thymus is a primary immune organ.It is the origin of the thymus-dependentlymphocytes and a regulatory organalso in the humoral immune response.One question, already dealt with by

Robert Good, is about the most primi-tive species with a functioning immunesystem. Good and his coworkers stud-ied the hagfish Eptatretus stouti, acyclostome, and could not find a thy-mus or induce antibodies. So theycalled the hagfish the "negative hero"of immunology. A few years laterRobert Raison in the lab of Bill Hilde-mann in Los Angeles studied the im-

10

Zusammenfassung: Aspekte der Entwicklung von Immunglo-bulinen und Antikorpern.Die vorliegende Ubersicht diskutiert die Evolution derIntmunglobuline und die Ausbildung del' Vielfalt del'Antikorper. Vogel und Siiugetiere sind heutige Typen vonEntwicklungslinien, die sicti vor mehr als 300 MillionenJahren getrennt haben. In den Immunglobulinen undAruikorpersystemen gibt es viele Ahnlichkeiten, die jedochoft auf Analogien beruhen. So ist der dominierende humora-le Immunglobulintyp der Vogel das IgY im Gegensat: zumIgG der Siiugetiere. Auch die Mechanismen, die zumAntikorperrepertoire fuhren, sind unterschiedlich. Bei denSiiugetieren sind es VOl'allem Rekombination der Genseg-mente und somatische Mutationen, die das groj3e Antikor-perrepertoire liefern, wdhrend bei Yogeln der Prozej3 derGenkonversion die grofste Bedeutung hat. So werden imImmunsystem der Vogel und Siiugetiere vergleichbareLeistungen durch unterschiedliche Strukturen bzw. Mecha-nismen erreicht.

mune reaction of the hagfish again, buthe adapted the animals to a higherwater temperature. After antigen injec-tion he found proteins with bindingactivity to the antigen and believed tohave induced antibodies (Raison et aI.,1978). But in 1994 he questioned hisprevious results after new experiments,assuming the induced proteins to becomplement components.Because published data about the

antibodies of other groups of cyclos-tomes, especially the lampreys, arevery controversial, hard facts about thehumoral immune response begin withcartilaginous and bony fishes and in-clude all other groups of vertebrates.The only immunoglobulin class, oc-

curing in all vertebrate groups fromfish to mammals, is the IgM. In somespecies, especially in bony fishes, itcan be found in different types, and forspecial functions.One question, discussed for many

years, concemes the first appearance oflow molecular weight antibody class inphylogeny. Throughout many years, innearly all papers about low molecular

weight immunoglobulins of non-mam-malian vertebrates, they were called IgGin analogy to the dominant humoralimmunoglobulin class of mammals. Butin the paper of Leslie and Clem (1969)about the low molecular weight immu-noglobulin of the chicken, the chickenimmunoglobulin was called IgY andshowed well defined differences be-tween that immunoglobulin and the IgGof mammals. Unfortunately, the conclu-sions of Leslie and Clem were not notedby the majority of later authors. Onlyduring recent years the name IgY hasbeen accepted for the dominant humorallow molecular weight immunoglobulinof amphibians, reptiles, and birds, as inthe review of WaIT et al. (1995).Already in 1977 we published data

about the low molecular weight immu-noglobulins of amphibians, reptiles,and birds in comparison to the immu-noglobulins of mammals (Hadge et al.,1977). These investigations about theimmunoglobulins of non-mammalianvertebrates have been extended in thefollowing years and produced the fol-lowing picture:

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:!in AMBROSIUS--~~------------------------------¥~2 Structure of IgY-immuno-globulins

There are well defined structural diffe-rences of IgY-type immunoglobulinsand the 19G. That includes the molarmass of the heavy chains (table 1) andthe carbohydrate content of the immun-oglobulins (table 2).On the other hand, the IgY-type

immunoglobulins are much less flexi-ble than IgG as shown by fluorescencepolarisation technique (table 3). Butthe comparison of the data of the IgYof frog, tortoise, and chicken show asignificant higher flexibility of thechicken IgY than the other IgY types.Also, the structures of the Fe part of

the immunoglobulin isotypes IgY andIgG are different. The Fe part of IgY isthinner than the Fe part of IgG anddoes not contain any hole in its middlepart, as was revealed for the IgG Fefragment (Cser et al., 1982). The ab-sence of the hole agrees with thesupposition that the excess mass in theH-chain is distributed in the Fe regionnear the centre of mass of the whole mo-lecule, and it also supports the observa-tion that chicken IgY is less flexible incomparison with mammalian IgG.Unfortunately, the comparison of

