The immunoglobulin genes of fish

Pergamon

Developmental and Comparative Immunology, Vol. 19, No. 1, pp. 1-12, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0145-305X/95 $9.50 + .00

0145-305X(94)00052-2

THE IMMUNOGLOBULIN GENES OF FISH Gregory W. Warr Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, S. C. 29425-2211 (Submitted October 1994; Accepted November 1994)

[3AbstractmThe current state of knowledge concerning the structure, organization, and functional expression of immunoglobulin genes in chondrichthyan and osteichthyan fish is presented.

[~Keywords--Antibodies; Immunoglobulins; Fish; Evolution; Gene expression; Gene organization. Introduction

The immunoglobulin (Ig) genes encode the antibodies, a family of defense proteins that appear to be unique to the vertebrates, being found in all classes except (possibly) the agnathan fish (1). Antibodies are synthesized by B-cells, in which the immunoglobulin genes are not only expressed in a tissue-specific manner, but undergo a complex series of chromosomal rearrangements (to generate a large repertoire of functional antigen-binding sites) and somatic mutations (to permit structural refinements of the antigen binding site), thus generating antibodies of high affinity. This series of genetic events has been studied and defined as the result of an enormous research effort focused, because of their relevance to human health, on mammalian species, predominantly the mouse. The amount of research carried out on fish Ig genes has been relatively small, Address correspondence to Gregory W. Warr.

even though it has obvious importance. Knowledge of Ig gene structure and function is necessary for an adequate understanding of fish immunity, for our understanding of the evolution of immunity, and for the potential to manipulate fish immunity by genetic means. In this brief review I have the goal of setting forth, in summary form, what is known of fish Ig genes: their organization, structure, and expression. In order to make this review as useful as possible I have tried to reference as many relevant articles as possible, especially those presenting original data, and including not only papers already published, but also some that are in press. The latter have kindly been made available to me by their authors. Space constraints have limited the discussion of many important points. For this I apologize.

Fish Antibodies

The chondrichthyan fish (sharks, rays, skates, and chimaeras) and osteichthyan (bony) fish possess, as do all vertebrates except the agnathans, a high molecular weight polymeric IgM (illustrated in Fig. 1). In addition, some (but not all) fish p o s s e s s l o w - m o l e c u l a r weight Igs. The low-molecular weight Igs possessed by fish are predominantly of four distinct structural types (see Fig. 1); l) a monomeric form of IgM (reviewed in 2); 2) a smaller form of mono-

ro

VII VL

Covalent (S-S) bond(s) between domains

IgM(AFc)

Pentameric IgM

IgX or IgR (skates and rays) IgY( AFc ) or IgN (lungfish)

Tetrameric IgM

Figure 1. Schematic representation of the structures of the immunoglobulins found in fish. The structures of the IgM(AFc) and the antibody of the lungfish are speculative in that they are based on the sizes of the heavy chains rather than on primary structural analyses. The position and number of interchain disulfide bonds are highly variable between the Igs of different species.

--

~11 CH ~ eL

[~l

!

Monomeric IgM

Fish Ig genes

3

The IgH Loci of Fish

meric IgM that can be d e s i g n a t e d IgM(AFc), (see, e.g., ref. 3); 3) a molecule called IgX or IgR that is typical of the rajiformes and primitive sharks (4-9); and 4) a molecule that has been termed IgN and may be IgY(AFc), in the lungfish (10-12). Fish antibodies (as proteins) are not strictly speaking the subject of this review, and the reader is therefore referred to a relatively recent article if more information on this topic is desired (2). However, it is clear that fish antibodies are, as in all ectothermic vertebrates, typically of much lower affinity and diversity than those of higher vertebrates such as birds and mammals (see, e.g., ref. 13 and references cited in Wilson and Warr, ref. 2). Therefore, much of the research on their Ig genetics has been directed toward trying to understand this functional deficiency. The Igs of fish are typical in containing equimolar heavy (H) and light (L) polypeptide chains. These polypeptides are encoded by separate loci and will therefore be considered separately.

All heavy chains are encoded by multiple genetic segments: the variable (V) domain by VH, D, and JH segments (14), and each constant (C) domain by an exon. One of the characteristic features of the Ig genes is their chromosomal rearrangement during the development of a B cell: a VH, a D, and a Ja segment become fused to form a unit encoding the VH domain (15). It would seem intuitively obvious that there should be more copies of the diversity-generating VH, D, and Jr~ segments than of the C-region genes. While this is the case with the typical vertebrate IgH loci, including those of the teleosts (16-20) and holosteans (21,22), it is not so in the case of the elasmobranchs (Fig. 2). The elasmobranchs are unique in having a distinctive multicluster or multiple locus arrangement, in which units of (VH-D-DJH-CH) are repeated dozens of times along the chromosome (4,5,23-28). This arrangement occurs for the heavy chain

VHI-n

1: TELEOSTS and HOLOSTEANS

Dl-n

_,_,_,_._,

IIII

JHl-n

=_m_m

I.t

i

t-

>

< unknown - probably greater than 1 Mb

( V-D-D-J-C ) 1....

