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Evolution of the immunoglobulin superfamily by duplication of complementarity Takeshi Matsunaga Since the original finding that the/jr microglobulin (/J2m) has considerable amino acid sequence homology to immunoglobulin(Ig) domains ~, the members of this Ig superfamily have been steadily increasing in number (see Ref. 2). Apart from Igs, M H C (major histocompatibility complex) class I and II antigens 3'4 and their functional partner, the T-cell antigen receptor 5'6, are the most important family members, as both are recognition molecules in the T-cell immune system. Other M H C class I antigens are encoded in the mouse Q a J T L region and are expressed in lymphoid cells or liver 7'8. The Thy- 1 antigen, expressed predominantly in neuronal cells and fibroblasts, also belongs to the superfamily but its function is unknown 9. In addition, receptor proteins for transport of IgM and IgA show a remarkable resemblance to Igs ~°. The last example may provide us with an insight into the mechanism of evolution of the Ig superfamily. One of the questions is how evolutionarily related proteins came to possess similar but diverse functions of immunological importance. A structural feature that characterizes the Ig superfamily is that they are composed of a globular domain or domains ~. Each domain is constructed from two sheets made of several antirparallel/J-helices of approximately 100 amino acid residues. The whole domain structure is stabilized by a disulfide linkage and many are glycosylated to various extents. Most members are composed of protein subunits of more than two domains each, whereas the Thy-1 and /J2m represent single domain molecules. I will argue in this article that pre-existing interdomain interactions based on mutual complementarity have been repeatedly used in the evolution of Ig superfamily molecules. T h e duplication of complementarity in receptors

for IgM and IgA transportation Newborn mammals are often protected from gastrointestinal infection by antibodies contained in milk passed from nursing mothers. IgA antibodies are made in lymphoid tissues and secreted into fluids such as milk and other secretions. This excretion employs cell receptors j~ which bind IgA as well as IgM. Thus, the receptors on glandular epithelial cells bind these Igs at the basolateral cell surface and the Ig-receptor complex is then transported across the cell in vesicles and discharged into the external circulation. The Ig-bound domain of the receptors- the secretory component ~2- is proteolytically cleaved off during this process. ,

Department of General and Oncologic Surgery and Beckman Research Institute of City of Hope, City of Hope National Medical Center, Duarte, California, USA. ~) 1985, Elscxie~ Science Publishers B.V., Amsterdam 0167 - 4919/85/$02.00

The sequence of the entire c D N A clone of the receptor for IgA/IgM transportation revealed that it is a single polypeptide containing six Ig superfamily domains, five resembling V~ 10. The sixth domain which constitutes intra-membrane and intra-cytoplasmic portions has also some sequences related to V,. Thus, the IgA transport receptor gene is probably evolutionarily related to V~. What does this mean? How could two structurally related proteins come to be functionally related as 'ligand' and 'receptors'? It has been suggested that preexisting inter-domain complementarity may have been reutilized for the specific binding between IgA/IgM and receptors 1°'13.This can be outlined as follows. Homodimer proteins exist, gene duplication and subsequent diversification can then create heterodimers. If one of the chains in the heterodimer is expressed in cells of another kind as a surface protein, then two subunit proteins would assume a 'ligand-receptor' relationship. The-'ligand' protein and 'receptor' protein would be structurally similar, since they share a common ancestral gene at the stage of homodimer. This mechanism is illustrated in Fig. 1. IgA/IgM transport receptor would be unnecessary in the absence of Igs to be transported by it, so a transport receptor gene probably evolved from V~ genes after the Ig gene family was established. When the ancestral transport receptor gene was born and expressed in different cells as receptors, inter-domain complementarity previously used for inter-chain association may have been reutilized to generate specific binding between IgA/IgM and receptors by a mechanism analogous to the one depicted in Fig. 1. IgA and IgM are known to form polymers withoutJ chain 14. A second possibility is that V, antibodies which happened to have specificity for IgA/IgM proteins became receptors. However, a selective pressure on combining sites to preserve antigen-binding specificity may be unstable Is compared with that responsible for chain association. In other words, conserved complementarity for chain-association would have a better chance of being utilized in ligand-receptor binding. In both cases, it can be concluded that receptor proteins which recognize (or that are recognized by) Igs can be created by duplication of complementarity from Igs themselves. Before I discuss an implication of this finding for the evolution of M H C proteins, it will be helpful to consider the nature of inter-domain interactions.

