IMMUNOGLOBULIN/FIBRONECTIN SUPERFAMILY

    One of the first problems that animals had to solve was how to be multicellular.  In a multicellular organism, cells must interact with each other.  Once primitive animal cells began to express the immunoglobulin domain on their cell surfaces, other cells expressing the same (or slightly modified) domains could interact with them.  As vertebrate animals began to live longer (and put off reproduction until later in life), the issue of distinguishing between one’s own cells and foreign cells became ever more important and important families of immunoglobulins within the immunoglobulin superfamily evolved such as MHC  proteins, antibodies, and T-Cell receptors.

   A very early duplication of ancestral immunoglobulin/fibronectin molecules produced fibronectin and immunoglobulin families.  The functional domains of each of these molecules became incorporated into a variety of multi-domain proteins which can have single or multiple fibronectin domains and single or multiple immunoglobulin domains.  There are many molecules (such as the CAMs, contactin, nephrin, myomesin, MERTK, PUNC, TIE2, ROBO1, CRLF1, contactin3) which contain both fibronectin and immunoglobulin domains (depicted in green below).

   A number of proteins in the fibronectin branch of this superfamily have been adapted to complex immune reactions.  Macrophage mannose receptors allow macrophages to recognize carbohydrates and lymphocyte antigen 75, which is expressed in a number of tissues, is used in antigen presentation by dendritic cells (Online Mendelian Inheritance in Man).

     The immunoglobulin branch of the immunoglobulin/fibronectin superfamily includes genes known in both vertebrates and invertebrates, including the simplest invertebrates.  An immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase in some sponges (Schacke, 1994). Two Ig-like molecules are known from fruit flies, another from squid, and from sponges (the C2 set of Ig domains) (Schacke, 1994a).

     Immunoglobulins perform a variety of functions in the human body including roles in the nervous system (such NCAM, the neural cell adhesion molecule, homologs of which exist in insects and the mollusk Aplysia) and the placenta (such as the family of pregnancy specific glycoproteins, PSG, whose mutations can cause complications in pregnancy).  Some immunoglobulin genes are expressed by leukocytes and other cells as well.  PECAM, for example, is expressed in plateletes, monocytes, neutrophils, and some T cells and can interact with collagen.  It is also expressed  in endothelial cell junctions which may contain about a million molecules of PECAM (OMIM).      Basigin is the protein which composes the OK blood group.   It is widely expressed and mutations in mice cause infertility and abnormalities of the CNS (in memory and sensation).  It is possible that primordial members of the immunoglobulin family were similar to basigin (Miyauchi, 1990).

     Included in the wide variety of human cells which express immunoglobulins are white blood cells which function in innate immune responses.  For example, Paired Immunoglobulin-Like Receptor a is expressed on monocytes, granulocytes, and dendritic cells while FCGR1A, FCGR1B, and FCGR1C are expressed on macrophages and monocytes.  CD47 is involved in the increased calcium concentration in the cytoplasm of cells once they have bound to the extracellular matrix.  Macrophages require this protein to fuse in order to become osteoclasts.  It is critical as a marker of self for red blood cells.  Red blood cells lack MHC proteins and would be destroyed by macrophages at the spleen and natural killer cells if they did not express CD47  (OMIM).

     Although acquired immunity is considered to be a characteristic of vertebrates, many higher invertebrates possess humoral proteins called agglutinins which function in the immune response by can clump foreign cells together. (Hoar, 1983).  Although tunicates lack acquired immunity, they do possess genes which participate in the acquired immune responses of vertebrates such as complement proteins, lectins, 2 interleukin receptors, opsonins, agglutinins, cytokines, and hemolysins. (Burighel, from Harrison, 1997, p. 269; Dehal, 2002).

