Shared antigenic determinants of immunoglobulins in phylogeny and in comparison with T-cell receptors

Comp. Biochem. PhysioL Vol. 105B,Nos 3/4, pp. 423--441, 1993 Printed in Great Britain

0305-1M91/93$24.00+ 0.00 © 1993Pergamon Press Ltd

REVIEW SHARED ANTIGENIC DETERMINANTS OF IMMUNOGLOBULINS IN PHYLOGENY A N D IN COMPARISON WITH T-CELL RECEPTORS JOHN J. MARCHALONIS,*VALERIE S. HOHMAN, HULYA KAYMAZand SAMUELF. SCHLUTER Department of Microbiology and Immunology, University of Arizona, Tucson, AZ 85724, U.S.A. (Tel. 602 626-6409; Fax 602 626-2100) (Received 8 January 1993; accepted 12 February 1993) Abstract--1. Immunoglobulins are a complex multigene family of proteins specified by genes encoding variable (V), sometimes diversity (D), joining (J), and constant (C) domains. 2. Cross-reactions involving conformational determinants related to the VHa system of rabbits occur on heavy chains of vertebrate species ranging from elasmobranchs to man. 3. Serological markers characteristic of # chains, the heavy chain of the IgM macroglobulins, occur on homologous heavy chains of species representing all vertebrate classes. 4. Serological markers characteristic of ? type heavy chains, the major isotype in man, are restricted to the mammals, but are found on representatives of even the most primitive mammals, the egg-laying monotremes. 5. Variable region markers characteristic of 2 light chains are shared by light chains of shark and man. 6. Certain idiotypic markers defined by combining site V region sequences are broadly distributed in evolution. 7. Use of synthetic peptides as antigens and in epitope mapping show that amino acid sequences from the third framework region of the variable domain are broadly shared among light chain in phylogeny and between light chains and T-cell receptor fl chains. 8. The "switch peptides" linking the V and C domains of light chains and T-cell receptors, specified by the C-terminal portion of the J segment and the N-terminus of the constant region, are exposed in the three-dimensional structure of immunogiobulin or Tcrs, show striking homology, and form broadly shared antigenic determinants characteristic of immunogiobulins. 9. Although the multigene nature of the immunoglobulins and the complexity of antigenic determinants expressed by these large proteins renders comparison among molecules difficult, serum immunoglobulins and the closely related T-cell receptors express numerous shared determinants defined on the basis of amino acid sequence homology and three-dimensional conformations.

INTRODUCTION

Immunoglobulins (Igs) are major defense and self-recognition proteins of vertebrates. The immunoglobulin family is a complex set comprising circulating antibodies that are highly conserved in all placoderm-derived vertebrate species (sharks to mammals; Litman et al., 1971; Marchalonis, 1977), cell surface antigen receptors of bone marrow derived lymphocytes or B cells (Marchalonis and Cone, 1973; Vitetta and Uhr, 1973), and antigen-specific receptors of thymus derived lymphocytes or T-cells (Hedrick et al., 1984a; Yanagi et al., 1984). These proteins are disulfide-bonded heterodimeric functional antigen binding units consisting of pairs of light and heavy chains in serum Igs and B cell receptors (mlgM or mlgD) or pairs of heterologous chains forming either ~/fl or ?/8 structures in T-cell receptors (Tcrs) (Kronenberg et al., 1986; Toyonaga and Mak, *To whom correspondence should be addressed.

1987). Igs and Tcrs contain characteristic peptide sequences termed variable (V), joining (J) and constant (C) regions that are specified by individual gene segments. In addition, Ig heavy chains and Tcr fl and chains have genes specifying a diversity (D) region. A unique feature of these molecules is that gene rearrangement is required for the formation of functional B cell surface Ig, serum antibody (Brack et aL, 1978), and Tcr molecules (Kronenberg et al., 1986; Toyonaga and Mak, 1987), with the result that B and T cells are clonally restricted in receptor expression. It has been known for many years that antigenic cross-reactions occur among Igs of diverse species (Nash and Mach, 1971; Esteves and Binaghi, 1972; Neoh et aL, 1973; Jefferis et aL, 1982). Reagents directed against Ig classes (isotypes) of man have been used to type serum antibodies of distant mammalian species such as cetaceans (Nash and Math, 1971) and even primitive egg laying mammals such as the echidna (Marchalonis, 1977). Isotype-related serological cross-reactions were detected between

423

424

JOHNJ. MARCHALONISet al.

IgM of man and chickens (Mehta et al., 1971) using relatively insensitive techniques. Likewise, a number of serological studies carried out in the early 1970s showed that antigen specific receptors of T-cells cross-reacted with Igs (Hogg and Greaves, 1972; Marchalonis and Cone, 1973; Cone et al., 1974; Warner, 1974; Binz and Wigzell, 1975). Although conservation in structural properties (Marchalonis and Edelman 1965; Litman et al., 1971) and usage of amino acids (Marchalonis, 1972) among immunoglobulins of diverse vertebrate species has been known for more than 20 years, it required the application of recombinant DNA technology to establish exact relationships among light and heavy chains of distant vertebrate species. The sequencing of Ig proteins in man and mouse was made possible by the occurrence of multiple myeloma, a cancer of the lymphatic system that generates malignant plasma cells showing clonal gene rearrangement and producing individual Igs. The serum Igs of primitive vertebrate species such as sharks show charge heterogeneity comparable to that of mammals (Marchalonis et al., 1988a), but monoclonal malignant plasma cells have not been found. Therefore, it was necessary to clone genes specifying individual heavy and light chains (Litman et al., 1985a,b; Litman and Hinds, 1987; Schwager et al., 1988; Schluter et al., 1989). A parallel application of recombinant DNA technology was required to allow isolation of Ig-related genes restricted to T-cells (Hedrick et al., 1984a,b; Yanagi et al., 1984) because the membrane proteins were heterogeneous and present in insufficient amounts for detailed chemical analyses. Sufficient derived amino acid sequence data now exist for Ig light and heavy chains of diverse species (Kabat et al., 1991; Schwager et al., 1991; Hohman

et al., 1992; Zezza et al., 1992) and for Tcr fl chains of man (Toyonaga and Mak, 1987), mouse (Kronenberg et al., 1986), rats (Williams and Gutman, 1989), cows (Tanaka et al., 1990) and chickens (Tjoelker et al., 1990) to allow interpretation of certain observed serological cross-reactions in terms of sequence homology and location in the predicted three-dimensional structure of the molecules (Edmundson et al., 1975; Schluter et al., 1989; Marchalonis et al., 1992a). The use of synthetic peptides duplicating continuous antigenic structures of Ig light chains (Marchalonis et al., 1992a) and Tcr fl chains (Marchalonis et al., 1992b; Kaymaz et al., 1993a,b) has proven to be useful in detecting and mapping determinants shared between light chains of sharks and man (Marchalonis et al., 1992c) and those shared by Tcr +6chains and 2 light chains (Kaymaz et al., 1993a,b). Here, we analyze the types of serological cross-reactions that have been observed among members of the Ig family. Where possible, we map these particular regions and discuss their relevance to the structure and antigenicity of members of this family. The situation is complicated because Igs are a multi-gene family comprising many different (V, J, D, C) gene segments. Moreover, Igs express both continuous antigenic determinants specified by relatively short stretches of peptide (Seiden et al., 1984; Schluter et al., 1987) and conformational determinants formed through the close spatial-juxtaposition of residues that are distal from one another in linear sequence (Tonnelle et al., 1983; Mag e et al., 1984; Sasso et al., 1988). The problem is further complicated by the size of the heavy chains, e.g. p chains consist of one variable and four constant domains with a total covalent protein mass of over 60 kDa, and these molecules usually contain approximately 10% or more of their mass as carbohydrates (Marchalonis, 1977).

IDIOTYPES

,w,,o.,,PT,

X Frl

o,.

,J sEGMW

CONSTANT

Fr3

CDR3 ISOTYPES MAJORITY OF ALLOTYPES VHa ALLOTYPES Fig. I. Schematic diagram mapping various characterized antigenic markers onto a linear model of a universal immunoglobulin structure. The structure consists of a variable region comprised of framework segments (Frl, Fr2 and Fr3), hypervariable segments or complementarity determining regions (CDR1, CDR2 and CDR3). CDR3 is specified by diversity (D) and (J) segments in V, and Tcr Vfl. The Fr4 is specified by the joining segment. In three-dimensional models the V and C domains are compact structures linked by the exposed "switch peptide".

Shared antigenic determinants of Igs and Tcrs Considerations f r o m evolution

immunoglobulin

structure and

Figure 1 depicts the segment structure of a universal Ig chain. In normal functional molecules, two such chains, either heavy/light in antibodies or ct/fl or y/t5 in Tcrs, are required to form a functional antigen-binding domain (V region) and effector unit (C region). The evolutionary distinction between heavy and light chains is an ancient one that is clearly established in the elasmobranchs (sharks and rays), the ancestors of which diverged from those of mammals more than 400 million years ago (Romer, 1966; Loomis, 1988). Comparison of amino acid sequence, structural properties, and serological comparisons indicate that the shark Igs are homologous to the IgM class of man (Marchalonis, 1977; Litman and Hinds, 1987) and the light chains thus far described are homologs of 2 chain (Schulter et al., 1989; Shamblott and Litman, 1989a,b; Hohrnan et al., 1992). Tcrs have only been characterized in mammals and birds (Tjoelker et al., 1990), but the chains are also constructed in accordance with this model and are the result of genetic processes involving gene segments homologous to those of the Igs (Kronenberg et al., 1986; Toyonaga and Mak, 1987). This diagram is an over-simplification for the constant regions because it represents only a single constant domain which is the situation only in Iglight chains and Tcrs. The Ig classes are defined by the presence of particular heavy chains (#, ~,, ct, 6 and e) specified by particular genes. Each consists of separate domains or homology units of approximately 100 amino acids (e.g. 4 for 7 chain and 5 for ~t chain). Dendograms based on comparisons of light chain constant regions generally reconstruct our common notions of phylogeny. 2 light chains emerged early in evolution while the x chains were present by the phylogenetic level of amphibians (Marchalonis and Schluter, 1989; Schluter et al., 1989; Hazer, 1990; Schwager et al., 1991; Aguilar and Gutman, 1992; Hohman et al., 1992; Zezza et al., 1992). Comparable analysis suggests that/~ type heavy chains are present in all placoderm-derived vertebrates with the C/~4 domain showing the strongest degree of conservation (Marchalonis and Schluter, 1989). By contrast, it is not possible to build a consistent phylogeny based upon full length multi-domain CH regions, because the individual domains show an evolution that is more dependent upon their position in the chain than on the identification of a particular isotype (Liu et al., 1976; Barker et al., 1980; Schwager et al., 1988; Marchalonis and Schluter, 1989). Overall, Ig C-region domains tend to be approximately 30% identical to one another, a value which meets Doolittle's criterion for inclusion within a family (1989), and there are particular regions that show strong conservation. V regions can show stronger identities. For instance, a shark V2 shows >50%