Inter-domain complementarity and antibodycombining sites First consider the complementarity between domains responsible for inter-chain interaction. (The term 'complementarity' used throughout this article is not strict and is used to mean a minimum area of protein

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homodimer

heterodimer

plasma membrane

gene ~duplication

Fig. 1. Mechanisms of evolution of a ligand-receptor relationship from a protein heterodimer

Many proteins are homodimers. Gene duplicationand diversificationcan create heterodimersor tetramers. Heterodimerscouldalso arise from two related monomers. One of the heterodimer subunits can acquire hydrophobicpeptide to become an integral membrane protein and can be expressed in a differentcelltype. In the case of transport receptors for IgM/IgA, this basic scheme shouldbe modifiedslightlysincemore than one complementaritymay be used by severaldomainsto create a ligand-receptorrelationship.

surface needed for two domains to interact or to bind.) IgG is composed of two identical heavy chains and two identical light chains. X-ray crystallographic studies showed that there are a n u m b e r of contact amino acid residues responsible for inter-chain interactions and these residues are in areas of complementarity between domains 16. The Vn and V L chains are held together by m a n y contact residues mainly located in framework regions 2 and 3 in both chains as well as by several other contact residues between Cul and CL domains. Similarly, there is sufficient complementarity between Cn2 and CH2, and CH3 and CH3 to allow these domains to interact with each other. These interactions are stabilized by inter-chain disulfide linkages. Furthermore, adjacent domains on the same chain can also interact with each other although to a lesser extent. In addition, light chains can form dimers without heavy chains. We have only a limited picture of M H C antigens. The M H C class I heavy chain is composed of three major domains (see Ref. 3). The N and C 1 domains are recognized by cytotoxic T cells. The C2 domain is most conserved among different alleles of the same species and among different species, fl~m, a light chain, associates noncovalently with the C2 domain. The M H C class II antigens and T-cell antigen receptors 4'5'6 are thought to form heterodimers, having two major domains on each chain (a chain and/3 chain). Thus, it is understood that members ofIg superfamily proteins retain subunit structure through complementarity between domains. What about the combining site of antibodies? In spite of the overall structural similarity of domains among different superfamily members, the Vn and V L domains of antibodies are distinguished from those of other members by an extra 0-helical loop which comprises most of the complementarity determining region (CDR) 2. Indeed, it is known from X-ray crystallographic studies that some of the antigen contact residues for several haptenic determinants reside in the C D R 2

region 16. The C D R ! and 3join the C D R 2 to form a complete antigen-bindingpocket. Recently, we proposed ~7 that the conserved framework 2 region adjacent to the extra loop o f C D R 2 forms a floor area of the antigen-binding pocket. We further suggested that the extra loop o f C D R 2 was generated by repeated duplication of three amino acid residues originally located in the framework 2 region 17. In addition, the heavy chain residues in M603 myeloma protein (anti-phosphocholine) has a contact residue for both antigen binding and binding with a VL domain ~8. Thus it would seem that the antibody-combining sites have some overlap with a C D R involved in inter-domain interaction. This suggests that inter-domain complementarity responsible for chain association can develop into other forms of complementarity, such as combining sites. Both M H C restriction19and the idiotype network2°are based on the concept of complementarity between combining sites of antibodies or T-cell receptors and determinants on other domains of the superfamily (i.e. the V domain of antibodies and the N and C1 domains of M H C class I antigen). For the present discussion it is suf-

MHC Class 1

MHC Class 2

I-c

receptors

)-'---(

Igreceptor, T cell

Thy-1

Fig. 2. Alternative phylogenetic tree of M H C evolution

Thy-1is structurallyrelatedto V domainsofIgs and can be foundin some invertebratespecies such as the squid27. Alternatively,a single light chain-likeprotein may have given rise to other Ig superfamily members3°. V domains with combining sites are present in primitive fish (cyclostomes).MHC classI and II evolvedfrom Igs or T-cellreceptors for reasons explained in the text.