ACQUIRED IMMUNITY

     The acquired immunity of higher vertebrates such as ourselves involves incredibly complex interactions between immunoglobulin bearing cells, such as T cells, B cells, and natural killer cells.  It also involves the ability to distinguish between self and nonself, which is mediated through other members of the immunoglobulin superfamily, the MHC proteins (major histocompatibility complex).  The components of these complex immune systems have simpler homologs in invertebrates and primitive vertebrates.

     Sponges can distinguish between self and non-self in that a sponge can reject a graft made from another sponge.  Wandering phagocytes combat microbes in sponges and some flatworms, indicating that a primitive immune resistance predated the evolution of circulatory systems.  Invertebrates do not have a lymphatic system or any aspects of acquired immunity, but certain parts of their immune systems do display characteristics observed in vertebrates.  Although there is no complement system, there is a prophenoloxidase system which involves a cascade, including enzymes which kill microbes and clot blood.  Although invertebrates do not possess antibodies, there are molecules called lectins (found in bacteria, plants, and all animals) which can cause foreign cells to clump by binding to sugar groups.

In tunicates, both macrophages and morula cells participate in the rejection of foreign cells.  The numbers of morula cells can increase four times within the first 2 hours of the reaction.  Morula cells are similar to vertebrate lymphocytes in this feature and in some morphological characterisitics (Rinkevich, 1998).

     Lectins are a group of cell surface proteins which, like the immunoglobulin domain, have been utilized by many animals for immune reactions  (Hofer, 2001).   Sponges possess lectins homologous to those found in vertebrates (Gamulin, 1994; Schacke, 1994a). In invertebrates, lectins bind to foreign particles and facilitate phagocytosis (as do antibodies in vertebrates).  The human protein collectin (related to the lectins) coats foreign particles before phagocytosis, just as antibodies do (OMIM).    Mannose-binding lectin (MBL) is produced by liver hepatocytes and functions in the blood (hepatocytes are pictured below). 

MBL can bind Staphylococcus aureus, many Streptococcus, E. coli, Klebsiella, Haemophilus influenzae type b, yeast, other fungi, and viruses such as  HIV, IAV, and RSV.  Although MBL binds to microbial  monosaccharides which also exist in higher animals, these monosaccharides do not occur in the repetitive manner in vertebrates in which they are found in many microbes.  Cancer cells and virally infected cells can change their cell surface sugars and MBL can bind to these cells and inhibit tumor growth.  Deficiency of MBL in humans lessens complement binding to microbes,  Thus, MBL behaves in a way similar to antibodies in many respects (Lu, 2002; Gadieva, 2001).  

          Transplantation reactions occur in urochordates.  Urochordates express a gene homologous to natural killer cell receptors on some of their blood cells.  In fact, this receptor (CD94) is considered a marker for natural killer cells in humans (Khalturin, 2003).  Nonspecific cytotoxic cells (NCC cells) in teleosts and amphibians are equivalent to natural killer cells in mammals and seem to possess evolutionarily conserved receptors (Harris, 1991).   Natural killer cell receptors are encoded in a leukocyte receptor cluster region (LRC) of mammalian chromosomes.  Teleost fish have homologs of mammalian natural killer cell receptors (C-type lectin receptors) that are located in a homologous gene cluster (Yoder, 2001; Sato, 2003).

OPSONIZATION

     The antibodies of higher vertebrates function in opsonization.  When antibodies bind to a cell or molecule, it signals white blood cells to perform phagocytosis.

Antibodies are not the only molecules which can serve as opsonins; lectins are the primary opsonins in tunicates.  Lectins also perform opsonization in mollusks and arthropods (Pearce, 2001).  In addition to attracting white blood cells through opsonization, lectin-bound microbes attract other factors in the innate immune system, the complement proteins.  (Lu, 2002).