2: ELASMOBRANCHS

....

_n I I I I - - I

( V-D-D-J-C ) n

:,

'-'D-

approx 10 - 20 kb

3: COELACANTH

vD • i

VD I1

VD ]I

J C Ill?

~190 nt

apart

Figure 2. Organization of the IgH loci of fishes. The three main types of IgH locus organization described to date in fishes are shown. These are referenced in the body of the text (modified from ref. 2).

4

G.W. Warr

genes of both IgM and IgX in the rajiform fish, and interestingly the clusters for these genes appear unlinked. Indeed, based on the results of fluorescence in situ hybridization, it has been suggested that the tx and x genes are arranged in higher order tandem arrays of clusters (6). Recent studies on the IgH locus of the coelacanth have revealed what is probably a third type of IgH locus organization in fish (Fig. 2), but characterization of this locus is still at a preliminary stage (29).

The IgL Loci of Fish Light chains differ from H chains, as polypeptides, in having only a single constant region domain in addition to the variable domain. The structure of the L

chain gene is also simpler (as shown in Fig. 3) in that I) it does not have to encode a transmembrane domain to be used in the expression of membranebound immunoglobulin (see the later section: secreted and membrane forms of fish Igs); 2) it contains only a single C-region exon; and 3) the VL domain is encoded only by VL and JL segments, the D region being absent. Although a variety of L chain gene organizations is seen in the vertebrates, one unusual aspect of all fish IgL genes described to date (3039) is that they show the multicluster type of organization (Fig. 3). In most fish whose genes have been thoroughly examined, the light chains fall into a number of distinct structural types or classes (35,40). Whether or not this phenomenon, analogous to the existence of kappa

Chondrichthyes: germline unjoined Ginglymostoma cirratum, K-like (type III) Heterodontusfrancisci, K-like (type III)

VL

JL

Heterodontusfrancisci, type I

CL

Chondrichthyes: germline joined

Carcharhinus plumbeus, ~, (type II)

>-->-

>

--q:==::l

1---

VL/JL

Heterodontusfrancisci, type II Hydrolagus colliei, type II

CL

Raja erinacea, type II Raja erinacea, type I

Osteichthyes: germline unjoined <

--)-

>

Gadus morhua

VL <

JL

CL <

--~

),

Ictalurus punctatus, G-type

VL

VL

JL

eL

Figure 3. Organization of the IgL loci of fishes. The classification of chondrichthyan genes into three types is that of Rast et al., (ref. 35). The arrows indicate relative transcriptional orientation based on those examples for which it has been determined.

Fish Ig genes

5

and lambda light chain classes in mammals, will prove to be the case with all fish is not yet clear.

Generation of Antigen Binding-Site Diversity The generation of diversity in antibodies has been exhaustively studied in the mammals, and to a lesser extent in birds. The mechanisms can include random selection of V (D) and J segments for rearrangement and fusion, imprecision in V(D)J joining, introduction of new bases at the junctions during V(D)J joining, gene conversion, somatic mutation, and the random selection of expressed H and L polypeptide pairs, as summarized in Table 1. In considering the generation of antigen binding site diversity in fish (reviewed in 27, 34, 51, 52), we are confronted by the fact that few species have been investigated, and most investigations have, of necessity, focused only on one small facet of the topic. The extent of our knowledge can perhaps best be appreciated by attempting to answer a number of key questions relevant to binding-site diversity.

How Many V-Region Encoding Segments Are in the Germline? The segment encoding the largest portion (about two thirds) of the VH domain

is the VH gene (or gene segment). V H genes typically exist in large numbers (over 100) within a species and exhibit wide sequence diversity. Within this background of diversity, however, the sequences of VH genes within a species tend to cluster into groups of related sequences. Such families of VH genes generally share 80% (or more) nucleotide sequence identity. Man and mouse each possess in excess of 100 V a genes that group into 7 families in the human (53,54) and 14 families in the mouse (55). VH families found in teleost species may have an evolutionary history of 150 million years or more (56-60). The two species of teleost most thoroughly studied in respect of their V n families are the channel catfish (Ictalurus punctatus) and the rainbow trout (Oncorhynchus mykiss). They have been shown to possess at least 6 and 9 VH families, respectively (59,61-63). The number of VH genes in I. punctatus, as estimated by Southern blot, is well over 100 (61,63). The V n genes in I. punctatus show relatively close spacing in the chromosome (about 3 - 4 kb between genes) and interspersion of members of different families (64). The examination of VH gene number has not yet been so thorough in O. mykiss, but from the observations on members of an individual gene family (56 and data cited in ref. 59)

