IMMUNOGLOBULIN EVOLUTION: CHEMICAL STUDY OF CLAWED TOAD (XENOPUS LAEVIS) HEAVY AND LIGHT CHAINS

NH₂, terminal amino acid sequence determinations of clawed toad (Xenopus laevis) immunoglobulins indicate that approximately 30% of the heavy chains and less than 5% of the light chains have unblocked NH₂ termini. The major amino acid sequence of the X. laevis 7S immunoglobulin heavy chains is the same as that of the 19S immunoglobulin heavy chains. Thus in the synthesis of the heavy chains, the V_H genes coding for unblocked heavy chains can associate with C_H genes of both the 19S and 7S classes. This association is particularly important in amphibians because, in contrast to mammals and birds, the majority of amphibian antibody-producing cells synthesize both 19S and 7S immunoglobulins and do not participate in the ‘genetic switch’ characteristic of lymphocyte differentiation in higher organisms. In X. laevis, the major amino acid sequence at the first twenty-four positions of the unblocked heavy chains shows approximately 54% difference from the prototype amino acid sequence of the mammalian V_HIII subgroup. Thus, the V_HIII gene(s) must have started to appear after the evolutionary divergence of the common ancestor of mammals and birds from the amphibian line. The amino acid composition of the X. laevis 7S immunoglobulin heavy chains differs from that of its 19S immunoglobulins as well as those of human IgG and IgA. These data support the concepts (a) that amphibian 7S and 19S immunoglobins belong to distinct classes and (b) that amphibian 7S immunoglobulin does not resemble mammalian IgG or IgA.     

Immunoglobulin gamma chains of a monotreme mammal, the echidna (Tachyglossus aculeatus): amino acid composition and partial amino acid sequence.

The echidna represents the lowest stage in phylogeny at which molecules clearly homologous to IgG antibodies appear to occur. We provide evidence that a fraction of gamma chains possess an unblocked N terminal sequence comparable to the VHIII sub-group of human gamma chains and that glycine is the C-terminal residue. Statistical comparison of amino acid composition of the component chains with other immunoglobulin heavy chains suggests that echidna gamma chains are more closely related to eutherian gamma chains than to the 7S Ig heavy chains from amphibia or aves. The results are consistent with our view that true gamma-type heavy chains did not appear in evolution until after the mammalian line diverged from the stem reptiles.     

IMMUNOGLOBULIN GAMMA CHAINS OF A MONOTREME MAMMAL,THE ECHIDNA (TACHYGLOSSUS ACULEATUS): AMINO ACID COMPOSITION AND PARTIAL AMINO ACID SEQUENCE

The echidna represents the lowest stage in phytogeny at which molecules clearly homologous to IgG antibodies appear to occur. We provide evidence that a fraction of γ chains possess an unblocked N terminal sequence comparable to the V_HIII sub-group of human γ chains and that glycine is the C-terminal residue. Statistical comparison of amino acid composition of the component chains with other immunoglobulin heavy chains suggests that echidna γ chains are more closely related to eutherian γ chains than to the 7S Ig heavy chains from amphibia or aves. The results are consistent with our view that true γ-type heavy chains did not appear in evolution until after the mammalian line diverged from the stem reptiles.     

7S immunoglobulins of a monotreme, the Echidna Tachyglossus aculeatus: two distinct isotypes which bind A protein of Staphylococcus aureus

7S immunoglobulins of a monotreme mammal, the echidna Tachyglossus aculeatus, bind to Staphylococcus aureus A protein coupled to an insoluble matrix. This binding supports the homology between the previously described echidna IgG and IgG molecules of higher mammals and provides a rapid, simple method for the isolation of this protein from echidna serum. In addition, another echidna 7S immunoglobulin became bound to protein A-Sepharose. The major protein A-binding immunoglobulin has a slow electrophoretic mobility and possesses a heavy chain comparable in mass to typical mammalian γ chains (52,000 daltons). We suggest the designation IgG2 for this immunoglobulin. The minor protein A-binding immunoglobulin is electrophoretically faster than IgG2 and can be resolved from the former by gradient elution from DEAE-Biogel. We propose the nomenclature IgG1 for this molecule although we cannot discount the possibility that it might represent another main class such as IgA. The γ1 heavy chain is distinguishable from the γ2 chain on polyacrylamide gel electrophoresis in sodium dodecylsulphate containing buffers. The γ1 chain possesses a nominal mass of 61,000 daltons. The intact molecule has an apparent mass of 177,000 Daltons and comprises two pairs of light and heavy chains.     