COMPLEMENT

     In higher vertebrates, antibodies which have bound to a microbe’s cell membrane can initiate the response of a cascade of blood proteins, referred to as complement proteins, which can generate a hole on the microbial membrane and kill it.  This complement cascade is an important component of adaptive immunity.  All gnathostomes possess adaptive immunity while jawless fish lack it.  Although the complement system is an important component of adaptive immunity, it also functions in innate immunity.  Jawless fish possess complement proteins which function in the lectin pathway.  Cartilaginous fish were the first vertebrates to develop a classical pathway of complement proteins and this pathway had developed components similar to the mammalian pathway by the evolution of bony fish.

     Complement pathways involve enzymes called proteases which cleave inactive proteins to produce their active forms.  Hemolymph coagulation is part of innate immunity in many invertebrates.  Horeshoe crab proteases involved in the coagulation cascade possess homologous sequences to those found in complement cascades.  Some ectotherms have duplications of complement genes and a more elaborate innate set of immune mechanisms while mammals have elaborated their adaptive immune mechanisms (Zarkadis, 2001).

     The complement system is thought to have evolved from a simple mechanism similar to that found in lectin and alternative pathways. 

Three proteins might have functioned in this system: a C3-like protein (magenta, in the above drawing), a serine protease similar to factor B (green), and a complement receptor on immune cells (red).  Complement proteins (factor B and C3) are known in echinoderms and complement-like proteins are known in more primitive invertebrates.  The complement system seems to have evolved from a simple pathway involved in opsonization before the evolution of antibodies (Zarkadis, 2001).  C3 is the main component in all 3 complement pathways.  In all three pathways, it is cleaved (by C3bBb in the alternative pathway and C4bC2a in the classical and lectin pathways) into two fragments, and exposes its thioester region which binds the target molecule (Nakao, 2003).   The thioester bond of C3 can bind to microbes serving in opsonization and as the site for the late complement proteins (C5 through C9 to generate a membrane pore) (Fujita, 2004). 

    Although primitive invertebrates possess the domains used by the complement system, a complement system did not evolve until the deuterostomes.  Primitive deuterostomes evolved the components of the complement cascade and the genome duplications early in the history of the vertebrates allowed the integration of several related pathways (Fujita, 2004).

The complement pathway can also be activated by the serum mannose binding lectin (MBL, a C-type lectin) to the carbohydrates of microbes.  The serine protease MASP (MBL-associated serine protease) activates complement C3 which can either result in opsonization or the assembly of the complement factors which put a hole in the microbial membrane (Vasta, 1999).

     Sponge molecules possess the SCR/CCP domains which are found in complement proteins (Zarkadis, 2001).  In primitive deuterostomes, it is not obvious that the lectin and alternative pathways have different functions (Zarkadis, 2001).  Elements of the lectin pathway, MBL and MASP, are known since protochordates.  Elements of the alternative pathway, C3 and factor B, are known in echinoderms and factor D in bony fish. (Zarkadis, 2001; Lundqvist, 1999;Pearce, 2001). The sea urchin protein SpBf is a complement protein which possesses SCR domains, a von Willebrand factor domain, and a serine protease domain.  Sea urchins possess complement C3 proteins which seem to function in opsonization and whose levels increase in response to infection.  Sea urchin proteins possess a thioester region, which in vertebrate complement proteins C3, C4, and C5, is the region which are exposed and bind target molecules in complement activation.  Some insect proteins have molecules similar to complements with thioester regions (Smith, 2002). 

     Urochordates possess proteins pertaining to both the alternative pathway (C3, Bf) and lectin pathway (MBL, MASP).  Although tunicates lack acquired immunity, they do possess complement genes, lectins, and 2 interleukin receptor genes which probably function in innate immunity (Dehal, 2002).  Tunicates seem to have homologs of complement receptors and complement proteins are known to mediate phagocytosis in bony fish (Zarkadis, 2001).