amino acid sequences is hardly suitablefor the elucidation of the relationshipsbetween the different immunoglobulinclasses occuring in the different verte-brate groups because the differencesare too great. It only points to IgY asthe ancestor of IgG and IgE (Warr etal., 1995). On the other hand, thephenomenon of immunological cross-reactivity could be used very success-fully for the identification and classifi-cation of immunoglobulins in speciesother than man which also correspondswith the nomeclature rules (WHO re-port, 1969; Ambrosius et al., 1978).The technique mostly used for themeasurement of antigenic cross-reac-tivity of proteins, is the inhibition ofbinding of specific antibodies to theradio-labelled antigen by the proteinsto be compared. Fig. 1 shows theresults of such an experiment, usingcarp anti-chicken IgY antibodies of H-chain specificity and different immu-noglobulin preparations as inhibitors(Ambrosius and midge, 1987). Be-cause in that system the point of 50 %inhibition is measurable, it can be used

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Table 1: Relative Molar Mass of the Heavy and Light Chains of Low MolecularWeight Immunoglobulins of Different Vertebrates (Six Estimations)

Species Ig Type

ManCowGuinea-PigChickenDuckGooseTortoise Agrion. horsf.Lizard Ophis. apodusFrog Rana esculenta

IgGIgGIgG 2IgYIgYIgYIgYIgYIgY

Molar Mass (in KD)

H Chains L Chains

52 (Ref. Value)52.551,762.862.764.365.564.564.0

23,023.022.024.525.024.025.5.21.0

Data from Hadqe et aI., 1980

Table 2: Carbohydrate Composition of Immunoglobulins of Different Vertebrates

Species Ig Type Hexos. Content (in %) Sialic acidHexosamines Total

Man IgG 1.3 1.0 0.1 2.4Guinea-Pig IgG 2 1.3 0.9 0.1 2.3Chicken IgY 3.2 1.8 0.2 5.2Duck IgY 4.5 1.7 0.2 6.4Tortoise IgY 3.2 1.1 0.2 4.6

Data from Hadqe et aI., 1980

Table 3: Rotational Correlation Time of DNS-Conjugates of Immunoglobu-lins of Different Vertebrates

DNS-Conjugate Oocalc (nsec) OhexP (nsec) Oh/Oo

Frog IgY 54 67 1.24Tortoise IgY 54 68 1.26Chicken IgY 61 43 0.69Human IgG 59 20 0.35

Data from Zagyansky, 1975

as quantitative value for the antigeniccross-reactivity which is nearly identi-cal with the structural relationship.Extensive experiments of Dietlind

Hadge and others in our laboratorywith alltogether 43 different antisera ofdifferent experimental animal speciesshowed that the dominant humoral lowmolecular weight antibodies of thenon-mammalian vertebrates, excludingfishes, are all of the IgY type. Table 4shows the data of experiments withIgY fragment-specific carp antibodies.The 50 % inhibition values are, in the

cases of IgY-type immunoglobulins,always in the range of 1-2000 which

points to a strong relationship to thechicken IgY. On the other hand, theIgG-type immunoglobulins do not in-hibit the antigen-antibody systemmeasurably. Therefore, no clear rela-tionship is detectable. IgG antibodiesare only found in mammals. The struc-tural difference between IgY and IgGare not surprising in light of the factthat the evolutionary lines of reptilesand birds separated from the one of themammals in the Permian or even earli-er, which means 300 million years ago.This is independent of the possibilitythat IgY may be the ancestor of IgG(Warr et al., 1995).

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AMBROSIUS ~r;")---------------------------h~~--

~0~,,~.

IN HI BiTtON ['f,]

16 250 1000 4000 16000 64000 25GOOO

INHIBITOR [ng]64

Figure 1: Inhibition of the binding of 125 I-chicken IgY to carp anti-chicken IgYantibodies by different immunoglobulins. S = chicken IgY, 1 = goose IgY, 2 = duckIgY, 3 = tortoise IgY, 4 = sheltopusik IgY, 5 = frog IgY, 6 = human IgG, 7 = bovineIgG, 8 = human IgD. From Ambrosius and Hadqe (1987).