425

identity to a human V2VI sequence (Hohman et al., 1992) and a Va isolated using a murine Vn gene probe has approximately 70% identity to the probe (Litman and Hinds, 1987). The time of emergence of Tcrs is uncertain, but their V regions and J segments are clearly within the Ig V region family (Beaman et al., 1987; Marchalonis and Schhiter, 1989). For example, the human YT35 fl chain sequence is approximately 35% identical in its VDJC sequence to the human 2 light chain Mcg (Kaymaz et al., 1993a). It has been argued that Tcr appeared early in Vertebrate or chordate evolution to form a pair in allo-recognition involving the major histocompatibility complex (Hildemann, 1979; Klein, 1989). Conversely, it has been argued that they appeared late in vertebrate evolution and may not be present in major groups of lower vertebrates such as elasmobranchs (Smith and Davidson, 1992). Cladistic analysis suggests that Tcr ct and fl V-regions emerged following the divergence of V n and VL (Beaman et al., 1987; Hubbard and Marchalonis, 1988). One interesting aspect that has proven important in recent serologic studies is that the fourth framework region specified by the joining segment has a generally conserved sequence of the form FG(*)GTRL in light chains and Tcr fl chains whereas heavy chains have the homologous sequence WG(*)GTTV. These structures represent a punctuation at the end of the third complementarity determining region and lead into the switch peptide bridging the variable and the first constant domain. Even though the Tcr fl chain has an Fr4 homologous to that of light chains (Marchalonis and Schluter, 1989), this chain also resembles heavy chains in ttfat a diversity gene segment (D) contributes to the variability in CDR3 (Toyonaga and Mak, 1987). General features o f shared immunoglobulin determinants in phylogeny

Table 1 lists examples of immunoglobulin antigenic determinants showing extensive distribution in phylogeny. Here we concentrate on markers that are shared between mammalian and non-mammalian species. The prime examples of these are cross-reactive VH determinants that can be either allotypic, framework or idiotypic. Human or murine idiotypes are usually restricted to the mammals (Riesen, 1979; Kennedy et al., 1983), but idiotopes detected by certain monoclonal antibodies react with phosphorylcholine binding antibodies of mammals and with non-immunoglobulin molecules that bind this hapten (Volanakis and Kearney, 1981; Vasta et al., 1984). There is extensive cross-reaction among IgG molecules which are present in all mammals (Neoh et al., 1973; Jefferis et al., 1982; Marchalonis et al., 1992c) and are restricted to this vertebrate class. However, #-chain-related determinants occur in elasmobranchs, teleost fish, amphibians, birds and mammals. Genes specifying immunoglobulins homologous to /~ chains have been isolated from

426

JOHN J. MARCHALONISet al.

Table 1. Examples of immunoglobulin antigenic determinants showing extensive distribution in phylogeny Antigenic marker Detection system Species distribution (1) Vaa allotype Rabbit anti-allotype (Vnal; VHa2; Rabbits (allotypes); isotypes: man, mouse, cane toad, VHa3) galapagos shark, stingray, trout, goldfish, turkey (Rosenshein and Marchalonis, 1985) (2) Va framework Mouse mAb to VH- of myeloma protein Strong: mouse, rat, guinea pig, rabbit, dog, cow, sheep, MOPC 15 goat, horse, baboon, man. Intermediate: chicken. Weak: reptiles, Xenopus, carp, goldfish (Eshhar et al., 1983) (3) TEPC 15 Id Murine mAbs to TEPC 15 idiotopes; TEPC 15, human C-reactive protein (Volanakis and broadly cross-reactive marker maps to Kearney, 1981), horseshoe crab (Limulus) lectin (Vasta CDR3/Fr3 junction et al., 1984) (4) Mouse IgGl "a" allo- Murine mAbs to mouse ), 1 "a" allotype Mouse: "a" allotype. + ; "b" allotype - ; type (C~'/Fc marker) Strong: guinea pig, dog, tiger, man, chimpanzee, monkey, horse, cow, sheep, pig, goat; Intermediate: chicken, duck. Weak: turtle, alligator, lizard: Non-reactive: hare, rabbit, opossum, wallaby, echidna, frog, Xenopus, catfish (Parsons and Herzenberg, 1981) Rabbit antisera to human ), isotype Eutherian mammals, marsupials, monotremes markers (5) Human or mouse y or mouse y-chain (echidna): Negative: non-mammals (Esteves and Binaghi, 1972; Marchalonis et aL, 1982c) Twenty-fivemAbs weretested (Jefferiset al., 1982);some Murine mAb to human Fc markers (6) Human showed restricted specificity in the mammals, others reacted with all mammals tested except the cat; two showed autoreactivityagainst mouse IgG; two including one of the autoreactive mAbs reacted with chicken IgY Rabbit antisera to human or murine # Chicken (Mehta et al., 1972); toad Bufo, galapagos (7) Mammafian#-specific shark, carp, mouse, human (Marchalonis et al., 1992c) chain determinants Lamprey # (weak), mouse ),, guinea pig ),, turkey (8) General heavy chain Rabbit antisera to shark/~ chain (weak), toad/l (weak), stingray #, shark (Rosenshein et specific al., 1985); hagfish (weak) (Varner et al., 1991) Shark light chain, human and mouse 2 myeloma light (9) 2 chain related proteins Rabbit antibody to shark light chain chains (Marchalonis et al., 1992c) Shark and human light chains (Marchalonis et al., (10) Light chain specific Rabbit anti-human x and 2 1992c) (non-isotype associated)

representatives of each of these species: sharks (Litman and Hinds, 1987), stingrays (Harding et al., 1990), catfish (Ghaffari and Lobb, 1989), the clawed toad X e n o p u s (Schwager et aL, 1988), the caiman (Litman et al., 1985b), and chickens (Dahan et al., 1983). The antigenic distinction between light and heavy chains is clearly distinguishable using antibodies directed against both mammalian and shark immunoglobulin chains. Molecules antigenically related to m a m m a l i a n 2 chains occur in elasmobranchs (Marchalonis et aL, 1992c), a result consistent with the derived amino acid sequence of shark light chains. Heavy or light chain isotype related determinants showing wide phylogenetic distribution occur that are specific for light chains but do not discriminate between x and 2 chains (Marchalonis et al., 1992c). Conversely, some antigenic markers specific for heavy chains react with heavy chains of distinct classes. These heavy chain-related determinants have been detected using both monoclonal antibodies to immunoglobulin constant regions (Parsons and Herzenberg, 1981, Jefferis et al., 1982) and polyclonal antisera produced against purified heavy chains (Marchalonis et al., 1992c) or Fc fragments (Esteves and Binaghi, 1972). Examples of these types o f shared antigenic determinants will be considered in detail below. Figure 1 illustrates the location of some characterized Ig antigen derminants. Immunoglobulin class

or isotype definition is usually considered to be a property of the constant domains. However, isotypespecific markers have been located within the framework regions of light chains (Marchalonis et al., 1992a). Allotypic determinants are genetic markers within a species which occur on light chains and heavy chains. These are usually restricted to the constant regions, but a major variable region allotypic system has been documented in the rabbit (Tonnelle et al., 1983; Mage et al., 1984), and evidence for Vn-associated allotypes in man has been reported (Wang et al., 1978). The rabbit Vna allotype system is extremely interesting in an evolutionary context because rabbit anti-allotype sera will detect corresponding antigenic structures on Igs of man (Knight et al., 1975), mice (MackelVandersteenhoven et al., 1984) and a range of nonmammalian species including birds, bony fish and elasmobranchs (Rosenshein and Marchalonis, 1985). Constant region allotypes of man have been detected in primates using human alloantisera (Alepa, 1969). Except for an allotypic determinant on murine I g G l of the " a " allotype detected by a murine monoclonal antibody (Parsons and Herzenberg, 1981), allotypes defined by constant region sequences do not seem to show a broad phylogenetic distribution comparable to that of the VHa marker system. The monoclonal antibody to mouse IgG1 " a " allotype reacted with Igs of many mammals including carnivores,

Shared antigenic determinants of Igs and Tcrs primates, perissodactyls and artiodactyls and to a lesser degree with Igs of birds and reptiles. It did not, however, react with Igs of lagomorphs, marsupials or monotremes or with Igs of amphibia and fish. This is an interesting result because in the mouse the monoclonal antibody is specific for an allotypic marker of the ~,1 heavy chain. However, it also reacts with Igs of the chicken, a species which does not possess IgG immunoglobulin (Leslie and Clem 1969). This illustrates the situation of a sharing of a heavy chain-restricted marker that does not assort in evolution as an isotypic marker. The idiotypic markers define individual Ig binding sites and involve the complementarity determining regions [CDR] (Davie et al., 1986). Corresponding regions have been identified in Tcrs on the basis of V-region sequence comparison and by positioning in proposed three-dimensional models of the Tcr chain (Novotny et al., 1986; Chothia et al., 1988; Marchalonis et al., 1992b; Kaymaz et al., 1993a,b). In addition, the Fr3 segment of Tcr Vfl shows considerable sequence variation and has been identified as a fourth hypervariable region (Davis and Bjorkman, 1988) although structurally this segment corresponds to the Fr3 of light chains (Marchalonis et al., 1992b). Consistent with sequence differences among individual Vfl gene products, both rabbit anti-peptide antibodies and human IgG autoantibodies to Fr3 segments (Marchalonis et al., 1992b) show a hierarchy of cross-reactions allowing discrimination among human and mouse Tcrs. In general, CDR3 shows greater variability than do the other CDRs and can be considered a "private" idiotype because of greater distinction among related V region structures. For example, V regions selected from a particular V region set could have extremely similar CDR1 and CDR2 regions, but differ considerably in the CDR3 because of the additional diversity generated in this region during rearrangement. The CDR2 region has a relatively low degree of diversity and has thus been considered a "public" or shared idiotype. Immunization with synthetic CDR2 (Meek et al., 1990) and CDR3 sequences (Seiden et aL, 1984; Goldfien et aL, 1985) has been effective in the generation of anti-idiotypic antibodies. In particular, immunization with a peptide predicted from the sequence of the joining segment (JH1) of the heavy chain of murine Igs binding phosphorylcholine (TEPC 15) has resulted in the production of antibodies directed against a set of idiotypic determinants (Seiden et al., 1984). This particular peptide region is of considerable interest, both in studies of heavy and light chain evolution and in comparison of Igs and Tcrs, because it contains the highly conserved Fr4 region that is characteristic of Igs that arise from rearranging genes (Schluter and Marchalonis, 1986; Marchalonis et al., 1988b,c; 1989). Idiotypic markers have been found to be associated either with heavy (Crews et al., 1981) or light chains (Carson et al., 1987) or with a

427

conformation requiring the presence of both variable elements (Marchalonis et aL, 1979a). The observation that some idiotypcs are expressed only in association with C# heavy chains (Morahan et al., 1983) suggests that the idiotype can be effected by structures not necessarily part of the idiotypedefining sequences. Problems in the assessment o f immunological crossreactions

A number of complications must be considered in assessing immunological cross-reactivities. The first issue is the question of what structure constitutes an antigenic determinant. A relatively small segment of peptide (approximately 6 amino acids) or carbohydrate is actually needed to fill the combining site (Kabat et al., 1991), but a larger segment is often required to ensure optimal binding (Ross et al., 1989). Another complication is that different animals can react to the same protein or pcptide in different manners. As illustrated in Fig. 2, rabbit and goat antisera that are specific for 2 chain isotypic determinants do not react identically with synthetic peptides duplicating the continuous antigenic structure of the 2 chain Meg (Marchalonis et al., 1992a). Both antisera react with major constant region determinants, but the rabbit reacts strongly with V2 Frl and Fr3 determinants that are not recognized by the goat antibody. The goat also reacts with a peptide from the variable region that is not detected by the rabbit serum. Furthermore, some animals will not produce antibodies to certain peptides under conditions which normally generate high levels of antibody. For example, we have been unable to obtain antibodies in mice or rats to a 16-mer corresponding to a Tcr Jfl sequence (Marchalonis et al., 1988b), most probably because antibodies against

2.4

2.0

1.6

~ 1.2

~ 0.8 0.4 0.0

• 0

, 2

•

a -

,

4

6

•

, 8

•

, 10

•

, 12

-

, 14

-

, 16

.