262 ficient to accept that either form of complementarity can be duplicated and reutilized by new molecules.

More members predicted? If the duplication of complementarity de scribed above can play a significant role in the evolution of Ig superfamily genes, can one predict more family members? There are several candidates, including Fc receptors on various lymphoid cells21, receptors for transepithelial transport of IgG in placenta22, T3 antigens which form a complex with h u m a n T cell antigen receptors and are considered to be important for T-cell activation23, and T4 and T8 antigens (Ly 1 and Ly 23 in mice) which may be involved in determining the relationships between T-cell subset and M H C antigen class in M H C restrictionz3. These proteins and receptors have an auxiliary but important role in the regulation of i m m u n e responses or have a protective function in various circumstances. It can be seen that evolution based on duplication of complementarity would generate a limited cascade by Ig superfamily members.

M H C may have evolved after Igs Tunicates, direct ancestors of vertebrates, exhibit colony-fusion incompatibility controlled by a single genetic locus24. This suggests that the vertebrate M H C system might have evolved from gametic self- non-self discrimination25. Although M H C can be detected in fish26, a search has not yet produced evidence for an M H C in invertebrates. For example, cDNA of mouse H - 2 K which contains the conserved C2 domains, were used to probe genomes of two invertebrate species, tunicares and earth worms, in Southern hybridization. Although these probes could detect one to several crosshybridization bands in vertebrate species studied (i.e. chicken, frog, rainbow trout) under relaxed hybridization conditions, the results with the two invertebrates were negative (T. Matsunaga, unpublished). The concept of duplication of complementarity offers an alternative view that M H C antigens may have been secondarily derived from Igs or, more likely, from T-cell receptors in a thshion analogous to the evolution of IgA/IgM transport receptors. According to this view, V domains with primordial combining sites first evolved from members of the Ig superfamily which are not M H C antigens but were present in invertebrates. The Thy-1 antigen is a good candidate 27. During the gene duplication and diversification, germline V genes with primordial combining sites may have been selected for specificity for antigens of common pathogens (e. g. phosphocholine) and against specificity for self-antigens (self-non-selfdiscrimination encoded in germ-line). This stage may then have been replaced by mechanisms for somatic generation of antibody repertoire in order to cope with the rapidly changing antigenic makeup of microorganisms (e.g. hemagglutinin of h u m a n influenza virus, coat proteins of African tryp~nosomes). Because antibody specificity is generated randomly, it would have been inevitable that anti-self clones were produced 2s tbr which suppression mechanisms would

Immunology Today, vol. 6, No. 9, 1985

have been required at a somatic level (self- non-self discrimination not encoded in germ-line). The stage would then have been set for the creation of the new mechanisms of clonal elimination and helper T cells for antibody production. Anti-self antibody clones that escaped the mechanism of clonal elimination would not be produced in a large amount without help from T cells. M H C class II molecules may have originated from constant domains of antibodies or T-cell receptors by duplication of complementarity. O n e possibility is that the inter-chain complementarity might have been used in combination with combining sites, with the latter solely responsible for antigen binding (i.e. two binding sites on single receptor). Parallel evolution between M H C and T-cell receptors 29 could have occurred by inter-chain complementarity. However, as I have already discussed, two separate complementarity regions may overlap. The mechanisms to suppress antiself clones may have been further reinforced by the emergence of suppressor/cytotoxic T cells, with M H C class I antigens derived from M H C class 2 molecules. A phylogenetic tree of this scheme is shown in Fig. 2. The evolution of i m m u n e system may have been completed in the primitive fish about 500 million years ago, owing to the efficient mechanisms of duplication of complementarity. In summary, I suggest that the guiding force for the evolution of the Ig superfamily is the duplication of interdomain complementarity. This explains the structural similarity of the family members and provides an alternative view of the evolution of M H C antigens, namely that they may have been derived from Igs.

Note added in proof suggestedthat the T-cell receptor may use complementaritylocated outsidethe putative combiningsitewhenit interactswith the MHC antigen31. The T8 antigen is another member of the Ig superfamilya~, whereas the T3 antigen does not seemto be 33. AfterthesubmissionofthispaperPattenetal.