Table 1. Mechanisms of Generating Diversity in Immunoglobullns. Mechanism 1. Combinatorial diversity 2. Junctional imprecision 3. Junctional diversity a 4. Secondary VH recombination b 5. Gene conversion c 6. Somatic hypermutation d 7. Heavy/light chain pairing

Comments The random selection for recombination of V(D)J elements. Almost certainly restricted in loci with the multicluster arrangement. Imprecise joining of V, (D), J segments Enzymatic addition and removal of P and N nucleotides at the VH/D, D/JH, and VLJL boundaries Replacement of the VH region of a rearranged VDJ segment. Mediated by a cryptic recombination signal sequence. Non-reciprocal exchange of sequences from VH or VL (pseudo)genes to functional V region genes. Significant use of this mechanism reported only for birds and rabbits: recent evidence that it may occur in mice. The introduction of point mutations into functional V(D)J segments; occurs subsequent to antigen stimulation of specific B cells The essentially random choice of individual heavy and light chain V regions expressed in a B celt.

References: a = 41-43; b = 44; c = 45-48; u = 49, reviewed by Manser (50).

6

it is likely that in excess of 100 V H genes will also be found in this species. The V H genes of several other teleosts have also been investigated, and generally reveal several VH families, with multiple members of the families. For example, the Atlantic cod (Gadus morhua) has at least three V , families (65), as does the goldfish, Carassius auratus (66), and the ladyfish, (Elops saurus) has been shown to possess at least 2 VH families (18). It can be predicted that with more thorough investigation additional families of VH genes will emerge in these teleost species. One aspect of V H genes that needs to be borne in mind is that many germline sequences prove to be pseudogenes (18,54,64,66,67), which can be expected to have some bearing on the functional VH diversity actually available to the organism. Even less is known of the D and JH segments of teleosts than of the VH genes. No genomic D region has been cloned and analyzed, and only for I. punctatus has the JH region been cloned and mapped (68). This species possesses 9 JH regions, as compared to 4 in the mouse and 6 in the human. From cDNA analysis it appears that O. mykiss possesses at least 6 JH and G. morhua at least 2 JH (62,65), although in the absence of genomic cloning these numbers can best be regarded as provisional estimates. In so far as D region diversity can be inferred from cDNA sequences, the evidence suggests that teleosts are not lacking in multiple, diverse D segments (62,63,65). In considering VH, D and JIa number and diversity in the elasmobranch fishes, much more information is available on the number of these segments than on their diversity. It is clear from the multicluster organization that many germline copies of V H, D, and JH exist, that each cluster typically has 2 D segments but only a single V H and JH, and that germline joining of certain V, D, and J segments has occurred (4-6,22-27,28,69).

G.W. Warr

Detailed studies of the homed shark (52) suggest that most of the VH genes in this species belong to a single family, although a single V H representing an additional family was reported. Since the V L and JL segments of all fish examined to date (Fig. 3) are arranged in multiclusters, certain generalizations can be made. While there are, from Southern blot analyses, many VL (and therefore also JL and C L) gene segments, in some species, such as H. francisci (homed shark) there appear to be fewer IgL than IgH clusters (30). In I. punctatus it has been reported that more than one VL can be associated with each JL--CL pair (37), and in the teleosts the V L genes appear to be in opposite transcriptional orientation to JL and CL (37,70) whereas in elasmobranchs VL, JL, and CL are all in the same transcriptional orientation (reviewed in 35). While VL sequences can be classified into different families in chondrichthyan species (35), the situation is currently less clear with teleost V L sequences. There are at least two families of VL sequences in O. mykiss and G. morhua (36,70), whereas a single family of VL sequences was reported for I. punctatus by Ghaffari and Lobb (ref. 37). The situation with respect to VL families in teleosts will become clearer when many more VL sequences have been analyzed, and when the genetics of the multiple isotypes of teleost light chain have been resolved. In concluding this section it is clear that fish are not at any obvious disadvantage, as compared to other vertebrates, in terms of the number and diversity of their Ig V-region encoding elements.