, 18

.

~ 20

Peptide

Fig. 2. Mapping of 2 chain specific epitopes detected by rabbit anti-human ).chain (O) or goat anti-human ). chain (I-q) using synthetic overlapping peptides modelling the covalent structure of the human ). light chain Meg (Marchalonis et al., 1992a). The Y axis gives the absorbanc¢ of the ELISA reaction; the X axis gives the pcptide number with # 1 corresponding to the N-terminus and # 20 corresponding to the C-terminus of the light chain. The antiscra were tested at a dilution 1:1000.

428

JOHNJ. MARCHALONISet al.

this sequence would knock out the murine immune system by binding 2 light chains and Tcr fl chains. By contrast, rabbits produce extremely high levels of IgG antibody to this peptide. The difference in capacity to respond by antibody production seems to be related to a difference at a critical residue position in the synthetic Jfl sequence. This phenomenon illustrates both great differences in the capacity of species to respond to particular antigens and the critical importance of single residues to the formation of antigenic determinants. Another important consideration is that great differences in sensitivity and in specificity can be found depending upon the type of assay used. A direct binding assay such as an Enzyme Linked Immunosorbent Assay (ELISA) is capable of detecting picogram quantities of antigen (Sehluter and Marchalonis, 1986) and can detect cross-reactions in peptides of length 10-16 amino acids that share 50% identity in sequence (Marchalonis et aL, 1984; Vasta et al., 1984). Competitive inhibition assays can be very sensitive, but they are more selective and dependent upon fine differences in sequence or conformation. In practice, we have used competitive inhibition assays using either intact Ig or peptide as inhibitors to determine the degree of conformational dependence of antibody binding to continuous peptide determinants (Marchalonis et al., 1992a,b). If the peptide inhibits at least as well on

a molar basis as the intact protein, we conclude that the conformation of the peptide was essentially the same as that in the intact structure. Examples of this are the N-terminal 16 residues of human 2 light chain or the carboxyl terminal 16 residues of the same protein. By contrast, V2 Fr3 and C2 markers show considerable conformational dependence. Western blot analysis (Rosenshein et al., 1985) has also proven useful in studies of cross-reactions among Igs of diverse species, between Tcr ct and fl chains of man and mouse (Dedeoglu et al., 1992) and in comparison of epitopes shared between Tcrs and Igs (Schluter and Marchalonis, 1986; Schluter et al., 1987). This assay detects essentially linear peptide determinants either in free peptide (Kay et al., 1990; Dedeoglu et aL, 1992) or in denatured Igs which have been treated with mercaptans to break intrachain and interchain disulfide bonds (Schluter and Marchalonis, 1986; Schluter et al., 1987; Marchalonis et al., 1988b). Antigenic determinants related to rabbit Vna allotypes shared by vertebrate classes

VHa allotypes of domestic rabbits occur in the variable regions of heavy chains of all classes of immunoglobulins. The system contains essentially three phenotypes designated al, a2 and a3 that are controlled by allelic genes at the A-1 locus (Mage

1.6

1

2

3

4

a H.C. ~C 1.21

i

L.C.

g

0.8

0.4

I

I

20

I

I

80 320 Reciprocal dilution

1280

Fig. 3. Binding in an ELISA of rabbit antiserum (AK350-2) against the rabbit Vaa3 allotype to intact 7S immunoglobulin and to isolated heavy and light chains of C. galapagensis (Z~) intact 7S Ig. (O) isolated shark heavy chain, (l'q) isolated shark light chains and (0) normal rabbit serum reactivity. Microtiter wells were coated with 1.0 gg of test antigen. Insert: a 12% SDS-polyacrylamide gel run under reducing conditions showing purified C. galapagensis 7S IgM and its isolated heavy and light chains. Lane 1, intact 7S IgM; lane 2, purified 7S IgM heavy chain; lane 3: purified 7S light chain; lane 4: tool. wt standards: (a) phosphorylase b (94,000), (13) BSA (67,000), (c) ovalbumin (43,000) (d) carbonic anhydrase (30,000), (e) soybean trypsin inhibitor (20,100), and (f) ~t-lactalbumin (14,400) [from Rosenshein and Marchalonis, 1985].

Shared antigenic determinants of Igs and Tcrs et al., 1984). The individual Vna aUotypes are specified by conforrnational determinants that correlate with amino acid residues located within the first and third frameworks of the heavy chain variable region (Mage et al., 1984; Tonnelle et aL, 1983). Knight et al. (1975) showed that markers which are allotypic in rabbits cross react with man where the determinants are isotypic; i.e. present in the serum of all individuals tested. We tested a panel of rabbit anti-allotype specific reagents (kindly provided by Dr Rose G. Mage of the National Institutes of Health). Although there was variability in the individual rabbit sera, the evidence is conclusive that Igs of man, mice, amphibians (the marine toad, Bufo marinus), birds (the turkey), teleost fish (trout, carp and goldfish), and elasmobranchs (stingray, carcharhine sharks) (Rosenshein and Marchaionis, 1985) expressed V.a conformational determinants defined by residues on the heavy chains. The specific association of the Vxa3 cross-reactive marker with the heavy chain of shark Ig is illustrated in Fig. 3. Although the complete sequence of the VH region of Galapagos shark Ig has not yet been obtained, comparison of the first 20 N-terminal residues (Frl) (Table 2) shows eight identities between the shark VH and the rabbit Vaa3 prototype. From position 13 to 20, the shark has six identities with the VHa3 sequence, two of which are known to be allotype correlated. This finding of a V region marker widely shared among heavy chains of vertebrate species parallels the established conservation of Va gene sequences, selected using the VH S107 probe, among vertebrate species ranging from sharks to mammals (Litman et al., 1985a,b; Wilson et al., 1988). This widespread sharing of conformational determinants suggests that the three-dimensional structure of the V region of vertebrate Igs must be largely conserved. It also generalizes the finding that a markpr which is allotypic within a given species can be is~typic in other species. Although V region allotypic:markers have not been observed in light chains, the" strong sequence similarities between

429

shark and human light chains, indicate that the folding of light chains in evolution has likewise been conserved. Sharing o f isotypic determinants among immunoglobulins of diverse vertebrate classes

Molecules occurring in the serum of primates and non-primate mammals can often be typed using reagents directed against human Igs (Nash and Math, 1971; Jefferis et al., 1982). For example, Neoh et al., (1973) were able to use Ouchterlony immune diffusion analysis to show that homologs of IgG, IgA, IgM, IgD and IgE occurred in virtually all primates. In addition, these workers applied the "principal of phylogenetic distance" showing that chicken antibodies raised against the human markers would pick up significant cross-reactions in virtually all mammalian species. Figure 4 illustrates the use of Ouchtedony diffusion and phylogenetic distance by comparing the precipitation reactions of chicken antibody to mouse (Fab')2 with purified IgGs of mice, rabbits and guinea pigs (Marchalonis et aL, 1979b). Not only are the intensities of bands consistent with phylogenetic relationships, but the spurring indicates antigenic deficiency of the guinea pig relative to the rabbit and the rabbit relative to the mouse. Using more sensitive techniques such as ELISA and Western blot analysis we have found that antisera we have raised against either human or mouse Igs as well as commercially obtained conventional and monoclonal class specific antibody cross-react extensively among mammalian species. The spiny anteater or echidna, one of the two living monotreme species, possesses 7, #, x, and 2 that are clearly related serologically to their respective human chains (Marchalonis et al., 1992c). We have obtained crossreactions with antibodies directed against human IgA and IgD markers, but the precise characterization of these molecules remains to be established. Interestingly, a commercial monoclonal antibody

Table 2. Comparison of N-terminal sequences (FR1) of shark and rabbit heavy chainsa l

C.galapagensis

2

3

4

5

6

E V V

VEES

~a2

V

S

G

E

G G

E !E S G

K

E

S

9 10 11 12 13 14 15 16 17 18 19 20 Ident|ttes

AEYG~ GDLV G R LV

T q

VHa3 Qo- S VNal 0o - S ~-

7 8

LF

m L

20/20

T L

8/Z0

T

T

L

6/20

D

T

C

K

V1

5/Z0

• Comparison of N-terminal sequences (FR1) of shark and rabbit heavy chains of different allotypes. • designates allotype-coqrelatad residues; • designates ellotypHesocieted reeldmm; data taken from IMageeta/., 1984; rabbit ellotypo esquence data taken from Tonnaile M m/., 1983. Residues in boxes indicate regions of homology with shark p-chain. - indicates deletion used to maximize homology, o indicates position is cyclized pyrrolidone carboxylic acid.

430

JOHNJ. MARCHALONISet al.

Fig. 4. Immunodiffusion analysis of chicken antibody (A) to mouse (Fab')2 fragment tested against mouse IgG (M) rat IgG (R) and guinea pig IgG (G). Immunoglobulins were used at a concentration of 1 mg/mh to human 2 shows almost quantitatively equivalent binding to purified echidna Ig light chains and the human molecule. This indicates that the limited determinant seen by the monoclonal on the echidna antibody and the human prototype must be essentially identical. A particularly interesting and thorough early study (Jefferis et al., 1982) tested murine mAbs to human IgG in a cell agglutination assay for their reactivities against Ig of mammals and the chicken. Each mAb exhibited a unique profile of reactivities suggesting that human IgG expresses an extremely large set of immunogenic epitopes. Some mAbs showed a restricted profile of cross-reactive determinants, whereas others showed a wide range of Igbinding activity, including activity against mouse IgG. Two of the 25 mAbs tested had this autoantibody activity, and one of these also bound chicken Ig. By contrast, polyclonal sheep antibodies to human Fcy showed a profile of binding reactivities reflecting the expected phylogenetic relationships among the mammalian species studied. Esteves and Benaghi (1972) had previously found that polyclonal rabbit antisera to mammalian IgGs gave results among Igs of man, pig, cow, sheep and goat that reconstructed expected phylogeny and did not react with avian (chicken and duck) Igs. These results using reagents of defined specificity support our previous conclusions that all mammals can make Igs of the

IgG type and that true y chains are apparently restricted to mammals (Atwell and Marchalonis, 1975). It should be emphasized here that the use of monoclonal antibodies has not resolved the problem of class relatedness among immunoglobulins because monoclonal antibodies often show unexpected crossreactions that are not related to class definition. As shown above, conventional polyclonal antibodies tend to give results generally consistent with phylogenetic schemes but individual monoclonal antibodies can detect limited determinants shared among otherwise distinct immunoglobulins of unrelated species. The sharing of IgM determinants based upon reactivity with # chains is not restricted to mammals. Shared determinants between chicken and mammalian g chains were previously reported (Mehta et aL, 1972). The cross-reactivity between # chains of man and avian species has been confirmed and extended to teleosts and elasmobranch species (Marchalonis et al., 1992c) using ELISA and Western blot analysis. The heavy chains of chicken, turkey, trout, goldfish, and certain sharks reacted with some rabbit antisera directed against either human or murine Igs. Antisera directed against human y chains gave no detectable reactivity with the shark Igs. Isotype specific reactions could be detected readily in ELISA and in Western Blot when unreduced Igs