       The lectin-based opsoinzation pathway seems to be the original complement pathway.  Tunicates seem to have a minimal complement system involving a lectin which binds to serine proteases (forming GBL-MASP complex), C3, and a C3 receptor on blood cells (Fujita, 2004).  Complement proteins α2M/C3/C4/C5 form a gene family.  Tunicates have 2 molecules similar to α2M and two which are similar to C3.  They also possess 3 linked Bf genes, nine ficolin-like molecules, and 2 C1q like molecules (Fujita, 2004).

     Hagfish possess a protein CLP (complement-like protein) which functions in immune defenses is structurally similar to a mammalian complement protein (Hanley, 1992).  Lampreys possess homologs of C3, MASP, factor B (which has 3 SCR domains like those of gnathostomes and unlike sea urchins which have 5), and probably possess complement receptors.  Both hagfish and lamprey C3 function in opsonization (Zarkadis, 2001).  Hagfish possess a protein CLP (complement-like protein) which functions in immune defenses is structurally similar to a mammalian complement protein (Hanley, 1992).  In mammals, the C1q complement binds immunoglobulin in the classical pathway.  Lampreys possess C1q, apparently as a part of the innate reaction (Matsushita, 2004).  Classical and lytic pathways not known in lampreys (Fujita, 2004).

     Sharks possess homologs of C3, C4, and MASP, (Zarkadis, 2001).  Of the classical pathway which involves C1, C2, and C4, C4 is known in sharks and bony fish.  Of the terminal lytic proteins C5-C9, C5 and C8 are known in sharks (Zarkadis, 2001). Factors C6, C7, C8, and C9 form a gene family (Fujita, 2004).  Complement factors C1r and C1s bind to form a heterotetramers with two subnits of each enzyme (Presanis, 2003).

     In teleosts, many of the complement proteins genes are duplicated resulting in a greater functional diversity than possessed in mammals (Nakao, 2003; Zarkadis, 2001).

     Most of the components of the classical complement pathway have paralogs which function in the lectin or alternate complement pathways, suggesting that large-scale gene duplications led to three pathways derived from a single ancestral pathway (Fujita, 2004).

The gnathostome complement pathways were established before the branching of cartilaginous fish from the other lineages.  The C1q which binds antibodies in the classical pathway is a lectin homolgous to those which activate the lectin pathway (Fujita, 2004).

    It seems that all of the genes of the natural killer cell complex have evolved from duplications of a primordial gene with a C-type lectin domain (such as that of the hepatic asialoglycoprotein receptor).  On human chromosome12p, there is a 2 Mb region which contains at least 18 genes for lectin-like receptors,   In this gene family, there is a subfamily of lectin-like genes which are expressed in monocytes, dendritic cells, and/or endothelia.  Thus the receptors required for NK function have functions outside NK cells (Hofer, 2001). 

MHC

     Acquired immunity is only found in vertebrates and depends on B and T cell receptors.  While the immunoglobulin receptors on B cells can recognize proteins, receptors on T cells recognize peptides which are attached to MHC molecules.   In general, MHCI proteins (composed of MHCIa and b2 microglobulin chains) bind peptides in the cytoplasm.  These peptides are produced when proteins are broken down in the cytoplasm and taken to the endoplasmic reticulum where they bind the MHC proteins.  The MHC I proteins “present” these peptides on the cell membrane, predominantly to cytotoxic T cells.  MHCII proteins (which are heterodimers composed of a and b chains) bind peptides in lysosomes and endosomes.  These peptides are typically obtained from the breakdown of foreign proteins, originating outside the cell.  MHCII proteins present their peptides to T Helper cells.   The MHC proteins are encoded by HLA genes (human leukocyte antigens) and have changed little in structure during their evolution.  b2 microglobulin can function by itself as a chemotactic factor and may have evolved before the MHC proteins. 

ANTIBODIES AND T CELL RECEPTORS (TCRs)

      One of the characteristics of jawed vertebrates is the possession of adaptive immunity which allows them to predict a great diversity of potential antigens to defend against, attack antigens with specific humoral proteins, and retain a memory of the antigens they have encountered.  In addition to the MHC and complement factors already considered, this system depends on two novel types of immunoglobulin known only in gnathostomes: antibodies and T cell receptors.  These receptors are common in the human lymphocytes pictured below.