How Much Diversity is Created in Fish Antibodies by Joining V, (D), J Segments? The process of V(D)J joining creates diversity by virtue of 1) the random selection of elements for recombination,

Fish Ig genes

and 2) the junctional imprecision and enzymatic addition/removal of bases which occurs at the junctions (Table 1). The contribution of these two processes can be considered separately. First, there is no evidence that combinatorial diversity is restricted in the IgH loci of teleostean fish, with their typical "translocon" arrangement. However, it is generally thought that in those loci with a multicluster arrangement (all fish IgL loci, and elasmobranch IgH loci) rearrangement will favor the elements within each cluster, thus restricting the potential diversity g e n e r a t e d by combinatorial means (see, e.g., 27, 34, 35, 52). The presence of two D regions in the elasmobranch IgH clusters does, however, add the potential for some additional combinatorial alternatives. The second issue, that of junctional imprecision and enzymically generated new junctional sequences, has not been thoroughly studied in many situations. Where it has been investigated, however, there is clear evidence for the generation of a great deal of junctional diversity in fish (26,52,59,62, 63,65,68).

Does Somatic Mutation Generate Variable Region Diversity in Fish? Ever since the restricted nature of antibody diversity was recognized in ectothermic vertebrates, there has been speculation that this restriction was the result of some inadequacy in the genetic mechanisms by which it could be generated (13). Suspicion fell heavily on the phenomenon of somatic mutation, which is known to be responsible in large measure for the affinity maturation so characteristic of the strong antibody responses of mammals. However, investigations of the amphibian Xenopus laevis and the shark H. francisci have demonstrated that somatic mutation does occur at significant levels in the functional, rearranged V(D)J regions of these species

7

(52,71). Thus, the failure of somatic mutation to produce high affinity antibodies in these species probably reflects problems in the antigen-driven selection of the potential higher affinity binding sites generated by these mutations (52,71).

Restricted Affinity and Diversity of Fish Antibodies: S u m m a r y

and Speculations Certain features of fish Ig genes are quite striking and likely to be reflected in the quality of antibodies that they can encode. For example, the arrangement of an Ig locus in the multiple cluster configuration (found so far for all IgL loci and the IgH loci of elasmobranchs) might be expected to restrict combinatorial diversity, even though the number of clusters may be very large. The possession of Ig heavy and light chain loci arranged exclusively in multiple clusters has fallen under suspicion as one of the causes of the typically highly restricted antibody responses of elasmobranchs, (see, e.g., 27,52). However, the argument has been made by some authors that, even in the case of germline V(D)J fusion, which can be found in both IgH and IgL loci of some elasmobranchs (26,28,33-35,72) the potential for generating diverse antigen binding sites is still very high (34). From the data available, there do not appear to be any obvious deficiencies in the genetic mechanisms associated with the generation of additional diversity during the recombination of V(D) and J elements of fish. Rather, the lack of affinity maturation seen in the antibodies of fish (and other ectothermic vertebrates) must have a different cause. The reported lack of organized germinal centers in the lymphoid tissues of lower vertebrates has been suggested as such a cause (71), because the germinal centers are the sites of antigen-driven selection of high affinity antibodies in the mammals (73). However, the possibility must also be consid-

8

ered that the relatively restricted responses of fish result from presently uncharacterized regulatory mechanisms. Poorly understood mechanisms can lead to clonal dominance and restricted antibody responses in mammals (74).

Secreted and Membrane Forms of Fish Igs All immunoglobulins appear to have the potential to exist in two alternate forms: as a soluble secreted molecule in blood and other fluids, and as a membrane-bound receptor for antigen on the surface of B cells. Ig heavy chains are thus synthesized in two forms: one, with a hydrophobic C-terminal region that can associate with the B cell membrane, and a second, with a hydrophilic C-terminal region that is utilized in the secreted form of the molecule. These two forms of the heavy chain are encoded by a single gene, and alternate pathways of prem R N A processing determine which mRNA is expressed. The alternate premRNA processing pathway involves, in a typical mammalian IX gene, utilization of a cryptic donor site in the CIX4 exon which splices to the transmembrane exons. This site is used in elasmobranch tx genes (25), but appears to have been lost in teleost IX genes. In teleosts, the membrane form of the IgM results when the exons for the hydrophobic transmembrane region splice to the end of the CIX3 exon, thus eliminating the entire CIX4 domain from the membrane IgM (20,75-80). The loss of the CIX4 domain of teleost fish membrane IgM heavy chains does not seem to prevent this molecule from acting as a functional antigen receptor on B cells, or from mediating allelic exclusion (81). It is not yet known whether this unusual pathway for producing the membrane form of IgM is p r e s e n t in the e a r l i e s t - d i v e r g e d basal teleost groups, such as the hiodontidae (e.g., the mooneye, Hiodon ter-

G.W. Warr

gisus) and the clupeidae, (e.g., the herrings).