Shared antigenic determinants of Igs and Tcrs were tested. By contrast, these reactions, particularly those of the heavy chain, were often lost when the samples were reduced and denatured during the analysis. This finding indicates that the class specific determinants are most probably conformational determinants. This observation is of some interest because detailed analysis of homology among constant region sequences (Barker et aL, 1980; Schwager et al., 1988; Marchalonis Schluter, 1989) suggests that heavy chains most probably did not evolve as single units, but rather the individual domains evolved independently according to the position within the chain. For example, the CH1 domains of heavy chains of various species show greater homology to one another than to an overall isotypic distinction. Likewise, the C-terminal domains, be they C73 or C/~4, share greater similarity to one another rather than the other domains of the chain than would be expected from the class nomenclature. The evolutionary comparisons indicate that the conserved isotype specific determinants are apparently conformational determinants rather than linear ones. cDNA sequences specifying light chain constant regions from two separate species of sharks have now been determined (Schluter et al., 1989a; Shamblott and Litman, 1989a,b; Hohman et aL, 1992). Although all of the gene sequences currently available show homology to human 2 chains, we would not be surprised if additional classes of shark light chains were discovered (Marchalonis et al., 1988a). In the case of the sandbar shark, we have now sequenced five separate C2 genes finding that they differ from one another by about 5% and must represent separate genes. Rabbit antibodies produced against purified polyclonal light chains of the sandbar shark were specific for light chains within shark species and in mammalian species. Furthermore, rabbit antibody to the shark light chain was capable of distinguishing between r and 2 chains of man. The rabbit anti-shark light chain reacts strongly by ELISA against shark light chains and appreciably with several human and murine 2 myeloma proteins. By contrast, it reacts extremely weakly with a human

431

and murine r myeloma proteins. The cross-reaction between shark light chains and human 2 chains is not surprising, because comparison of the derived light chain sequence of the sandbar shark with human 2 chains indicates that these molecules are approximately 40% identical (Schluter et al., 1989; Hohman et al., 1992). In fact, there are runs of amino acid sequence where the shark and human molecules are identical at 13 of 16 positions. We have recently used a set of nested, overlapping synthetic peptides to map the antigenic determinants on the human 2 molecule Meg which are detected using rabbit antisera specific for human 2 chains (Marchalonis et al., 1992a). Using the same methodology, we have determined the exact location of peptide antigenic determinants shared between human and shark light chains recognized by antisera directed against shark light chains which cross-react with human 2 chains. Thus far, we have identified two major shared antigenic determinants and two minor ones that correspond to linear stretches of sequence (Marchalonis et aL, 1992c). The major cross-reactive peptides (Table 3) lie within the third framework segment of the variable region (residues 67-82) and the fourth framework segment (residues 100-115) which is specified by the joining segment gene. These same regions are also strongly antigenic in the reaction of the isotype specific rabbit antibody to human 2 chain, thereby showing that class specific determinants are not restricted to constant regions (Fig. 2). The degree of identity in the Fr4 peptide is approximately 60%, and the similarity is even stronger because residues that we have found to be antigenically equivalent, such as valine and isoleucine, valine and leucine, and lysine and arginine, occur in corresponding positions (Marchalonis et al., 1988b). The Fr4/J region sequence is universally conserved in Igs and Tcrs that arise from rearranging gene segments (Marchalonis et al., 1988b,c). This region will be discussed in more detail below in the context of Tcr and Ig light chain cross-reactions. The minor peptide determinants detectable in ELISA show less sequence identity, but clearly align between the shark and human sequences. Once again, even though the linear

Table 3. Antigenic cross-reactive peptides of shark 2 and human 2" --.Fr3-

SbS (Clone 5.1)

ISJSISISJNIV N H

I r NV

Fr4-

S ~--

Mcg pep.11 (100-115)

V~-~T~-~VT~-~QMKA

SbS (Consensus)

] FI_~RIG T KIL NIV L GINLPJR S

• The human peptides bound to rabbit antiserum raised against intact, isolated shark light chains (Rosenshe~n & Marchalonis, 1987). SbS sequence data are from Hohman et aL, (1992).

432

JOHNJ. MARCHALONISet al.

sequence identity is as low as 25%, antigenically equivalent residues are found at critical positions. Our previous studies indicate that positively charged residues such as lysine and arginine are critical to forming Ig antigenic determinants and tend to be equivalent in properties (Marchalonis et al., 1988b). The linear peptide approach developed will miss certain conformational determinants. It would not, for example, pick up the shared V.a determinants because these are formed by residues in Frl and Fr3 that are too distant in linear sequence. However, clear evidence has been found for antigenic cross-reactions between light and heavy polypeptide chains, respectively, of sharks and man. These data are consistent with amino acid sequences and with predicted three dimensional structures of shark light chains (Schluter et al., 1989). They emphasize a structural conservation within the Ig family and allow the definition of boundaries where one would expect antigenic cross-reactions among related but divergent molecules.

Expressions of combining site (idiotypic markers) by antibodies of different species and non-immunoglobulin molecules There are examples of idiotypic markers shared between antibodies of man and mouse such as those occurring in the autoimmune disease SLE (Takei et al., 1987) and myasthenia gravis (Dwyer et al., 1983). In addition, a cross-reactive idiotype defined by rabbit antibodies to hepatitis B surface antigen was detected in sera of rabbits, mice, guinea pigs, swine, goats and chimpanzees (Kennedy et al., 1983). This marker, however, was absent from chicken antibodies to the same antigen. Human antibodies to phosphorylcholine (PC) can bear a determinant cross-reactive with the murine PC-binding myeloma protein TEPC-15 (Riesen, 1979). A remarkable finding by Volanakis and Kearney (1981) was that C-reactive protein, an acutephase protein which binds phosphorylcholine, likewise possesses a determinant which is cross-reactive with the TEPC-15 idiotype. For many years we have been interested in a lectin (limulin) of the ancient arachnoid Limulus polyphemus and have carried out serological and structural studies of this sialic acid-binding molecule from a living fossil (Marchalonis, 1977; Marchalonis and Edelman, 1968). The Limulus lectin is comprised of 18 subunits of Mr 22,000 associated via non-covalent interactions. It has no significant homology with Igs, but is related to C-reactive protein based upon sequence homology (Nguyen et al., 1986) and serological crossreactivity (Vasta et al., 1984; Ying et al., 1992). Furthermore, certain monoclonal antibodies specific for TEPC-15 idiotopes show clear reactivity with both Limulus lectin and C-reactive protein (Vasta et al., 1984). Limulus lectin can be considered to share idiotopes with TEPC-15, but it is clearly not

equivalent because not all of the monoclonal antiidiotype antibodies reacted with the limulin molecule. A number of other acute-phase proteins such as cq-acid glycoprotein and lectins of different specificities such as Didemnum lectin (which binds galactose) were uniformly negative with the monoclonal antibodies. Furthermore, the capacity of the monoclonal antibodies to react with Limulus lectin could be competed out using dinitrophenolphosphorylcholine, thereby indicating that the antiidiotype and the hapten were competing for the combining site, as expected in the classical reaction of idiotypes with antibody. However, neither C-reactive protein nor Limulus lectin shows significant overall sequence homology to vertebrate Igs. Moreover, it would be difficult to conceive of any sort of direct evolutionary homology between Limulin and Igs. Despite this, we carried out a computer analysis using the program RELATE, considering sequence stretches of six and 10 amino acids in order to identify short stretches of significant identity which might be expected to account for the observed serological cross-reaction between antibodies to the TEPC-15 idiotype (Marchalonis et al., 1984). The only stretch of significant identity between the sequenced portion of Limulus lectin and C-reactive protein is that corresponding to residues 59-71 in Limulin and residues 52-64 in C-reactive protein. There is identity in nine out of 13 positions in this stretch (Marchalonis and Schluter, 1990). The same analysis of TEPC- 15 and C-reactive protein identified a stretch of C-reactive protein sequence corresponding to residues 83-92 in C-reactive protein and residues 60-68 in TEPC-15, which was 30% identical. Comparison of TEPC-15 and Limulus lectin identified an overlapping stretch of sequence that showed 50% identity corresponding to residues 64-71 in TEPC-15 and 47-54 in Limulus lectin. Although the shared residues are not identical in the comparisons between Limulus lectin and C-reactive protein with TEPC-15, the stretches overlap and the binding of monoclonal anti-idiotype can be blocked by hapten. The segments of shared sequence correlated with the shared antigenicity and ligand binding, and the lengths correspond to the size (6-15 residues) stated by Tainer et al. (1984) to be optimal for production of anti-peptide antibodies that react with the intact protein. The region of TEPC-15 identified (SASVKGRFIVSR) corresponds to the juncture of the CDR2 and Fr3 segments.

Immunoglobulin and T-cell receptor epitopes defined by synthetic peptide specified by joining segment minigenes One peptide region of immunoglobulins and Tcrs that shows particularly strong homology is the Fr4 segment encoded by J mini-genes. Table 4 presents a phylogenetic comparison of peptide sequences specified by J2, Jr, Jn, Jfl and J0c mini-genes. The Jfl and J0c sequences fall into a grouping with the Jx and

Shared antigenic determinants of Igs and Tcrs J1 because the initial residue of the Fr4 region is phenalanine (F) and the sixth residue is positively charged, either arginine (R) or lysine (K) which appear to be antigenically equivalent in determining the reactivity with rabbit antibodies (Marchalonis et al., 1988b). The N-terminal Fr4 residue of heavy chains is tryptophan (W) and the sixth residue tends to be uncharged. Seiden et aL (1984) showed that antibodies directed against the synthetic peptide corresponding to the JH of murine TEPC-15 (JH 1) reacted with a set of idiotypes defined by the variable portion of the peptide. In the phylogenetic context, antisera to the JH peptide reacted in ELISA with the immunizing peptide, with the murine proteins MOPC 104E and TEPC-15, with the human myeloma protein MCE [JH2] (Mackel-Vandersteenhoven et al., 1985), and with heavy chains of shark immunoglobulin (Rosenshein et al., 1985). Rabbit antisera to the JHI peptide (murine TEPC 15) also reacted in Western blot analyses with components of human and murine T-cells (Mackel-Vandersteenhoven et al., 1985). The extensive homology in derived amino acid sequences between Tcr and Igs (Hedrick et al., 1984a,b; Yanagi et al., 1984) was consistent with earlier results of antigenic studies suggesting that Tcrs were new classes of Ig distinct from characterized B cell antibodies (Cone et aL, 1974; Binz and Wigzeli, 1975; Marchalonis, 1975). Alignments of sequences enabled the production of peptides to Tcr and Igs that might be antigenically cross-reactive and