PHYLOGENETIC EMERGENCE OF IMMUNOGLOBULIN CLASSES DISTINCT FROM IgM

The low molecular weight immunoglobulin from the marine toad (Bufo marinus) was found to differ markedly from classical mammalian IgG immunoglobulin. Higher intact molecular weight of 168,000 ± 4000 was the result of larger heavy chains (61,400 ± 2300) than mammalian γ chains (50,000 daltons). The light chains were of normal molecular weight (23,000). Polyacrylamide gel electrophoresis in SDS buffers was shown to be a simple and reliable way of determining the type of heavy chain present in immunoglobulins from different species, despite the fact that the mobility of the chains in this system does not accurately reflect their molecular weight. Using this technique it was shown that the heavy chain from the LMW Ig present in Xenopus laevis, an anuran amphibian, and the chicken, a representative of the birds, showed similar properties to the toad LMW Ig heavy chain. In addition, the LMW Ig heavy chain from the possum (Trichosurus vulpecula), a marsupial, showed properties which were characteristic of classical γ heavy chains. We conclude that the LMW Ig present in anuran amphibians does not belong to the IgG class as had been previously thought. This Ig class appears to have been retained in the reptiles and birds. The appearance of IgG appears to be a relatively recent event, recurring at a time after the divergence of the mammalian line from the stem reptiles.     

Studies on Xenopus laevis immunoglobulins

The anuran amphibian Xenopus laevis has been shown to produce two classes of antibodies to HGG, BSA and Hc. These antibodies were characterized by gel filtration on Sephadex G-200 as `19S'' and `7S'' immunoglobulins. In the course of immunization, antibody activity could be initially detected in the `19S'' immunoglobulin fraction, followed by the appearance of the activity in the `7S'' immunoglobulin fraction at a later stage of immunization. A switch-over from `19S'' to `7S'' activity was not observed. Both immunoglobulins were composed of heavy and light polypeptide chains. The `19S'' protein had heavy chains with a molecular weight of 74,500, similar to human μ-chain (73,900). The `7S'' protein differed from human IgG in respect to the molecular weight of its heavy chain which was shown to be 64,500. Light chains of both immunoglobulins of Xenopus were found to have a molecular weight of 26,700, similar to human immunoglobulin light chains (25,000).     

Amino acid sequence of the constant region of immunoglobulin light chains from Rana catesbeiana

Little is known about the detailed structure of immunoglobulins in non-mammalian vertebrates. We have determined the amino acid sequence of the constant region of immunoglobulin light chains from the bullfrog, Rana catesbeiana. There appears to be one major type of light chain in this species. However, at position 153, about half of the chains have lysine, the remainder having arginine. Variation at this same or at an adjacent position is responsible for allotypic or isotypic variation in human kappa and lambda chains. At three positions (118, 119, and 181), residues that occur in all known kappa and lambda chains in other species are replaced in the Rana catesbeiana sequence. One of these unusual substitutions--the replacement of proline by cysteine at position 119--allows the formation of an extra intrachain disulfide bond within the constant domain, between positions 119 and 214. This bond appears to replace the usual disulfide bridge between heavy and light chains so that, in the immunoglobulins of this species, the light chains are not covalently bonded to heavy chains. The sequence of the Rana catesbeiana constant region is compared to sequences of a variety of mammalian light chain constant regions. We consider the implications of these comparisons for the timing of the divergence of kappa and lambda chains relative to the divergence of the lineages leading to amphibians and to mammals and birds.     

Structural studies of the Xenopus 19S immunoglobulin and 7S immunoglobulin and two immunoglobulin-like proteins.