     Lymphocytes can generate a diversity of 10¹⁶ different receptors and each lymphocyte carries 10⁵  copies of one receptor (Laird, 2000).  Where did these complex proteins come from?  Antibodies and T cell receptors are assembled from smaller chains, known as constant (C), variable (V), diversity (D), and joining regions (J).  Some of these components evolved long before antibodies and T cell receptors.

Variable immunoglobulin domains are known in cell surface molecules of plants and fungi in addition to a diversity of animals, including the most primitive animals, the sponges (Muller, 2001; Muller, 2001a).  The V and C regions of immunoglobulins resulted from the duplication of an ancestral region of about 110 amino acids (Schluter, 1997).  The ancestral Ig/TCR receptor probably had a V domain and a C domain. The C1 domain of antibodies and TCRs is known in MHC molecules I and II, tapasin, and SIRPS (Du Pasquier, 2004). 

An antibody is depicted in the following illustration.

     The J chain of vertebrate antibodies is thought to function in the dimerization of antibody subunits and their transport.  The gene for the J chain is expressed in a number of protostome invertebrates as well as in diverse groups of vertebrates.  In invertebrates, it is expressed on macrophage-like blood cells and epithelial surfaces.  Since these animals lack antibodies, it appears that the J chain functions in other capacities as well (Takahashi, 1996).  The J chain is expressed one week before the mu chain in human fetal development, consistent with the suggestion that it serves multiple functions (Takahashi, 1996).

     Vertebrates possess two types of immunoglobulin which are thought to be similar to the ancestral immunoglobulin which gave rise to antibodies and TCR genes, the JAM/CTX and the nectin/poliovirus receptor (PVR) families.  These proteins serve as cell adhesion molecules and virus receptors (such as polio, Coxsackie virus, and reoviruses).  Apparently, one ancestral pair of these genes was linked and subsequently duplicated to produce the four paralagous linkage groups known in the human genome.  JAM, CTX, and nectin molecules can form dimers, as can the subunits of antibodies and T cell receptors (Du Pasquier, 2004).  Drosophila possesses a protein with V and C chains showing homology to nectin (Du Pasquier, 2004). Tunicates, among the most primitive chordates, possess JAM, CTX and PVR genes which possess both a V and a C domain (Du Pasquier, 2004).

     Jawless fish have no organized lymphatic tissue and lack both a spleen and thymus.  Lampreys and hagfish seem to lack antibodies, T Cell receptors, MHC proteins, and RAG. (Zarkadis, 2001).

They do, however, possess serum heterodimeric proteins which resemble both antibodies and T cell receptors (Varner, 1991).  These receptors are formed from two different heavy chains attached to two different light chains, unlike the antibodies and TCRs of vertebrates.  While hagfish can reject foreign skin grafts, they do so with very modest increases in the amount of antibody-like moelcules in their blood (0.3% of serum protein compared to 50% in sharks) (Varner, 1991).  T cell receptors are depicted below.

Hagfish also possess proteins recognized by anti-IgM antisera (which bind IgM antibodies of higher vertebrates (Rumfelt, ).

     Lamprey blood contains cells which are morphologically indistinguishable from mammalian lymphocytes although no MHC, T cell receptor, or antibodies are known from lampreys.  These cells express genes associated with mammalian lymphocytes and occur in tissues (such as the intestines) where lymphocytes are common in higher vertebrates (Mayer, 2002).  Lymphocyte-like cells in lampreys are small with little cytoplasm and produce lymphocyte transcription factors (Spi and Ikaros), CD45, BCAP, and CAST (which in mammals are primarily expressed in lymphocytes), CD98 and CD9 (which mammals use in lymphocyte proliferation and migration), proteasome subunits (PSMB4, PSMB7, 26S subunit pUb-R3, PSMA2, PSMA6, and PSMF1), and ABC9 (similar to the ABC proteins which mammals use to transport peptides to the MHC)., the complement protein C1q, and a number of other genes expressed in mammalian lymphocytes (hepsin, sygin 2, RAMP4, and talin) (Mayer, 2002).  Lampreys not only have a Spi gene (a gene which is specific to the differentiation of lymphocytes in higher vertebrates), it is expressed on the surface of the lymphocyte-like cells (Shintani, 1999). 