Table 4. Comparison among J region sequences

J A (Hu BL2)

exposed in the 3-dimensional structure of the molecules. The Jfl peptide A N Y G Y T F G S G T R L T V V containing CDR3 residues A N Y G Y T and the Fr4 sequence FGSGTRLTVV was chosen on the basis of extensive identities in comparisot~ among Igs and Tcrs. Extensive searches of computer data bases showed that the "J region" is characteristic of a family consisting of Igs and Tcrs because nearly all molecules containing stretches showing > 50% identity were from this cluster. Similar computer searches using conserved peptides from V and C domains did not result in unique association with the Ig/Tcr family. This finding, coupled with the discrimination between JH and Jfl/JL sequences suggested that antibodies directed against J segment peptides would be highly discriminatory and useful probes for immunoglobulins in evolution and in analysis of distinct T-cells. We have not been able to detect reactivity with anything other than members of the immunoglobulin family of molecules in studies of sera of animals ranging from lampreys to man and in extracts of lymphoid and non-lymphoid cells (Hubbard et al., 1989;, Dedeoglu et al., 1992). Affinity purified rabbit antibody against the Jfl peptide reacts strongly in both the denaturing Western blot assay (Schluter and Marchalonis, 1986) and non-denaturing assays such as ELISA (Marchalonis et aL, 1988) and immunofluorescence cell staining studies (Shankey et al., 1989). The reagent is a useful universal beterologous probe for the detection of the rearranging members of the immunoglobulin family. As shown in Western blot analyses (Fig. 5), the anti-Jfl antibodies bound to both Tcr fl chains (mouse: lane b) and to numerous Ig light chains (mouse: a, b,; trout: c, d; shark: e, f; toad: h; mouse r: i; human: j) as well as to a small subset of human heavy chains (Schluter and Marchalonis, 1986; Schluter et al., 1987; Marchalonis et al., 1988a,b). Analysis of whole sera (lanes a, c, e and j) of various species shows that, except for reaction with some human heavy chains, light chains are the only components detected. The turkey IgM (g) was completely unreactive. The only components detected in the detergent lysate of the mouse spleen cells (b) were the ~t/fl Tcr from T-cells and light chains from B cells. The antibody used here was affinity purified on Jfl peptide covalently bound to Sepharose. Although affinity purification can remove high affinity antibodies, the procedure allows one to use a defined reagent. Tcrs and Igs share epitopes defined by joining segments. This conclusion was substantiated by showing that antiJfl antibodies could be affinity purified on a monoclonal immunoglobulin light chain (human r, Gun) affinity column (Schluter et al., 1987) with the purified antibodies reacting with both Jfl peptide and the light chain. Not all the anti-Jfl activity bound to the x light chain, but the purified antibodies retaining reactivity for the Jfl peptide indicate that shared and T-cell specific determinants are expressed by the same

EFOOOT LTQLI: --,Fr4

a A (Hu Newm)

WVFGGGTKLTVL~ W FGGGTKLTVL

O A (Nu NIO4E) a A (Pig)

)FGG

0 A (Chicken)

GI

a A (Shark-SB)

st F L~ FGG sj F 6 | L~FG~

a r (Hu Roy)

a r (Hu Ti) a K (MU SI07) J K (Rabbit)

Or (Dog Gom)

YIIFG|

a H (Hu TEPCI5)

MYF_~A

a H (Hu HcE)

GGF

a H (Xenopus)

FTL|V

G|

j H (Shark-Her)

YYF|F

IGU

a B (Hu YT35)

ANYGY~

GS

a a (Hu PY14)

SASKII

~S

il

433

434

JOHNJ. MARCHALONISet al.

a

b

c

d e f ghi

j Ig heavy chain -~- T cell receptor 13chain Ig light chain

Fig. 5. Analysis of reactions of affinity purified rabbit antibody against Jfl peptide on immunoglobulins and T-cell receptors as assessed by Western Blot analysis (Schluter et aL, 1987). Lanes: a, mouse serum; b, detergent extract of mouse spleen; c, trout serum; d, trout IgM; e, shark serum; f, shark IgM; g, turkey IgM; h, toad IgM; i, TEPC 15 (monoclonal murine IgA/K); j, human serum. Although there are many components in serum as visualized by protein staining (not shown), the affinity purified antibody to the Jfl peptide reacts with either few components which correspond to immunoglobulin or Ter chains or does not react at all (lane g--turkey IgM).

16-mer peptide. This finding of antigenic complexity in J region peptides parallels the observation of Seiden et al. (1984), who found that a number of distinct idiotypic specificities were carried by the JH1 peptide. The specificity for the peptide itself is indicated by the fact that all of the binding of anti-J/~ to light chain, Tcr molecules, or to the peptide itself were inhibited by free peptide. Since the use of immunochemical data alone to show relationships between proteins can be misleading, we have attempted to fully characterize the binding properties of the anti-Jfl antibodies by assessing their reactivity with various monoclonal myeloma proteins and with synthetic peptides of known sequence (Marchalonis et al., 1988b). The anti-J~ antibodies show very high specificity for an epitope defined by the conserved Fr4 signature sequence (F) (G) (*) (G) (T) (R or K) (V or L). Substitutions at residue position 3 (marked by *) had little effect on the reactivity. The nature of the residue at position 6 is critical and must be either of the two positively charged amino acids arginine or lysine. Substitution with neutral or negatively charged residues drastically reduces reactivity with the antibodies, even with peptides identical at all other positions to the immunizing J~ peptide. Rabbits, the species that produced these antibodies, tend to have a negatively charged residue, glutamic acid (E), at this position. This helps to explain the apparent paradox that rabbits can raise antibodies to a self component. Consistent with these sequence dependencies for antigenicity, immunoglobulins of chicken and turkey failed to react with the anti-J/~ serum. The anti-J/~ bound the synthetic Ja peptide illustrated in Table 4 above as would be expected from similarities in the a Fr4 sequence by comparison to/~ and light chain sequences. Reaction of anti-J/~ with heavy chains is rare but sometimes occurs because a small subset of human Va sequences [two of more than 300 listed by

Kabat et al., (1991)] have Fr4 regions with arginine at the critical position; viz, VnlI SUP-T1 VH-JH FGSGTRLSIR and Vail, TS2, WGPGTRVTV. Serological cross-reactions between Tcrs o f man and mouse

It is reasonable to expect that antigenic crossreactions will occur between Tcr chains of man and mouse because extensive identity occurs in comparison of amino acid sequences of various V regions and also in the comparison of C/~ regions. For example, human V/~8 and mouse V/~ll are 64% identical; human C/~ and mouse C/~ are 80% identical; human Va 13 and murine Va2 are 70% identical; and human Ca and mouse Ca are 66% identical. We (unpublished observations) have used the progressive alignment algorithm of Feng and Doolittle (1987) to analyze interspecies relationships among Tcr V/~ genes and found that certain human and murine V/~ sequences can be grouped into clusters showing greater than 50% identity with one another. One of these consists of human V/~6, 8, and 18 which form a group with murine V/~ll (Marchalonis et al., 1992b). Mouse V/~5 forms a cluster with human V/~ 1 and V/~5. Mouse V/~8 forms a similar grouping with human V~ 15, mouse V/~7, and human V/~3. It can be concluded that the prototypes of these clusters emerged before the ancestral divergence of humans and mice. In Western blot analysis, Tcr a chains of man and mouse showed strong cross-reaction (Dedeoglu et al., 1992) using hamster monoclonal antibody to the murine a chain developed by R. T. Kubo (National Jewish Hospital, Denver, CO). A similar result was found for a hamster monoclonal antibody to murine /~ chain. Strong cross-reactions were also obtained in Western blot analysis of rabbit antibodies directed against the J~ peptide and a conserved constant region peptide around cysteine

435

Shared antigenic determinants of Igs and Tcrs 2.4"

147. Comparisons of the human and murine Jfl sequences are as follows: ---CDR3 Human Jfl

-

-

.

F

r

4

.

-

2.01.6-

-

A~]Y G Y T F G S G T R L T~V I

I

I

0

I

Murine Jfl(D6) Q[N[S D Y T F G S G T a L L[~ I

<

The comparison of the conserved constant region peptides is as follows:

1.2-

0.80.40.0

•

0

Murine Cfl

2

,

•

4

,

6

L V C L A R G F F P D H V E

Human and murine Jfl sequences are 69% identical and contain the critical arginine in the Fr4 segment. The Cfl peptides are identical except for a threonine at position 7 in the human and an arginine in the mouse. The observed cross-reactions are thus consistent with shared sequence. Other cross-reactions would also be predicted based upon overall sequence homologies among Tcr fl chains of man, mouse, rat, and rabbit. A rabbit antiserum was prepared by Bonyhadi et aL (1987) against a synthetic peptide (residues 159-187) predicted from gene sequence of the human Tcr chain that immunoprecipitated the Tcr ~/6 complex of sheep T-cells (Mackay et al., 1989). Although the sequence of sheep Tcr 7 chain is not known, the antibody was directed against a conserved region of Tcr-C7 because the corresponding murine sequence was 94% identical to that of the human prototype.

Shared epitopes between Tcr fl chains and light chains defined by synthetic peptides On the basis of serological cross-reactions between antigen receptors of T-cells and serum immunoglobulins (Hogg and Greaves, 1972; Marchalonis et al., 1972; Roelants et al., 1973; Warner, 1974) coupled with observations that the two types of molecules differed in functional properties (Cone et al., 1974; Warner, 1974; Binz and Wigzell, 1975), it was proposed that the T-cell molecules were a type of immunoglobulin distinct from antibodies produced by B cells (Marchalonis, 1975). Direct proof of the relatedness of antigen specific T-cell receptors to immunoglobulins was achieved by the cloning and sequencing of the genes specifying these molecules (Hedrick et al., 1984b; Yanagi et al., 1984). Kaymaz et al. (1993a,b) analyzed cross-reactivity between Tcr f chains and immunoglobulin light chains by synthesizing a set of nested, overlapping 16-mer peptides that duplicated the sequence which corresponds to the continuous VDJC sequence of the Tcr fl chain gene clone YT35 and determined the capacity of rabbit antisera directed against either human or murine immunoglobulins to react with the

•

,

.

,

.

,

•

,

. . . . .

8 10 12 14 16 18 20 22 Peptide

Fig. 6. Mapping ofTcr fl chain epitopes detected by reaction of rabbit antisera against human 2 light chain (r-l), anti-r light chain (O), or ~ heavy chain (O) with the nested set of synthetic overlapping peptides duplicating the covalent structure of the human T-cell receptor fl chain YT35 (Kaymaz et al., 1993a,b). The Y axis gives the absorbence of the ELISA reaction; the X axis gives the number of the particular peptide with 1 corresponding to the N-terminus of the Vfl region and 22 corresponding to the C-terminus of the constant domain. The reaction was carried out using the sera at a dilution to 1:500.

peptides. As shown in Fig. 6, rabbit antisera specific for human x and 2 chains bind appreciably to certain peptides, and this reactivity is not present in antibodies specific for human ~, heavy chain or normal rabbit serum. In addition, a rabbit antiserum produced against polyclonal murine IgM (predominantly containing x chain) also reacts with the same characteristic peptides, most notably, peptide f3, f18, fl 11, and f 17. Based upon titrations, the strongest binding is to peptide Bill. The antiserum to the murine immunoglobulin reacts with both peptides f17 and f18. In studies involving a panel of rabbit anti-immunoglobulin sera the most consistent immunoglobulin cross-reactive binding was with peptide fl 11. This peptide is the "switch peptide" linking the V and C domains. It contains the antigenically active segment of the Fr4 peptide as well as the beginning of the constant region. In 3-dimensional structure, it is an exposed region forming an elbow bend connecting two compact domains. An alignment of this region comparing the switch peptide of the Tcr fl Chain with that of the human 2 chain Mcg is fill fl

S

__RL

VEDL

2

T

KV

LG-QP

AN

T

illustrated above. The switch peptide of the Tcr fl chain is slightly longer than the corresponding region of the 2 chain, but the homology is unmistakable. A second shared region that warrants special consideration is the portion of the Vfl region defined by

436

JOHN J. MARCHALONIS et al.