Table 4: Antigenic Structure Comparison with Carp anti-Chicken

IgY (Fc) IgY (Fab)

Immunoglobulins 50 % Inhibition

Chicken IgYGoose IgYDuck IgYTortoise IgYFrog IgYHuman IgGBovine IgG

13653

30066

> 30.000> 30.000

1133767

2.2001.495

> 100.000> 100.000

Personal Data from D. Hadqe

Contrary to IgG, the mammalian IgAhas a high antigenic cross-reactivitywith IgY. This could be shown byexperiments with many antisera againstchicken IgY and its fragments as wellas against turkey IgY in our laboratory.Tables 5 and 6 show some results withH chain-specific rabbit anti-chickenIgY antibodies and carp anti-chickenIgY (Fc) antibodies, respectively. In allexperiments the antigenic cross-reac-tivity of mammalian IgA with chickenor turkey IgY was in the same range asthat of the IgYs of other amphibian,reptilian, or avian species. Therefore,we conclude, that the mammalian IgAis the direct descendant of the IgY, thedominant humoral antibody type ofnon-mammalian vertebrates.

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Table 5: Antigenic Structure Compari-son with Rabbit anti-Chicken IgY

Immunoglobulins 50 % Inhibition

Chicken IgYTurkey IgYHuman IgAFr

Human IgAHecGoose IgYPorcine IgATortoise IgYDuck IgYCarp IgMChicken 19BHuman IgG

133

250450

1.3501.5001.7501.900

Data from Hadqe and Ambrosius, 1984

31gB in birds

In 1972 several authors found a third Igclass in the blood serum and in thesecretions of chickens with H chainsclearly different in structure and anti-genicity from H chains of IgM and IgY(Bienenstock et aI., 1972; Lebacq-Verheyden et aI., 1972; Orlans andRose, 1972). Owing to its preferentialoccurrence in secretory fluids like sali-va, bronchial, intestinal, or oviductsecretions, and since that third chickenIg is nearly the only immunoglobulincomponent in the bile, it has beenpresumed to be homologous to mam-malian IgA. Since we were interestedin the phylogenetic status of that "so-called chicken IgA", we studied theantigenic structure similarity of thatimmunoglobulin to mammalian IgA,IgG, IgM, and non-mammalian IgYsusing a series of double-antibody RIAsystems and antisera against the "so-called chicken IgA" as well as againsthuman IgA (Ambrosius and midge,1982; Hudge and Ambrosius, 1983;Ambrosius and midge, 1987; midgeand Ambrosius, 1988). In not one ofthe systems a measurable antigeniccross-reactivity of the "so-called chik-ken IgA" with one of the other immun-oglobulin types could be found. This isin agreement with the fact, that thistype of the "so-called chicken IgA" isto be found only in gallinaceous birds.Probably it is a special immunoglobu-lin type which evolved within thatgroup of birds. It should not any longerbe called IgA because it is not ahomologue to mammalian IgA. Wepropose to use the term 19B in conside-

Table 6: Antigenic Structure Compari-son with Carp anti-Chicken IgY (Fe)

Immunoglobulins 50% (Inhibition)

Chicken IgY 1Porcine colostrum IgA 100Goose IgY 200Tortoise IgY 200Duck IgY 300Human IgAHec 450Human colostrum IgA 500Chicken 19BHuman IgG

Data from Hadqe and Ambrosius, 1984

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:;:~ AMBROSIUS

--h~~-----------------------------¥ct

ration of the fuct that it is a biliaryimmunoglobulin of some birds.

4 Origin of antlhody-diversity

The efficiency of an antibody systemdepends not only on the quantity ofantibody production. Very important isalso the quality of the produced anti-body types. Here, the affinity plays aleading part. As we could demonstratein studies with tortoises, chickens, andrabbits the produced antibodies of IgYas well as IgO class show the sameincrease in affinity following the firstand second immunisation which is wellknown from the fully matured immuneresponse (Ambrosius et al., 1972; Fie-big, 1972; Fiebig and Balcerova,1975). A condition for that process is agenetic mechanism which delivers ahigh number of differing antibodieswithin a short time. In mammals thatmechanism is well known for someyears (fig. 2).