Acknowledgement I thank Dr SusumuOhno for discussionsduring the preparationof this manuscript.

References 1 Peterson,P. P., Cunningham,B. A., Berggard, I. and Edelman,G. (1972)Proc. NatlAcad. Sci. USA 69, 1697-1701 2 Jensenius,J.C. andWiltiams, A.F.(1982)Nature(Lond.)300,583-588 3 Ploegh,H. L., Orr, H. T. andStrominger,J.L. (1981)Cell24, 287-299

4 Kaufman,J. F., Auffrey,C., Korman,A.J., Shackelford,D. A. and Strominger,J. (1984)Cell 36, 1-13 5 Yanagi,Y., Yoshikai,Y., Legget, K., Clark, S. P., Aleksander,I. and Mak, T. W. (1984)Nature (Lond.) 308, 145 149 6 Hedrick,S. M., Nielson,E. A., Kavaler,J., Cohen,D. I. and Davis, M. M. (1984) Nature (Lond.) 308, 153 158 7 Solski,M.J., Uhr,J. W., Flaherty,L. andVitetta, E. S. (1981)J. Exp. Med. 153, 1080-1091 8 Kress,M., Cosman,D., Khoury,G. andJay, G. (1983)Cell34, 189-196 9 Cohen,F. E., Novotny,J., Sternberg,M. J., Campbell,D. G. and Williams,A. F. (1981)Biochem.J. 195,31-40 10 Mostov, K.E.,Friedlander, M. andBlobel, G.(1984)Nature(Lond.)308, 37-43 11 Mostov,K. E., Kraehenbuhl,J.P. andBlobel,G. (1980)Proc. NatlAmd. Sci. USA 77, 7259-7261 12 South,M. A., Cooper, M. D., Wollheim,F. A., Hong,R. and Good, R. A. (1966)J. Exp. Med. 123,615-627 13 Williams,A. (1984)Nature(Lond.)308, 12 13 14 Eskeland,T. and Brandtzaeg,P. (1974)Immunochemistry 11, 161 163 15 Ohno,S., Epplen,J. T., Matsunaga,T. and Hozumi,T. (1981)Prog. Aller~7 28, 8-39

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16 Amzel,L. M. and Poljak, R. J. (1979)Ann. Reo. Biochem. 1979, 48, 961-997 17 Ohno, S. Matsunaga,T. and Lee, A. D. (1984)&and. J. Immunol. 20, 997-1008 18 Kazim,A. L. and Atassi,Z. (1981)Biochem.J. 187,661-666 19 Doherty, P. C., Blanden, R. V. and Zinkernagel, R. M. (1974) Transplant. Rev. 29, 89-124 20 Jerne, N. K. (1974)Annal. Immun. (Inst. Pasteur) 125C, 373-384 21 Leslie,R. G. Q. (1982)Immunol. Today, 3,265-267 22 Johnson,P. M. and Brown,P. G. (1981)Placenta2,355-370 23 Reinherz,E. L., Meuer, S. C. and Schlossman,S. F. (1983)Immunol. Today 4, 5-9 24 Scofield,V. L., Schlumpberger,J.M., West,L. A. andWeissman,I. L. (1982)Nature (Lond ) 295,499-502 25 Burnet,F. M. (1971)Nature(Lond.) 232,230-235

)

26 Shinohara, N., Sachs, D. H., Nonaka, N. and Yamamoto, H. (1981) Nature (Lond.) 292,362-363 27 Williams, A. F. and Gagnon, J. (1982) Science216,696-703 28 Matsunaga, T. and Ohno, S. (1980) The Immune System: Festschrift in honor of Niels Kaj Jerne on the occasion of his 70th birthday, 1, 76 80, Karger, Basel 29 Jerne, N. K. (1971)Eur.J. Immunol. 1, 1-9 30 Hill, R. L., Delaney, R., Fellows, R. E. and Lebovitz, H. E. (1966)Proc. NatlAcad. Sci. USA 56, 1762-1769 31 Patten, P., Yokata, T., Rothband,J., Chien, Y-H., Arai, K. and Davis, M. M. (1984)Nature (London)312, 40-45 32 Littman, D. R., Thomas, Y., Maddon, P . J . , Chess, L. and Axel, R. (1985) Cell 40,237-246 33 Van den Elsen, P., Shepley, B. A., Boist, J., Coligan, J. E., Markham, A. F., Orkins, S. and Terhorst, C. (1985) Nature (London)312,413-418

New directions in research

Is fl2-microglobulin required for MHC class I heavy chain expression?