Table 5. Alignment of framework 3 homol~y and antigenic regions of Tcr and light chains 164)1851

BII A

(B2-59)

[Hcg

G~SIV

186)

EKKERFS

I L ESASTNQTSHIY LCAI

ID R F S G

(), numbering lystam tl that of Kabet tit a/. ~11971) for Tar//chains. Residues 64, 85, and 86 ere charactm'istic of/11 chain#. The position of paptid(~l/17 and/18 are indicated by horizontal lines. Homologies indicated are those shared with either or both of the//1 axamptu (based upon Kaymaz et a/. ~ 1993a1.

antigenic segments of peptides fl 7 and fl 8 that correspond to Fr3. Table 5 aligns Vfl, V2 and VK segments in this region. The individual amino acids contributing to the antigenicity would be concentrated in the region of residues SFSTLKIQPSEPRDSA, but the other residues are probably necessary to give the proper conformation. Antibodies directed against this region of the 2 light chain Mcg show a strong conformational dependence (Marchalonis et al., 1992a). The degree of direct sequence homology among Tcr fl chains and immunoglobulin light chains is clear. In addition, the fact that antisera directed largely against mouse x chains show strong binding to both peptides f17 and f18 is consistent with the degree of sequence identities between murine x chains and the Vfl prototype sequence. The peptide immunochemical results, coupled with the identification of specific regions of sequence correspondence between Tcr fl and the characterized 2 light chain Mcg, allowed the development of a 3-dimensional model of human fl chain consistent with its role in antigen recognition and in response to super antigens (Marchalonis et al., 1992b; Kaymaz et al., 1993a). In this model, it is of interest that Fr3 segments would not be involved in forming the combining site for antigen but would be exposed on the outer face of the folded Vfl domain. Moreover, residues lying in the Fr3 region have been implicated in the reaction of T-cells to superantigens (Pullen e t a l . , 1990). Both regions antigenically detected here would be accessible for interaction with molecules in the solvent. SUMMARY AND CONCLUSIONS

Although there are a number of carefully performed antigenic analyses using polyclonal and monoclonal antibodies as well as synthetic peptide

antigens, the complexity of the Ig system with its large number of V-regions and C-regions limits the number of definitive conclusions regarding shared Ig epitopes that can be reached at this time. Nevertheless, some firm generalizations can be made that apply both to serum antibodies and to Tcrs. This review does not consider non-immunoglobulin members of the immunoglobulin "superfamily" (Williams et al., 1985; Williams and Barclay, 1988). Kaufman and his associates have recently described serological analyses of MHC products of diverse vertebrate species (1990), and recombinant DNA analyses support the existence of MHC antigens in elasmobranchs (Kasahara et al., 1992), teleosts (Hashimoto et al., 1992), amphibians (Kasahara etal., 1992), and chickens (Xu et al., 1989). Products of the immunoglobulin "superfamily" tend to be marginally related to the rearranging immunoglobulins (Marchalonis et al., 1984; Matsunaga and Moil, 1987). Furthermore, they have not been reported to cross-react antigenically with immunoglobulin; e.g. fl2-macroglobulin is homologous to Igy chain but does not react with chicken antisera to Igs (Marchalonis et al., 1980). A fundamental problem to be addressed in analyzing the evolution of multi-genic systems by antigenic or sequence analysis is the necessity that the correct molecules are compared to one another. For example, of the hundreds of Vn genes expressed by the mouse, one gene family specifying antibodies directed against phosphorylcholine is widely expressed throughout vertebrate species to the extent that a murine Vn gene probe, VHS107V, can be used in cross-hybridization experiments to identify homologous sequences in elasmobranchs (Litman et al., 1985a; Litman and Hinds, 1987), reptiles (Litman et al., 1985b), and teleosts (Wilson et al., 1988). However, this does not hold for all the V H sequences. Moreover, it does not

Shared antigenic determinants of lgs and Tcrs

apply to light chain sequences. In order to isolate genes specifying light chains from two separate species of sharks, it was necessary to produce antibodies against the purified shark light chains and use these to screen expression vector libraries produced from lymphoid tissue (Shamblott and Litman, 1989a; Schluter et al., 1989). T-cell receptors crossreact antigenically with immunoglobulins, but the genes specifying them were isolated using a subtractive hybridization technique to detect T-cell-specific sequences (Hedrick et al., 1984b; Yanagi et al., 1984). Once the gene sequences of Tcrs and shark light chains were obtained, it was readily apparent that these molecules were homologous to the characterized rearranging immunoglobulins. Therefore, it was feasible to define regions of antigenic sharing between Tcr and Igs. Recent application of synthetic peptide technology has facilitated the localization of epitopes shared between light chains of carcharhine sharks and man (Marchalonis et al., 1992c) and markers shared between human 2 light chain and Tcr fl chain (Kaymaz et al., 1993a,b). In this context, it is important to distinguish between antibodies made against the intact protein that react with individual synthetic peptides and antibodies made against synthetic peptides that are then tested for reactivity against the intact product. We have used the first approach to demonstrate and map the linear epitope shared between human and shark 2 chains and human 2 and Tcr fl chains. The second approach is useful for developing reagents against "universal determinants" shared by Tcrs and light chains (Schluter and Marchalonis, 1986; Marchalonis et al., 1988b) or to illustrate cross-reactions between Tcrs of man and mouse (Dedeoglu et al., 1992) or of sheep and man (Mackay et al., 1989). F o r examples, peptides predicted from the sequence of the joining segment have allowed the generation of antibodies showing cross-reactions among light chains and Tcr fl and ~ sequences, but which are not reactive with the JH regions. The studies reviewed here provide antigenic evidence to buttress conclusions based upon sequence and physicochemical approaches which indicate that immunoglobulins are a highly conserved family of proteins (Litman et aL, 1971; Marchalonis, 1972, 1977; Marchalonis and Schluter, 1989). Despite the existence of a large set of V region structures, these share common structural features defined by the frameworks as well as some highly conserved idiotypic markers. Constant regions seem to have undergone a more pronounced evolutionary divergence because of the appearance of distinct heavy chain isotypes in different vertebrate classes as the result of independent duplications of the ancestral heavy chain (Atwell and Marchalonis, 1975), and also because of position effects in the location of domains within the complete heavy chain (Liu et aL, 1976; Barker et aL, 1980; Schwager et aL, 1988; Marchalonis and CBP(B)105-3/4--B

437

Schluter, 1989). The switch peptide linking the V and C domains is specified by the J-minigene segment and would be exposed to the solvent. These J-region pep tides can be classed into two broad groups; one rep resenting shared features between Tcrs and Ig light chains, and the second being characteristic of Ig heavy chains. Of all the peptide sequences considered, this region appears to be the most characteristic in defining Igs. It also defines the major cross-reactive determinants shared among immunoglobulin chains in evolution and in comparison of immunoglobulins with Tcrs.

Acknowledgements--This work was supported in part by Grant # D C B 9106934 from the National Science Foundation, Grant # GM 42437 from the National Institutes of Health, and Grant #CA42049 from the National Cancer Institute. We would also like to thank Ms Diana Humphreys for her assistance in the preparation of this manuscript. REFERENCES

Aguilar B. A. and Gutman G. A. (1993) Transcription and diversity of immunoglobulin lambda chain variable genes in the rat. Immunogenetics (in press). Alepa F. P. (1969) Antigenic factors characteristic of human immunoglobulin G detected in the sera of non-human primates. Ann. N.Y. Acad. Sci. 162, 170--176. Atwell J. L. and Marchalonis J. J. (1975) Phylogenetic emergence of immunoglobulin classes distinct from IgM. J. Immunogenetics 1, 367-391. Barker W. C., Ketcham L. K. and Dayhoff M. O. (1980) Origins of immunoglobulin heavy chain domains. J. molec. Evol. 15, 113-127. Beaman K. D., Barker W. C. and J. J. Marchalonis (1987) Molecular biology of T lymphocyte recognition elements. In Antigen-Specific T Cell Receptors and Factors (Edited by Marchalonis J. J.) Vol. II, pp. 105-126. CRC Press, Boca Raton, FL. Binz H. and Wigzell H. (1975) Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. J. exp. Med. 142, 197-211. Bonyhadi M., Weiss A., Tucker P. W., Tigelaar R. E. and Allison J. P. (1987) Delta is the Cx-gene product in the ~/6 antigen receptor of dendritic epidermal cells. Nature, Lond. 333, 574-578. Brack E., Hirama M., Lenhard-Schuller R. and Tonegawa S. (1978) A complete immunoglobulin gene is created by somatic recombination. Cell 15, 1-14. Carson D. A., Chen P. P., Fox R. I., Kipps T. J., Jirik F., Goldfine R. D., Silverman G., Radoux V. and Fong S. (1987) Rheumatoid factor and immune networks. Annu. Rev. Immunol. 5, 109-126. Chothia C., Bosell D. R. and Lesk A. M. (1988) The outline structure of the T-cell ct/fl receptor. EMBO J. 7, 3745-3755. Cone R. E., Feldman M., Marchalonis J. J. and Nossal G. J. V. (1974) Cytophilic properties of surface immunoglobulin of thymus-derived lymphocytes. Immunology 26, 49-60. Crews S., Griffin J., Huang H., Calame K. and Hood, L. (1981) A single VH gene segment encodes the immune response to phosphorylcholine: somatic mutation is correlated with the class of the antibody. Cell 25, 59-66. Dahan A., Reynaud C. A. and Weill J. C. (1983) Nucleotide sequence of the constant region of a chicken # heavy chain immunoglobulin mRNA. Nucleic Acids Res. 11, 5381-5389.

438

JOHN J. MARCHALONISet al.