Xenopus laevis 19S and 7S immunoglobulins (Ig) were extensively reduced and alkylated, their H and L chains spearated and their molecular weights determined. Two kinds of L chains of molecular weight 25,000 and 27,000 were revealed by SDS-polyacrylamide gel electrophoresis. In addition two Ig-like proteins consisting of heavy chains only, of 19S H-type and with similar molecular weight, were detected in Xenopus serum ans isolated. These proteins share common antigenic determinants with Xenopus 19S Ig heavy chains and are devoid of light chain determinants.     

A biclonal origin of two monoclonal proteins, IgG3(kappa) and IgA1(lambda), from a single patient.

The cellulose acetate electrophoretic pattern of the serum from patient Sik disclosed two distinct peaks, representing two monoclonal proteins. On immunoelectrophoresis the two M-components were found to differ in heavy chain class as well as in light chain type, IgG3(kappa) and IgA1(lambda). Serum immunoglobulin levels remained relatively constant over a period of 7 years and no clinical symptoms of a malignant deterioration occurred. It was found that the isolated M-components did not share idiotypic antigenicity. Bone marrow cells synthesizing the monoclonal proteins were identified by means of the immunofluorescent technique using isotypic as well as idiotypic antisera. Two distinct monoclonal cell populations were observed, containing either the IgG3(kappa) or the IgA1(lambda) monoclonal protein. The alpha 1-chain belonged to the VHIII subgroup, whereas the gamma 3-chain was found to be blocked. Subsequent sequence determination showed the gamma 3-chain to belong to the VHIII subgroup. It was concluded that the two M-components in the serum of patient Sik resulted from two independent neoplastic transformations.     

Major immunoglobulin classes of the Echidna (Tachyglossus aculeatus)

The Australian echidna responds to the antigen Salmonella adelaide flagella by producing antibodies characterized by mol. wt of 900,000 and 150,000. After cleavage of interchain disulphide bonds, both the high and low mol. wt immunoglobulins can be resolved into light and heavy polypeptide chains. In both cases, the light chains resemble those of other vertebrate immunoglobulins in size (22,500 Daltons) and electrophoretic mobility. The 900,000 Dalton immunoglobulin contains heavy chains similar to human μ chains in size (70,000 Daltons) and electrophoretic mobility. The 150,000 Dalton immunoglobulin contains a different class of heavy chain, similar in size (50,000 Daltons) and electrophoretic mobility to human γ chains. Proportional mass contributions of the light and heavy chains to the intact molecule suggest the structure of the intact molecules could be represented by (L₂, μ₂)₅ and (L₂, γ₂) for the high and low mol. wt immunoglobulins respectively. These configurations are similar to those described for human γM and γG immunoglobulins.The results are relevant to theories of the evolution of the different classes of immunoglobulins. While the echidna is distinctly more primitive than eutherian mammals and still retains structural features characteristic of reptiles, its major immunoglobulin classes are very similar to human IgM and IgG. The striking similarities between the γ-like heavy chain of the echnidna and human IgG heavy chains suggest that the echidna may be the first species in which a γ chain gene directly homologous to mammalian γ chain genes is expressed.     

Immunoglobulins of the marsupial Setonix brachyurus (Quokka). Characterization of three major serum classes, IgG2, IgG1 and IgM

Three immunoglobulin classes, IgG2, IgG1 and IgM, have been identified in the serum of the marsupial Setonix brachyurus (quokka). Each of these classes has been isolated in pure form and partially characterized physicochemically. These immunoglobulins differed in their electrophoretic mobility, molecular size, carbohydrate content and in the antigenic determinants of their heavy chains. IgG2 exists in two major subclasses, one of which (IgG2a) was isolated as a homogeneous preparation. The results of immunoelectrophoretic analyses also suggest the likely presence of subclasses within the IgG1 class. This molecular complexity of the quokka humoral immune system is much greater than that of birds and ectotherms, but is comparable to that shown by eutherian mammals.     

Isolation and partial characterization of immunoglobulin from a urodele amphibian (Necturus masculosus)

The primitive amphibian species, Necturus maculosus, a urodele, possessed serum immunoglobulin characterized by a mol. wt of approximately 900,000. Upon reduction of disulphide bonds and analysis under dissociating conditions, this molecule was resolved into polypeptide chains resembling light chains and μ-type heavy chains and having mol. wt of 22,000 and 70,000 respectively. The Necturus immunoglobulin was antigenically related to the IgM-like immunoglobulins of the toad (Bufo marinus) and the Xenopus. Unlike these anuran amphibians, however, the Necturus did not possess detectable amounts of low molecular weight immunoglobulins.