One of a number of 200-1000 Vgene segments recombine with one ofmostly 5 J segments and in the case ofH-chains one of 20-50 D segments.Therefore, only the process of recom-bination produces a diversity of about1000 (L-chains) or 100.000 (H-chains)immunoglobulin chains. The associati-on of L- and H-chains can yield 108different antibodies. That number canincrease by a factor of about 100 inconsequence of imprecise DNA recom-bination which leads to changes in theamino acids at the junction sites of theV, D, and J gene segments, and also bypoint mutations. These somatic mutati-ons, which primarily occur after stimu-lation by antigen, lead to a selection formutations of antibodies with higheraffinity for the antigen.Chicken, the only analysed species

with dominant IgY production, devel-ops its antibody repertoire by a differ-ent strategy (fig. 3) (Bezzubova andBuerstedde,1994).The chicken light chain locus con-

tains only a single functional V and Jgene segment. 25 pseudo- V gene seg-ments are homologous to the V genebut lack the common transcription reg-ulatory and signal recognition sequenc-es. The single V and J gene segmentsrearrange by VJ recombination andcreate only a limited diversity at the

ALTEX 13, SUPPLEMENT 96

Murine kappa chain locus

V200 (--------------------~ V, J, ~---) Js C• It----BJ~ • I~~r_ f-

Germ-lino ~

VJ-RecombinatlonJ,

cL

--I1I1-IIII"-1{ ~~!!8888I---I'''''-~-I---.. }-~_II--

Somatic HypermutationJ,

cL V J--I.HIII •• ~{,~,~~~~~m---.~~~m~I~r-~_~

Figure 2: Diversification of the mouse kappa chain locus. The locus consists ofapproximately 200 V segments, 5 J segments, and a C gene segment. VJrecombination joins one of the V gene segments to one of the J gene segments.Following antigen stimulation somatic mutations are introduced into the rearran-ged V gene.

Chicken light chain locus

.V25 ~--------~ .V1

------{~ •.III!!ImGerm-line

V J CIi •V

VJ-RecomblnatlonJ.

L C•Gene Conversion

J.v J C

L,..~,----11- -a--_4_---I'I-- __ 1--

Figure 3: Diversification of the chicken light chain locus. The locus contains onlya single V, J, and C gene segment downstream of a pool of 25 pseudogenes. VJrearrangement joins the V and J gene segment. Afterwards, gene conversionwithin the bursa of Fabricius introduces sequence substitutions in the functionallyrearranged V gene.

junction of the V and J gene segments.Afterwards, gene conversion introduc-es sequence substitutions in the func-tionally rearranged V gene. Blocks ofpseudo gene sequenzes appear in the Vgene. The conversion tracts comprisefrom 10 to more than 120 bp, and asingle V gene can receive segmentalexchange from up to six differentpseudogenes.

Presently, we can not accuratelycalculate the antibody repertoire ofchicken. But it may be in the samerange as that of mammals. There is nosign that the specificity of chickenantibodies of IgY class differ from thespecifity of mammalian antibodies ofIgO class. Chickens can recognisemammalian proteins or pep tides asforeign and produce antibodies. Mam-

13

---------- ---------

AMBROSIUS

mals sometimes cannot recognisemammalian material as foreign anti-gens and produce no antibodies or onlyin very low quantities. Therefore,chickens can be excellent experimentalanimals for the production of antibod-ies of diagnostic value in human andveterinary medicine.

References

Ambrosius, H., Asofsky, R., Binaghi, R. A.et al. (1978). Proposed rules for thedesignation of immunoglobulins of ani-mal origin. Bull. WId. Hlth. Org. 56,815-817.

Ambrosius, H., Frenzel, E.-M. und Fiebig,H. (1972). Untersuchungen zur Strukturund Affinitat der Antikorper von Schild-kroten. Ann. Immunol. Hung. 16, 15-36.

Ambrosius, H. and Hadge, D. (I 982). Aphylogenetic view of avian immunology.Folia BioI. (Praha) 28, 1-21.

Ambrosius, H.and Hadge, D. (1987). Chik-ken Immunoglobulins. Vet. Immunol. Im-munopathol. 17,57-67.

Bezzubova, O. Y. and Buerstedde, J. M.(1994). Gene conversion in the chickenimmunoglobulin locus: A paradigm ofhomologous recombination in higher eu-karyotes. Experientia 50, 270-276.

Bienenstock, J., Perey, D. Y. E., Gauldie, J.and Underdown, B. J. (1972). Chickenimmunoglobulin resembling IgA. 1. lm-munol. 109,403-406.