Davie J. M., Seiden M. V., Greenspan N. S., Lutz C. T., Bartholow T. L. and Clevinger B. L. (1986) Structural correlates of idiotopes. A. Rev. Immunol. 4, 147-165. Davis M. M. and Bjorkman P. J. (1988) T-cell antigen receptor genes and T-cell recognition. Nature 334, 395-401. Dedeoglu F., Hubbard R. A., Schluter S. F. and Marchalonis J. J. (1992) T-cell receptors of man and mouse studies with antibodies against synthetic peptides. Expt. Clin. Immunogenet. 9, 95-108. Doolittle R. F. (1989) Similar amino acid sequences revisited. Trends Biochem. Sci. 14, 244-250. Dwyer D. S., Bradley R. J., Urquhart C. K. and Kearney J. F. (1983) Naturally occurring anti-idiotypic antibodies in myasthenia gravis patients. Nature 17, 611~515. Edmundson A. B., Ely K. R., Abola, E. E., Schiffer M. and Pangiotopoulos N. (1975) Rotational allomerism and divergent evolution of domains in immunoglobulin light chains. Biochemistry 14, 3953-3961. Eshhar Z., Gigi O., Givol D. and Ben-Neriah Y. (1983) Monoclonal anti-V n antibodies recognize a common V H determinant on immunoglobulin heavy chains from various species. Eur. J. ImmunoL 13, 533-540. Esteves M. B. and Binaghi R. A. (1972) Antigenic similarities among mammalian immunoglobulins. Immunology 23, 137-145. Feng D. F. and Doolittle R. F. (1987) Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. molec. Evol. 25, 351-360. Ghaffari S. H. and Lobb C. J. (1989a) Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains. J. Immunol. 143, 27302739. Ghaffari S. H. and Lobb C. J. (1989b). Cloning and sequence analysis of channel catfish heavy chain cDNA indicate phylogenetic diversity within the IgM immunoglobulin family. J. Immunol. 142, 1356-1365. Goldfien R. D., Chen P. P., Fang S. and Carson D. A. (1985) Synthetic peptides corresponding to the third hypervariable region of human monoclonal IgM rheumatoid factor heavy chains define an immunodominant idiotype. J. exp. Med. 162, 756-761. Harding F. A., Cohen N. and Litman G. W. (1990) Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea. Nucleic Acids Res. 18, 1015-1020. Hashimoto K., Nakanishi T. and Kurosawa Y. (1992) Identification of a shark sequence resembling the major histocompatability complex class I e 3 domain. Proc. natn. Acad. Sci. USA 89, 2209-2212. Hazer D. J. (1990) Immunoglobulin 2 light chain evolution: Igl and Igl-like sequences from three major groups. Immunogenetics 32, 157-174. Hedrick S. M., Nielsen E. A., Kavaler J., Cohen D. I and Davis M. M. (1984a) Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins. Nature 308, 153-158. Hedrick S. M., Cohen D. I., Nielsen E. A. and Davis M. M. (1984b) Isolation of cDNA clones encoding T-cellspecific membrane-associated proteins. Nature 308, 149-153. Hildemann W. H. (1979) Immunocompetence and a11ogeneic polymorphism among invertebrates. Transplant. 27, I-3. Hinds K. R. and Litman G. W. (1986) Major reorganization of immunoglobulin V H segmental elements during vertebrate evolution. Nature 320, 546-552. Hogg N. M. and Greaves M. F. (1972) Antigen-binding thymus-derived lymphocytes. II. Nature of the immunoglobulin determinants. Immunology 22, 967-980. Hohman V. S., Schluter S. F. and Marchalonis J. J. (1992) Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and

implications for the evolution of light chains. Proc. natn. Acad. Sci. USA 89, 276-280.

Hubbard R. A. and Marchalonis J. J. (1988) Molecular biology of T cell recognition elements. EOS, No. 4, Vol. VIII, 248-256. Hubbard R. A., Speidel M. T., Marchalonis J. J. and Cone R. E. (1989) A monoclonal antigen-binding T cell immunoprotein: Antigenic relatedness to T cell receptor fl chain Frl V and J peptide segments: physicochemical distinctiveness from classical immunoglobulins and T cell receptor heterodimers. Mol. Immunol. 26, 447-456. Jefferis R., Lowe J., Ling N. R., Porter P. and Senior S. (1982) Immunogenic and antigenic epitopes of immunoglobulins 1. Cross-reactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species. Immunology 45, 71-77. Kabat E. A., Wu T. T., Perry H. M., Gottesman K. S. and FoeUer C. (1991) Sequences of Proteins of Immunological Interest, 5th Edn. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD. Kasahara M., Vazquez M., Sator K., McKinney E. C. and Flajnik M. F. (1992) Evolution of the major histocompatibility complex: isolation of class 11 A cDNA clones from the cartilaginous fish. Proc. natn. Acad. Sci. USA 89, 6688~692. Kaufman J., Skjoedt K., Salomonsen J., Simonsen M., Du Pasquier L., Parisot R. and Riegert P. (1990) MHlike molecules in some non-mammalian vertebrates can be detected by some cross-reactive xenoantisera. J. Immunol. 144, 2258-2272. Kay M. M. B., Marchalonis J. J., Hughes J., Watanabe K. and Schluter S. F. (1990) Definition of a physiologic aging autoantigen using synthetic peptides of membrane protein Band 3: localization of the active antigenic sites. Proc. natn. Acad. Sci. USA 89, 5734-5738. Kaymaz H., Dedeoglu F., Schluter S. F., Edmundson A. B. and Marchalonis J, J. (1993a) Reactions of antiimmunoglobulin sera with synthetic T-cell receptor peptides: implications for the three-dimensional structure and function of the Tcr fl chain. Int. Immunol. (in press). Kaymaz H., Dedeoglu F., Schluter S. F., Edmundson A. B. and Marchalonis J. J. (1993b) Cross-reactions of anti-immunoglobulin sera with synthetic T-cell receptor fl peptides: mapping on a 3-dimensional model. In Immunobiology of Proteins and Peptides VII (Edited by Atassi M. Z.), Plenum Press, New York. Kennedy R. C., Ionescu-Matin I., Sanchez Y. and Dreesman G. R. (1983) Detection of interspecies idiotypic cross-reactions associated with antibodies to hepatitis B surface antigen. Eur. J. Immunol. 13, 232-241. Klein J. (1989) Are invertebrates capable of anticipatory immune responses? Scand. J. Immunol. 29, 499-505. Knight K. L. , Malek T. R. and Dray S. (1975) Human immunoglobulin with a' allotypic determinants of rabbit immunoglobulin heavy chains. Nature 253, 216-217. Kronenberg M., Siu G., Hood L. E. and Shastri N. (1986) The molecular genetics of the T-cell antigen receptor and T-cell antigen recognition. A. Rev. Immunol. 4, 529-591. Leslie G. A. and Clem L. W. (1969) Phylogeny of immunoglobulin structure and function. III. Immunoglobulins of the chicken. J. exp. Med. 130, 1377-1382. Litman G. W., Rosenberg A., Frommel D., Pollara B., Finstad J. and Good, R. A. (1971) Biophysical studies of the immunoglobulins. The circular dichroic spectra of the immunoglobulins--a phylogenetic comparison. Int. Arch. Allergy appl. Immunol. 40, 551 575. Litman, G. W., Berger L., Murphy K., Litman R., Hinds K. and Erickson B. W. (1985a) Immunoglobulin Vrt gene structure and diversity in Heterodontus, a phylogenetitally primitive shark. Proc. natn. Acad. Sci. USA 82, 2082-2086.

Shared antigenic determinants of Igs and Tcrs Litman G. W., Murphy K., Berger L., Litman R., Hinds K. and Erickson B. W. (1985b) Complete nucleotide sequences of a 3 Vn genes in Caiman, a phylogenetical ancient reptile: evolutionary diversification in coding segments and variation in the structure and organization of recombination elements. Proc. natn. Acad. Sci. USA 82, 844-848. Litman G. W. and Hinds K. R. (1987) The immunoglobulin VH system of Heterodontus, a phylogenetically primitive elasmobranch. In Evolution and Vertebrate Immunity: The Antigen-Receptor and MHC Gene Families (Edited by Kelsoe G. and Schulze D. H.), p. 35. University of Texas Press, Austin, TX. Liu Y. S. V., Low T. L. K. Infante A. and Putnam F. W. (1976) Complete covalent structure of a human IgA1 immunoglobulin. Science 193, 1017-1020. Loomis W. F. (1988) Four Billion Years: an Essay on the Evolution of Genes and Organisms. Sinuar Associates, Sunderland, MA. Mackay C. R., Beya M. F. and Matzinger F. (1989) ~/~ cells express a unique surface molecule appearing late during thymic development. Eur. J. Immunol. 19, 1477-1483. Mackel-Vandersteenhoven A., Moseley J. M. and Marchalonis J. J. (1984) Partial characterization of T cell components to defined VH(Vr)-markers. Cell. lmmunol. 88, 147-161. Mackel-Vandersteenhoven A., Vasta G. R., Waxdal M. J. and Marchalonis J. J. (1985) Segmental homology between T cell receptors and immunoglobulin variable regions: Evidence that antisera to synthetic JH1 peptide react with murine and human T cell products. Proc. Sac. exp. Biol. Med. 178, 476-485. Mage R. G., Bernstein K. E., McCartney-Francis N., Alexander C. B., Young-Cooper G. O., Padlan E. A. and Cohen G. H. (1984) The structural and genetic basis for expression of normal and latent Vaa allotypes of the rabbit. Molec. Immunol. 21, 1067-1081. Marchalonis J. J. and Edelman G. M. (1965) Phylogenetic origins of antibody structure. I. Multichain structure of immunoglobulins in the smooth dogfish (Mustelus canis). J. exp. Med. 122, 601-618. Marehalonis J. J. and Edelman, G. M. (1968) Isolation and characterization of a natural hemagglutinin from Limulus polyphemus. J. molec. Biol. 32, 453-465. Marchalonis J. J. (1972) Conservatism in the evolution of immunoglobulin u-chains. Nature (New Biol.) 236, 84-86. Marchalonis J. J., Cone R. E. and Atwell J. L. (1972) Isolation and partial characterization of lymphocyte surface immunoglobulins. J. exp. Med. 135, 956-971. Marchalonis J. J. and Cone R. E. (1973) Biochemical and physiological properties of lymphocyte surface immunoglobulin. Transplant. Rev. 14, 3-49. Marchalonis J. J. (1975) Lymphocyte surface immunoglobulins: molecular properties and function as receptors for antigen. Science 190, 20-29. Marchalonis J. J. (Ed.) (1977) Immunity in Evolution. Harvard Press, Cambridge. Marchalonis J. J., Warr G. W., Smith P., Begg G. S. and Morgan F. J. (1979a) Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten. Biochemistry 18, 560-565. Marchalonis J. J., Warr G. W., Bucana C. and Hoyer L. C. (1979b) The immunoglobulin-like T-cell receptor. I. In situ demonstration of immunoglobulin Fabregion determinants of rodent T- and B-lymphocytes using chicken antibodies. J. Immunogenet. 6, 289-310. Marchalonis J. J., Warr G. W., Wan A.-C., Burns H. and Burton R. C. (1980) A Fab-related surface component of normal and neoplastic human and marmoset T cells: demonstration, functional analysis and partial characterization. MoL Immunol. 17, 877-891.