The finding raises the evolutionary possibility that the γ-like heavy chains of advanced amphibians may represent the results of a gene duplication independent of that which was responsible for the emergence of the γ heavy chain of mammals.

     

Subclasses of porcine immunoglobulins G. Production of subclass specific antisera with S-sulfonated gamma chains

Using antisera made against porcine S-sulfonated heavy chains two subclasses of porcine IgG could be distinguished on the basis of antigenic determinants located on the Fc fragment. The antisera prepared against the porcine gamma chains reacted with other mammalian IgG proteins, but not with avian, amphibian or fish immunoglobulins. Antigenic determinants responsible for cross-reactivity are further localized on the Fc′ fragment of the porcine IgG molecule. Comparable antisera, specific for porcine light chains, did not cross-react with IgG from any of the other species tested. Porcine IgG and F(ab′)₂ fragment reacted with both the anti-heavy and anti-light chain sera, while the Fab fragment reacted only with the anti-light chain sera. Important antigenic determinants are thus located on the light chains and the Fc portion of the heavy chain of the porcine IgG molecule.     

Reassessment of the heavy chain deletion in human immunoglobulin Sac in the light of current theories of immunoglobulin gene assembly

IgG Sac is an immunoglobulin with large deletions in both heavy and light chains. Reexamination of the amino acid sequence of the γ chains of this immunoglobulin has led to the conclusion that the deletion in these chains, like that in the light chains, is the result of a pre-translational event, and not the result of in vivo proteolytic degradation. These chains differ from other γ heavy chain disease proteins in that the post-deletion normal sequence begins with the J segment rather than with the hinge region, and in that they are covalently associated with light chains.     

MOLECULAR PROPERTIES OF T LYMPHOMA IMMUNOGLOBULIN

Immunoglobulin (Ig) released into the medium by monoclonal continuously cultured murine T lymphoma cells of the lines WEHI-22 and WEHI-7 was isolated by serological precipitation or solid-phase immunoadsorption techniques. The intact immunoglobulin had an electrophoretic mobility on sodium dodecyl-sulphate (SDS) containing polyacrylamide gels comparable to that of IgG (mass 150,000). This mobility was significantly faster than that of ‘7S’ IgM of murine B lymphocyte surfaces. The T lymphoma immunoglobulin consisted of a pair of heavy chains linked by disulphide bonds and light chains non-covalently bound to the heavy chains. The isolated heavy chains migrated slightly faster than the μ chains of MOPC 104E IgM. Some, but not all, antisera directed against μ chains of normal mouse serum IgM bound T lymphoma immunoglobulin apparently via a cross-reaction localized to the F_d fragment. These data indicate that immunoglobulin of T lymphoma cells and, presumably normal T lymphocytes, represents an immunoglobulin isotype which is distinct from those immunoglobulins found on the B cell surface.     

Amino Acid Sequence Homology between HLA-A,B,C Antigens, β2-Microglobulin and Immunoglobulins

Papain-solubilized HLA-A,B,C antigen heavy chains have been cleaved by combined acid and CNBr treatment to yield three large fragments. A 14,000-dalton peptide comprises the NH2-terminal portion of the molecule, less a five-membered peptide. The 14,000-dalton fragment is followed in the linear sequence by a 9000-dalton peptide connected through an aspartyl-prolyl bond to the COOH-terminal 11,000-dalton fragment. The 9000- and 11,000-dalton fragments contain disulphide bridges that are immunoglobulin-like inasmuch as they encompass some fifty-five to sixty amino acid residues. The NH2-terminal portion of the HLA antigen heavy chain is devoid of cysteine. NH2-terminal amino acid sequence analyses do not reveal homologies between the 14,000- and 9000-dalton fragments, beta2-microglobulin, and the constant immunoglobulin domains. However, the NH2-terminal sequence of the 11,000-dalton fragment is as homologous to beta2-microglobulin and the constant immunoglobulin domains as they are to one another.     