It is generally accepted that fl2microglobulin (//~m) is required for the cell surface expression of all M H C class I heavy chains, in a manner analogous to the requirement of light chains for I g M heavy chain expression 1. This belief is based on studies showing the failure of the h u m a n D a u d i and murine R1 cell lines to express class I heavy chains on their surface even though they are clearly detectable in the cytoplasm 2'3. In both cases, this lack of expression has been clearly linked to an absence of fl2m synthesis whichresults from defective/32m genes 4'5. Furthermore, it has been shown that in a mutant cell line that expresses a form of H L A - A 2 that does not bind fl2m, the H L A - A 2 is neither expressed on the cell surface nor undergoes oligosaccharide processing 6 analogoustoths H L A heavy chains of Daudi cells 7. (It had previously been shown that glycosylation is not necessary for H L A (Ref. 8) or H-2 (Ref. 9) class I heavy chain expression.) Even X e n o p u s oocytes, which faithfully reproduce the intracellular transport of h u m a n class I heavy chains, require h u m a n fl2m to translocate class I heavy chains from the endoplasmic reticulum to the trans-Golgi compartment m. The requirement for/32m is probably due to the fact that without it class I heavy chains adopt random conformations l* that do not have appropriate recognition structures for proper intracellular transport. However, data have now been reported which challenge the necessity of/~2m for class I heavy chain expression .2'.3. These studies showed that unlike the parent cell line, EL4/NY, a mutant cell line E L 4 / M a r did not express the H - 2 K b antigen but retained expression of the H-2D b antigen. Nevertheless, using F A C S analysis, microcytotoxicity testing and immunoprecipitation of a surface-labeled cell lysate,//2m could not be detected on the mutant cell surface .2'13. Northern blot analysis showed that no H - 2 K b m R N A was made but there was m R N A for H - 2 D b and fl2m. In fact, it could be demonstrated that//2m protein was present inside the mutant cell. The authors concluded that 'the apparent

absence of//2m on the surface of the cell line suggests that there may be some feature of the H-2D b molecule that allows it to be expressed in the absence of detectable //2m '13. They speculated that perhaps the glycosyl unit attached to the C2 domain .4 could replace the requirement for/J2m. Potter et al. 12.13 apparently do not favor the possibility that the fl2m m a y dissociate rapidly from the H-2D b heavy chain on the cell surface and, hence, m a y have been undetectable in their investigations. However, considerable data supports this possibility. Initial work '5 on the characterization of the H-2D b molecule clearly demonstrated a very low avidity of the D b heavy chain for /32m. H-2D b precipitated from cell lysates which had been stored frozen had no associated/32m whereas material precipitated from fresh lysates had 15% of the anticipated amount of//2m. Similarly the H-2L d molecule, which is identical in amino acid sequence to H-2D b (Ref. 16) in the fl2m binding domain (C2) 'y, often yields low or undetectable amounts of f12m upon immunoprecipitation 18a9. However, others have clearly demonstrated that H-2L a is capable of binding ]~2m (Ref. 20). It is unlikely that the dissociation of H-2D u or L d from flzm results from antibody displacement during immunoprecipitation since freshly prepared H-2D b clearly has//2m associated with it 15. Therefore, since the association of H-2D b and//2m is quite labile compared with other class I molecules, it seems that//2m may be bound to the H-2D u heavy chain during transport to tile cell surface but that it rapidly disassociates at the cell surface. A high concentration of intracellular fl2m (Ref. 10) or other factors may enhance intracellular association until transport to the surface is completed. Several other points brought up by Potter et al. ,2,13are worth discussing. F r o m experiments with serum-free media, they state 13 that the failure to detect murine//2m on the E L 4 / M a r cell surface cannot be explained by its displacement with bovine //2m present in fetal calf © 1985,ElsevierSciencePublishm~B.V., Amsterdam 0167 4919/85/$02.00