439

Marchalonis J. J., Vasta G. R., Warr G. W. and Barker W. C. (1984) Probing of the boundaries of the extended immunoglobulin family of recognition molecules: jumping domains, convergence and minigenes. Immunol. Today 5, 133-142. Marehalonis J. J., Schluter S. F., Rosenshein I. L. and Wang A. C. (1988a) Partial characterization of immunoglobulin light chains of careharhine sharks: evidence for phylogenetic conservation of variable region and divergence of constant region structures. Dev. comp. ImmunoL 12, 65-74. Marchalonis J. J., Schluter S. F. Hubbard R. A., McCabe C. and Alien R. C. (1988b) Immunoglobulin epitopes defined by synthetic peptides corresponding to joiningregion sequence conservation of determinants and dependence upon the presence of an arginyl or lysyl residue for cross-reaction between light chains and T cell receptor chains. Molec. ImmunoL 25, 711-784. Marchalonis J. J., Schluter S. F., Hubbard R. A., Diamanduros A., Barker W. C. and Pumphrey R. S. H. (1988c) Conservation of immunoglobulin variable and joining region structure and the design of universal antiimmunogiobulin antibodies reactive with antigen-binding T cell receptors. Int. Rev. lmmunol. 3, 241-273. Marehalonis J. J. and Schluter S. F. (1989) Evolution of variable and constant domains and joining segments of rearranging immunoglobulins. FASEB J. 3, 2469-2479. Marchalonis J. J. and Schluter S. F. (1990) Origins of immunoglobulins and immune recognition molecules. Bio Science 40, 758-768. Marchalonis J. J., Dedeoglu F., Kaymaz H., Schluter S. F. and Edmundson A. B. (1992a) Antigenic mapping of a human 2 light chain: correlation with 3-dimensional structure. J. Protein Chem. 11, 129-137. Marchalonis J. J., Kaymaz H., Dedeoglu F., Schluter S. F., Yocum D. E. and Edmundson A. B. (1992b) Human autoantibodies reactive with synthetic autoantigens from T-cell receptor fl chain. Proc. natn. Acad. Sci. USA 89, 3325-3329. Marchalonis J. J., Schluter S. F., Yang H.-Y., Hohman V. S., McGee K. and Yeaton L. (1992c) Antigenic cross-reactions among immunoglobulin of diverse vertebrates (elasmobranchs to man) detected using xenoantisera. Comp. Biochem. Physiol. 101A, 675-687. Marchalonis J. J., Kaymaz H., Schluter S. F. and Yocum D. E. (1993) Natural occurring human autoantibodies to defined T-cell receptor and light chain peptides. In Immunobiology of Proteins and Peptides VII (Edited by Atassi M. Z). Plenum Press, New York (in press). Matsunaga T. and Mori N. (1987) The origin of the immune system. The possibility that immunoglobulin superfamily molecules and cell adhesion molecules of chicken and slime mould are all related. Scand. J. Immunol. 25, 485-495. Meek K., Takei M., Dang H., Sanz I., Dauphinee M. J., Capra J. D. and Talal N. (1990) Anti-peptide antibodies detect a lupus related interspecies idiotype that maps to H chain CDR2. J. Immunol. 144, 1375-1387. Mehta P. D., Reichlin M. and Tomasi T. B. Jr. (1972) Comparative studies of vertebrate immunoglobulins. J. Immunol. 109, 1272-1277. Morahan G., Berek C. and Miller J. F. A. P. (1983) An idiotypic determinant formed by both immunoglobulin constant and variable regions. Nature 301, 720-722. Nash D. R. and Mach J. P. (1971) Immunoglobulin classes in aquatic mammals. Characterization by serologic crossreactivity molecular size and binding of free secretory component. J. ImmunoL 107, 1424-1430. Neoh S. H., Jaboda D. M., Rowe D. S. and Volier A. (1973) Immunoglobulin classes in mammalian species identified by cross-reactivity with antisera to human immunoglobulin. Immunochemistry 10, 805-813.

440

JOHN J. MARCHALONISet al.

Nguyen N. Y., Suzuki A., Boykins R. A. and Liu T. Y. (1986) The amino acid sequence of Limulus C-reactive protein. Evidence of polymorphism. J. biol. Chem. 261, 104-156. Novotny J., Tonegawa S., Saito H., Kranz D. M., Eisen H. N. (1986) Secondary tertiary and quaternary structure of T-cell specific immunogiobulin-like polypeptide chains. Proc. natn. Acad. Sci. USA 83, 742-746. Parsons M. and Herzenberg L. A. (1981) A monoclonal mouse antiallotype antibody reacts with certain human and other vertebrate immunoglobulins: genetic and phylogenetic finds. Immunogenetics 12, 207-219. Pullen A. M., Wade T., Marrack P. and Kappler J. W. (1990) Identification of the region of T-cell receptor chain that interacts with the self-superantigen MIs-1 a. Cell 61, 1365-1374. Riesen W. F. (1979) Idiotypic cross-reactivity of human and murine phosphorylcholine-binding immunoglobulins. Eur. J. Immunol. 9, 421-425. Roelants G. E., Forni L. and Pernis B. (1973) Blocking and redistributed ("capping") of antigen receptors on T- and B-lymphocytes by anti-immunoglobulin antibody. J. exp. Med. 137, 1060. Romer A. S. (1966) Vertebrate Paleontology 3rd Edn. University of Chicago Press. Rosenshein I. L. and Marchalonis J. J. (1985) Broad phylogenetic expression of heavy-chain determinants detected by rabbit antisera to VHa allotypes. Mol. Immunol. 22, 1177-1183. Rosenshein I. L., Schluter S. F., Vasta G. R. and Marchalonis J. J. (1985) Phylogenetic conservation of heavy chain determinants of vertebrates and protochordates. Dev. comp. Immunol. 9, 783-795. Rosenshein I. L. and Marchalonis J. J. (1987) The immunoglobulins of carcharhine sharks: a comparison of serological and biochemical properties. Comp. Biochem. Physiol. 86B, 737-747. Sakano H., Maki R., Kurosawa Y., Roeder W. and Tonegawa S. (1980) Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature 286, 676~83. Sasso E. H., Barber C. V., Nardella F. A., Yount W. J. and Mannik M. (1988) Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C~2C~,3 Interface region contains the major determinants. J. Immunol. 140, 3098-3107. Schluter S. F. and Marchalonis J. J. (1986) Antibodies to synthetic joining segment peptide of the T-cell receptor fl chain: serological cross-reaction between products of T-cell receptor genes antigen binding T-cell receptors and immunogiobulins. Proc. natn. Acad. Sci. USA 83, 1872-1876. Schluter S. F., Rosenshein I. L., Hubbard R. A. and Marchalonis J. J. (1987) Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide. Biochem. biophys. Res. Commun. 145, 699-705. Schluter S. F., Hohman V. S., Edmundson A. B. and Marchalonis J. J. (1989) Evolution of immunogiobulin light chains: complementary DNA clones specifying sandbar constant regions. Proc. natn. Acad. Sci. USA 86, 9961-9965. Schwager J., Mikoryak C. A. and Steiner L. A. (1988) Amino acid sequence of heavy chains from Xenopus laevis IgM deduced form cDNA sequence implications for evolution of immunoglobulin domains. Proc. natn. Acad. Sci. USA 85, 2245-2249. Schwager J., Burckert N., Schwager M. and Wilson M. (1991) Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity. EMBO J. 10, 505-511.

Seiden M. V., Clevinger B., McMillan S., Srouji A., Lerner R. and Davis J. M. (1984) Chemical synthesis of idiotopes. Evidence that Antisera to the same JH~ peptide detect multiple binding site-associated idiotopes. J. exp. Med. 159, 1338-1350. Shamblott M. J. and Litman G. W. (1989a) Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes. Proc. HatH. Acad. Sci. USA 86, 5684-5688. Shamblott M. J. and Litman G. W. (1989) Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest co-evolution of immunoglobulin gene organization. EMBO J. 8, 3733-3739. Shankey T. V., Schluter S. F. and Marchalonis J. J. (1989) Flow cytometric analysis of human lymphocytes using affinity purified antibody to T cell receptors synthetic J region. Cell lmmunol. 118, 526-531. Smith L. C. and Davidson E. H. (1992) The echinoid immune system and the phylogenetic occurrence of immune mechanisms in deuterostomes. Immunol. Today 13, 356-361. Tainer J. A., Getzoff E. D., Alexander H., Houghten R. A., Olson A. J., Lerner R. A. and Hendrickson W. A. (1984) The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein. Nature 312, 127-134. Takei M., Dang H. and Talai N. (1987) A common idiotype expressed on a murine anti-SM monoclonal antibody and antibodies in SLE sera. Clin. exp. Immunol. 70, 546-554. Tanaka A., Ishiguro N. and Shinagawa M. (1990) Sequence and diversity of bovine T-cell receptor E-chain genes. Immunogenetics 32, 263-271. Tjoelker L. W., Carlson L. M., Lee K., Lahti J., McCormack W. T., Leiden J. M., Chen, C. H., Cooper M. D. and Thompson C. B. (1990) Evolutionary conservation of antigen recognition: the chicken T-cell receptor fl chain. Proc. natn. Acad. Sci. USA 87, 7856-7860. Tonnelle C., Cazenave P. A.-A., Brezin C., Moinier D. and Fougereau M. (1983) Structural correlates to the immunoglobulin heavy chains al00 allotype. Molec. Immunol. 20, 753-761. Toyonaga B. and Mak T. W. (1987) Genes of the T-cell antigen receptor in normal and malignant T-cells. A. Rev. Immunol. 5, 585420. Varner J., Neame P. and Litman G. W. (1991) A serum heterodimer from hagfish (Eptatretus stoutii) exhibits structural similarity and partial sequence identity with immunoglobulin. Proc. Ham. Acad. Sci. USA 88, 1746-1750. Vasta G. R., Marchalonis J. J. and Kohler, H. (1984) Invertebrate recognition protein cross-reacts with an immunoglobulin idiotype. J. exp. Med. 159, 1270-1276. Vitetta E. S. and Uhr J. W. (1973) Synthesis transport dynamics and fate'of cell surface Ig and alloantigens in murine lymphocytes. Transplant. Rev. 14, 50-75. Volanakis, J. E. and Kearney J. F. (1981) Cross-reactivity between C-reactive protein and idiotype determinants on a phosphocholine-binding murine myeloma protein. J. exp. Med. 153, 1604-1614. Wang A. C., Mathur S., Pandey J., Siegal F. P., Meddaugh C. R. and Litman G. W. (1978) Hv(l), a variable region genetic marker of human immunoglobulin heavy chains. Science 200, 327-329. Warner N. L. (1974) Membrane immunoglobulins and antigen receptor on B and T lymphocytes. Adv. lmmunol. 19, 67-216. Williams A. F., Barclay A. N., Clark M. and Gagnon J. (1985) Cell surface glycoproteins and the origins of immunity. In Proceedings of the Sigrid Juselius Symposium:

Shared antigenic determinants of Igs and Tcrs Gene Expression During Normal and Malignant Differentiation (Edited by Anderson L. C., Gahnberg C. G. and Ekblom P.), p. 125. Academic Press, New York. Williams A. F. and Barclay A. N. (1988) The immunoglobulin superfamily---domains for cell surface recognition. A. Rev. Immunol. 6, 381-405. Williams C. B. and Gutman G. A. (1989) T-cell receptor fl-chain genes in the rat: availability and pattern of utilization of V gene segments differs from that in the mouse. J. Immunol. 142, 1027-1035. Wilson M. R., Middleton D. and Wart G. W. (1988) Immunoglobulin heavy chain variable region gene evolution structure and family relationships of two genes and a pseudo-gene in a teleost fish. Proc. natn. Acad. Sci. USA 85, 156-159.

441

Xu Y., Pitcovski J., Peterson L., Auffray C., Bourlet Y., Gerndt B. M., Nordskog A. W., Lamont S. J. and Warner C. M. (1989) J. Immunol. 142, 2122-2132. Yanagi Y., Yoshikai Y., Leggett K., Clark S. P., Alexander I1. and Mak T. W. (1984) A human T cell-specific eDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nature 308, 145-149. Ying S.-C., Marchalonis J. J., Gewurz A. T., Siegel J. N., Jiang H., Gewurz B. E. and Gewurz H. 1992 Reactivity of anti-human CRP and SAP monoclonal antibodies with Limulin and Pentraxins of other species. Immunology 76, 324-330. Zezza D. J., Stewart. S. E. and Steiner L. A. (1992) Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of x and 2 chains. J. Immunol. 149, 3968-3977.