Cser, L., Gladkih, I. A., Hadge, D. andAmbrosius, H. (1982). X-ray small-anglescattering study of general structure ofchicken immunoglobulin Y. ImmunologyLeu. 4, 15-19.

Fiebig, H. (1972). Vergleichende Untersu-chung der Affinitiit von Anti-Hapten-Antikiirpern von Vertretern verschiede-ner Wirbeltierklassen. Univ. Leipzig:Dissertation.

Fiebig, H. (1973). Die Entwicklung derAntikorperaffinitat in der Phylogenese.Allerg.1mmunol. 19,248-255.

Fiebig, H. and Balcerova, J. (1975). Studieson the affinity of anti-DNP antibodies ofthe chicken inbred lines. Folia Biol. 21,365-366.

Hadge, D. and Ambrosius, H. (1983).Evolution of low molecular weight im-munoglobulins - III. The immunoglobu-lin of chicken bile - not an IgA. Mol.lmmunol. 20, 597-606.

Hadge, D. and Ambrosius, H. (1984).Evolution of low molecular weight im-munoglobulins - IV. IgY-like immuno-globulins of birds, reptiles and amphibi-ans, precursors of mammalian IgA. Mol.lmmunol. 21,699-707.

14

Hadge, D. and Ambrosius, H. (1986).Evolution of low molecular weight im-munoglobulins. V. Degree of antigenicrelationship between the 7S immunoglo-bulins of mammals, birds, and lowervertebrates to the turkey IgY. Developm.Compo Immunol. 10,377-385.

Hadge, D. and Ambrosius, H. (1988).Comparative studies on the structure ofbiliary immunoglobulins of some avianspecies. II. Antigenic properties of bilia-ry immunoglobulins of chicken, turkey,duck and goose. Developm. Compo Im-munol. 12,319-329.

Hadge, D., Fiebig, H. and Ambrosius, H.(1977). Strukturelle Untersuchungen anlmmunglobulinen niederer Wirbeltiere.1. Uber die Evolution der niedermoleku-laren Immunglobuline. Wiss. Z. KMULeipzig 26, 67-80.

Hadge, D., Fiebig, H. und Ambrosius, H.(1980a). Evolution of low molecularweight immunoglobulins. 1. Relationshipof 7S immunoglobulins of various verte-brates to chicken IgY. Developm. CompoImmunol. 4, 501-513.

Hadge, D., Fiebig, H., Puskas, E. andAmbrosius, H. (1980b). Evolution oflow molecular weight immunoglobulins.II. No antigenic cross-reactivity of hu-man IgD, human IgG and IgG3 tochicken IgY. Developm. Compo Immun-01. 4, 725-736.

Lebacq-Verheyden, A. M., Vaerman, J. P.and Herernans, J. F. (1972). A possiblehomologue of mammalian IgA in chik-ken serum and secretions. Immunology22, 165-175.

Leslie, G. A. and Clem, L. W. (1969).Phylogeny of immunoglobulin structureand function. III. Immunoglobulins ofthe chicken. 1. Exp. Med. 130, 1337-1352.

Orlans, E. and Rose, M. E. (1972). An IgA-like immunoglobulin in the fowl. Im-munochemistry 9, 833-838.

Raison, R. L., Hull, C. J. and Hildemann,W. H. (1978). Characterization of immu-noglobulin from the Pacific hagfish, aprimitive vertebrate. Proc. Natl. Acad.Sci. U.S.A. 75, 5679-5682.

Vainoi, O. and Imhof, B. A. (1995). Theimmunology and developmental biologyof the chicken. Immunol. Today 16, 365-370.

Warr, G. W., Magor, K. E. and Higgins, D.A. (1995). IgY: clues to the origins ofmodern antibodies. lmmunol. Today 16,392-398.

WHO (1969). An extension of the nomen-clature for immunoglobulins. Bull. WId.Hlth. Org. 41,975-978.

Zagyansky, Y. A. (1975). Phylogenesis ofthe general structure of immunoglobulins.Arch. Biochem. Biophys. 166,371-381.

Correspondence addressProf. Herwart AmbrosiusUniversitat LeipzigFakultat fur Biowissenschaften,Pharmazie und PsychologieInstitut fur Zoologie/ImmunbiologieTalstraBe 33D-04103 Leipzig

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