Antigenic determinants of heavy chain variable regions: immumological typing of the human immunoglobulin VHIII subgroup.

An antigenic determinant of the VHIII variable region subgroup was defined by means of a heterologous specific antiserum using a hemagglutination inhibition procedure. The specificity of this antiserum was established in inhibition experiments with proteins either of known primary structure or belonging to a definite VH subgroup. A series of IgG, IgA, IgM and IgD monoclonal proteins was examined for the presence of this VHIII subgroup antigenic determinant. The data showed that 50% of the IgG, 62% of the IgA, 55% of the IgM and 41% of the IgD were VHIII-positive, and that certain "blocked" monoclonal immunoglobulins belonged to this subgroup. A preferential association of the VHIII antigenic determinant with the IgG1 and IgG3 subclasses was observed among IgG myeloma proteins while the preferential association was only observed with the IgG1 subclass when anti-Rh antibodies were studied. The VHIII subgroup exhibited nonallelic behavior.     

A novel IgA-like immunoglobulin in the reptile Eublepharis macularius

The appearance of antibody genes over evolution coincided with the origin of the vertebrates. Reptiles are of great interest in evolution since they are the link between the amphibians, birds, and mammals. This work describes the presence of a gene in the reptile leopard gecko (Eublepharis macularius) where phylogenetic studies suggest that it is the gene orthologue of immunoglobulin A (IgA) and immunoglobulin X (IgX) in Xenopus. Messenger RNA samples taken from different tissues showed expression of this antibody in intestinal tissue. Data on the structure deduced from the sequence of nucleotides showed an antibody with four domains in the constant region. There is a sequence of 20 amino acids in the C terminus similar to the secretory tail of immunoglobulin M (IgM) and IgA. A detailed analysis of the sequence of amino acids displayed a paradox, i.e., domains CH1 and CH2 showed a clear homology with domains CH1 and CH2 of immunoglobulin Y (IgY) while domains CH3 and CH4 were homologous with domains CH3 and CH4 of IgM. This homology pattern is also seen in Xenopus IgX and bird IgA. The most logical explanation for this phenomenon is that a recombination between the IgM and IgY gave rise to the IgA.     

Xenopus laevis 19S immunoglobulin. Ultrastructure and J chain isolation.

Electron microscopy examination of the 19S immunoglobulin of Xenopus laevis revealed a hexameric structure with a central core. The molecules measured 360-430 A across the span of the arms and the average diameter of the central region was 140 A. A polypeptide, homologous to human J chain, was isolated by chromatography on DEAE-cellulose from the reduced and alkylated X. laevis hexameric macroglobulin. This polypeptide had a fast mobility in alkaline-urea gel electrophoresis, with distinct antigenicity, as compared to heavy and light chains. It shared common antigenic determinants with human J chain.     

Secretory immune system of the duck ( Anas platyrhynchos ). Identification and expression of the genes encoding IgA and IgM heavy chains

IgA has not previously been identified in waterfowl. Studies instead revealed physical and antigenic similarities between duck bile immunoglobulin (Ig) and serum IgM. Here, a differential screening approach was used to clone, from a duck spleen library, the cDNA encoding the heavy (H) chains of IgM and the Ig, identified here as IgA, occurring in duck secretions. Phylogenetic comparisons of inferred amino acid sequences of entire H chain constant (C) regions and of individual domains revealed that the duck μ chain was closest to chicken μ (54 % overall identity), and duck α was closest to chicken α (50 % identity). Comparison of the μ and α C regions revealed areas of up to 65 % amino acid similarity within the C4 domains, accounting for the previously noted antigenic overlap of duck IgM and IgA. Messages for α and μ were detected in duck lymphoid organs but the α message was most abundant in the respiratory, alimentary and reproductive tracts. The α message first appeared around 14 days of age and reached adult levels of expression only at 35 – 50 days. The results indicate that the duck has a mucosal immune system which utilizes IgA; however, the delayed expression and secretion of duck IgA explains the susceptibility of ducklings to mucosal pathogens. Since the waterfowl are among the most primitive extant birds, the recognition of IgA in the duck supports the conclusion that IgA occurs throughout the class Aves and also existed in the common ancestors of birds and mammals.