Regulation of Ig Gene Transcription in Fish The conventional understanding of Ig gene expression is that it requires B-cell specific promoter and enhancer elements, and that the process of V(D)J rearrangement brings a promoter sufficiently close to an enhancer that efficient transcription of a functional gene can be initiated (reviewed in 82). A large research effort has been focused on the nature of transcriptional control in the mammals (mostly man and mouse), the nature of the transcription factors involved, and the specific sequence motifs to which they bind (see, e.g., 82-84). Our knowledge of fish Ig gene transcriptional control is, in contrast, very sparse. Most of the information available consists of analyses of sequences flanking and within the Ig genes for transcription factor-binding motifs that match the consensus for those motifs already described in the mammals (see, e.g., refs. 2,6,64,77,85). This almost certainly has some merit when considering the promoters, which are invariably in the upstream (5' flanking) region of the V H and VL genes. The minimal motifs for B-cell specific promoter function are the octamer (ATGCAAAT) and a TATA box (86), which appear to be identifiable in the 5' flanking region of all fish Ig genes so far examined, except for the elasmobranch IgH clusters (see, e.g., 6,26). The regulation of expression of multicluster loci, to avoid simultaneous expression of multiple genes (especially where germline joining has occurred), poses interesting questions to which the answers are not yet clear. The Via promoter region of teleosts contains some motifs similar to those described in mammalian VH promoters, although their organization and arrangement differ (cf refs. 18,64,66,82). Functional studies have shown that the

Fish Ig genes

9

5' region of a teleost (goldfish, C. auratus) VH gene can function as a tissuespecific promoter in both catfish and mouse B cells (87). In mammals, IgH enhancers are found in the D region, the JH to C~ intron, and 3' of the constant region genes. Transcription factor binding motifs have been identified (by inspection of sequences) in the Jn to C~ introns of many fish IgH genes, of both elasmobranch and teleost species (see, e.g., 2,6,77,85). However, in one case where the enhancer in a fish Ig gene (the IgH locus of I. punctatus) has been studied functionally, activity was found to localize to the region surrounding and immediately 3' of the TM2 exon, a unique site for a vertebrate IgH enhancer. This enhancer showed B-cellspecific expression in both mouse and fish systems, despite possessing only some of the sequence motifs identified as essential for enhancer function in the mammalian IgH locus (87). However, reciprocal cross-species activity of vertebrate Ig enhancers may be a common pattern: Michard-Vanh6e et al. (ref. 88) have shown B-cell-specific expression driven by a mammalian Ig promoter/ enhancer pair in transgenic O. mykiss. It is not known if the enhancer discussed above is the only one in the IgH locus of I. punctatus, or if these results obtained

with it will prove to be typical of the teleosts and other fish.

Concluding Comments The last 10 years have seen a great deal of success in defining the structure and organization of fish Ig genes. Investigators will soon understand much more about the expression of fish Ig genes (particularly the nature of the transcriptional control elements) and the relationship between immunogenetics and the functional immune responses displayed by fish. This information should provide insight into the manner in which the antibody response of fish has adapted to the particular evolutionary challenges it has faced. It should also provide the opportunity for the experimental manipulation of fish antibody responses. Acknowledgements--I wish to thank Drs. Ivar Hordvik, Jacques Charlemagne, Gary L i t m a n , Takeshi M a t s u n a g a , a n d Chris Amemiya for providing prepublication copies of manuscripts and unpublished data. The research of the author has been supported by awards from the National Science Foundation (DCB 9106316 and MCB 9406249). I thank Ms. Kathy Wiita for her excellent help and patience in the preparation of this MS.

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and complexity in the skate, Raja erinacea. Nucleic Acids Res. 18:1015-1020; 1990. Anderson, M.; Amemiya, C.; Luer, C.; Litman, R., Rast, J.; Niimura, Y.; Litman, G. Complete genomic sequence and patterns of transcription of a member of an unusual family of closely related, chromosomally dispersed Ig gene clusters in Raja. Int. Immunol. 6:16611670; 1994. Kobayashi, K.; Tomonaga, S.; Kajii, T. A second class of immunoglobulin other than IgM present in the serum of a cartilaginous fish, the skate, Raja kenojei: Isolation and characterization. Mol. Immunol. 21:397-404; 1984. Kobayashi, K.; Tomonaga, S. The second immunoglobulin class is commonly present in cartilaginous fish belonging to the order Rajiformes. Mol. Immunol. 25:115-120; 1988. Kobayashi, K.; Tomonaga, S.; Tanaka, S.

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