     In addition to antibodies such as IgM and IgW, sharks synthesize the antibody-like molecules IgNARC (new antigen receptor in cartilaginous fish) and NAR (new shark antigen receptor) (Schluter, 1997).  The NAR immunoglobulin (new antigen receptor) known in nurse sharks functions as an independent molecule without forming a dimer with other immunoglobulins.  Its variable region seems to be equally related to both antibody and TCR variable regions and may be derived from the ancestor of both (Roux, 1998).  Sequence comparisons suggest that NAR  separated from the antibody group of molecules before the classes of antibodies were produced.  (Wilson, 1997)  One set of fish proteins are referred to as NITRs or novel immune-type receptors.  NITRs may be similar to the ancestral antigen receptor given that they possess a V, J, and C domain (Kasahara, 2004). 

     In sharks, B cells make 4 classes of antibody, including IgM, which humans also produce.  Shark antibodies can be secreted and can function on the cell membrane.  While higher vertebrates create antibody diversity by possessing large numbers of antibody parts in gene clusters which will be randomly shuffled 50 V, 30 D, 6 J, 8 C), sharks possess more than 100 clusters which do not contain this diversity (1 V, 2 D, 1 J, 1 C) and are not reshuffled (Hohman, 1993).   To express it another way, sharks possess (V_H-D-D-J_H-C_H)_n while bony fish and tetrapods possess (V_H)_n-(D)_n-(J_H)_n-(C_H)_n.  (Magor, 1999). 

The antibody light chains in sharks and teleost fish form a number of clusters which share a V-J-C organization (Pitstrom, 2002).  One type of immunoglobulin in sharks, IgW displays characteristics which might be expected from a primordial immunoglobulin (Berstein, 1996)  In addition to IgM, lungfish possess immunoglobulins similar to the IgW previously known only in sharks.  Thus, IgW and IgM must have duplicated early in the evolution of gnathostomes (Ota, 2003).

     Both B and T cells are known in bony fishes.

While the shuffling of the components of immunoglobulins are essential to the aquired immunity of higher vertebrates, immunoglobulins which aren’t shuffled are also capable of immune function in vertebrate groups ranging from sharks to mammals.  Mammals possess families of receptors such as paired Ig-like receptors (PIR), Ig-like transcripts (ILT; also known as leukocyte inhibitory receptors LIR), and killer inhibitory receptors (KIR) which can interact with MHC I and II proteins.  These Ig-like molecules do not rearrange themselves (like Ig and TCR receptors) yet they function in immune responses (Laird, 2000).

     In summary, the complex immune reactions of higher vertebrates depend on a variety of genes in the immunoglobulin and lectin superfamilies.  Both of these gene families were present in the most primitive animals, as were phagocytic cells and some ability to distinguish between self and nonself.  Many of the molecular components required for the acquired immunity of higher vertebrates are present in invertebrates (especially primitive chordates) which lack acquired immunity.  Many of the components of the complex humoral and cell mediated reactions of higher vertebrates are present in jawless and cartilaginous fish whose immune responses are simpler than those of higher vertebrates.   The most complex aspects of the human immune system supplement, rather than replace, other components which are homologous to the immune mechanisms of more primitive animals (such as the fibronectin-containing macrophage mannose receptors, lectin-containing receptors, and immunoglobulin-like receptors whose components are not rearranged).