Cloning and structural analysis of two highly divergent IgA isotypes, IgA1 and IgA2 from the duck billed platypus, Ornithorhynchus anatinus.

To trace the emergence of modern IgA isotypes during vertebrate evolution we have studied the immunoglobulin repertoire of a model monotreme, the platypus. Two highly divergent IgA-like isotypes (IgA1 and IgA2) were identified and their primary structures were determined from full-length cDNAs. A comparative analysis of the amino acid sequences for IgA from various animal species showed that the two platypus IgA isotypes form a branch clearly separated from their eutherian (placental) counterparts. However, they still conform to the general structure of eutherian IgA, with a hinge region and three constant domains. This indicates that the deletion of the second domain and the formation of a hinge region in IgA did occur very early during mammalian evolution, more than 166 million years ago. The two IgA isotypes in platypus differ in primary structure and appear to have arisen from a very early gene duplication, possibly preceding the metatherian eutherian split. Interestingly, one of these isotypes, IgA1, appears to be expressed in only the platypus, but is present in the echidna based on Southern blot analysis. The platypus may require a more effective mucosal immunity, with two highly divergent IgA forms, than the terrestrial echidna, due to its lifestyle, where it is exposed to pathogens both on land and in the water.

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes.

BACKGROUND: The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital. RESULTS: We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species. CONCLUSION: Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.

TCR gamma chain diversity in the spleen of the duckbill platypus (Ornithorhynchus anatinus).

TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.

High variability in complementarity-determining regions compensates for a low number of V gene families in the lambda light chain locus of the platypus.

Based on the analysis of a panel of variable (V) region sequences from the Australian duck-billed platypus and the Australian short beaked echidna, the monotremes were found to express a highly diversified Vlambda repertoire. High variability was observed both in sequence and in length of all three CDR regions. However, all monotreme sequences were found to form a separate branch on a distance tree, and the monotremes appear to express only two Vlambda gene families. The appearance of all Vlambda gene segments in one branch on the distance tree gives further support for the notion that deletions of entire V region clans or families, followed by successive rounds of gene duplications may be a relatively common phenomenon during vertebrate evolution. Four different constant region sequences were also identified and a preferential use of certain J segments to each constant region was observed. A more detailed picture of the locus was obtained by analysis of genomic DNA by Southern blot and PCR. The organization of the lambda locus involves multiple V and several constant region genes with one or several joining segments positioned upstream of each constant region, similar to the organization in mouse and man. An mRNA frequency analysis shows that the lambda light chain accounts for more than 90% of the light chain transcripts in the spleen. The abundance and the high variability indicate that light chain diversity at the lambda locus contributes significantly to the antigen-binding repertoire in monotremes. A high lambda to kappa light chain ratio also indicates that variability in the CDR regions is more important for the repertoire size than the total number of V gene families.

The complexity of expressed kappa light chains in egg-laying mammals.

Complementary DNAs encoding immunoglobulin light chains were isolated from two monotreme species, Ornithorhynchus anatinus (duckbill platypus) and Tachyglossus aculeatus (echidna). The sequences of both the variable and constant regions of these clones had greater similarity to IGK than to other light chain classes and phylogenetic analyses place them squarely within the mammalian IGK group, establishing them as monotreme IGK homologues. The constant region sequences of all clones were essentially identical within each species and, along with Southern blot results, the data are consistent with a single IGKC in each species. The expressed IGKV repertoires from both platypus and echidna were randomly sampled and there appear to be at least four platypus and at least nine echidna IGKV subgroups. The IGKV subgroups are highly divergent within species, in some cases sharing as little as 57% nucleotide identity. Two of the IGKV subgroups are present in both species, so there is some degree of overlap in the germline repertoires of these two monotremes. Overall the complexity seen in platypus and echidna IGK light chains is comparable with that of other mammals considered to have high levels of germline diversity and is in contrast to what has been found so far for monotreme IGL.

Cloning of IgE from the echidna (Tachyglossus aculeatus) and a comparative analysis of epsilon chains from all three extant mammalian lineages.

In continuation of our evolutionary studies of immunoglobulin (Ig) expression, we present here the cloning of IgE from a monotreme, the short-beaked echidna (Tachyglossus aculeatus). Including echidna IgE, 15 epsilon chain sequences have been isolated and each of the three mammalian lineages (placentals, marsupials and monotremes) is now represented by at least two sequences. Phylogenetic analyses based on all available epsilon chains and a selection of other mammalian Ig isotypes (IgM, IgA and IgG) were generated using three different algorithms. The resulting trees strongly support the Theria hypothesis, which states that the monotreme lineage was the first of the three extant mammalian lineages to appear in evolution. Furthermore, to increase our understanding of IgE we have done a detailed comparative analysis, with focus on primary structure, potential N-glycosylation, charge distribution and conservation of residues in the putative receptor-binding site. The overall structure of IgE, i.e. four constant domains and the positions of putative disulfide-bridge formations, are conserved, as is an N-glycosylation site in the third constant domain. An increased homology was observed in the putative receptor-binding site, which suggests an important function for the IgE/Fc epsilon RI interaction. IgE has been found exclusively in mammals, but it is present in all extant mammalian lineages. This, together with the overall conservation of structure, indicates that IgE appeared as a separate isotype early in mammalian evolution and that structural maintenance may have a selective advantage.

Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna).

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.

Antibody response to sheep red blood cells in platypus and echidna.

There is limited information regarding the kinetics of antibody responses exhibited by the platypus and the echidna in response to a T cell dependent antigen. In this preliminary study a platypus, an echidna and a rabbit were inoculated with sheep red blood cells to compare their antibody responses and kinetics. The antibody titres, produced by the platypus and echidna, were less than those elicited in the rabbit. Furthermore, the echidna and platypus exhibited a weak secondary response. This was most likely due to a failure of the platypus and echidna to undergo the characteristic IgM to IgG isotype switch following second antigen exposure. The conformational structure of these antibodies may differ from eutherian antibodies. This was further supported by a heat sensitivity experiment that indicated that these antibodies are more labile than rabbit immunoglobulins and therefore structurally less stable.

Evolution of the major histocompatibility complex: Isolation of class II beta cDNAs from two monotremes, the platypus and the short-beaked echidna.

Extant mammals are composed of three lineages: the eutherians, the marsupials and the monotremes. The majority of the mammalian major histocompatibility complex (MHC) data is based on the eutherian mammals, which generally have three classical MHC class II beta chain gene clusters - DRB, DQB and DPB, as well as the non-classical DMB and DOB. Marsupial DMB, DAB and DBB have been characterised. Confusion still surrounds the relationship of the marsupial DAB and DBB genes with the classical eutherian class II clusters. Here we present the first monotreme MHC class II beta chain sequences. Four MHC class II beta chain sequences were isolated from a spleen cDNA library from the short-beaked echidna, and one from a spleen cDNA library from platypus using a brushtail possum DAB probe. Given the non-orthologous relationship of the monotreme sequences with marsupial and eutherian beta chain clusters, we recommend that the five new monotreme sequences be assigned the nomenclature 'DZB', signifying the description of a new mammalian beta chain cluster. Our analysis suggests that all mammalian beta chain sequences (except DMB) evolved from a common ancestor. Maximum likelihood analysis places the monotreme beta chain sequences at the base of the mammalian clade, indicating their ancestral status. However, within the mammalian clade, monophyletic clades are not robust, and elucidation of the order of gene duplication that gave rise to the present-day gene clusters is not yet possible.

Characterization of beta(2)-microglobulin coding sequence from three non-placental mammals: the duckbill platypus, the short-beaked echidna, and the grey short-tailed opossum.

To further characterize genes of immunological importance from non-placental mammals, cDNAs encoding beta(2)-microglobulin (beta(2)m) were isolated from two prototherians, the platypus and an echidna, and one metatherian, a grey short-tailed opossum. In addition, a second allele of beta(2)m was identified in another metatherian species, the brushtail possum. Analysis of the deduced translations revealed conservation of key residues in these molecules over a long evolutionary history. The types of nucleotide substitutions present among the various taxa are also consistent with purifying selection at this conserved locus. An evolutionary tree of beta(2)m was constructed that supports the classic view of evolution with prototherians as the basal mammalian group.

Echidna IgA supports mammalian unity and traditional Therian relationship.

IgA is found only in birds and mammals where it is the principal immunoglobulin class found in secretions, providing protection at mucosal surfaces. The structure of IgA in birds is different from that of marsupials and eutherians. The avian heavy-chain constant region of IgA (Ca) consists of four domains, while marsupial and eutherian Ca consists of three domains plus a hinge. Here we describe the cloning and characterization of the heavy chain of IgA from the short-beaked echidna, Tachyglossus aculeatus, and report that monotreme Ca is composed of three domains plus a hinge, making it similar to its therian counterparts. The amino acid sequence identity of echidna Ca is approximately 47% with the therians and 30% with birds. Phylogenetic analysis of the Ca sequences provides strong support for the Theria hypothesis, which proposes that monotremes diverged prior to the separation of marsupial and eutherians, and directly contradicts the results of the mitochondrial data, which support a "Marsupionta" relationship which has marsupials and monotremes closer to each other. The characterization of the heavy chain of IgA from monotremes, in conjunction with the recent description of monotreme IgG and IgE nucleotide sequence, confirms that the "second big bang" of immunoglobulin evolution predated the divergence of extant mammals.

Characterisation of echidna IgM provides insights into the time of divergence of extant mammals.

The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cmu)was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cmus, and approximately 30% amino acid identity with Cmu from birds and reptiles. Phylogenetic analysis of mammalian Cmu provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cmu sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130mya, and Australian and American marsupials separated approximately 65mya.

The major histocompatibility complex in monotremes: an analysis of the evolution of Mhc class I genes across all three mammalian subclasses.

We report the isolation and characterization of cDNA clones of expressed, functional major histocompatibility complex class-I ( Mhc-I) genes from two species of monotremes: the duck-billed platypus and the short-beaked echidna. The cDNA clones were isolated from libraries constructed from spleen RNA, clearly establishing their expression in at least this one peripheral lymphoid organ. From the presence of conserved amino acid residues, it appears the expressed sequences encode molecules that likely function as classical Mhc-I. These clones were isolated using monotreme Mhc-I processed pseudogenes as probes. These processed pseudogenes were isolated from genomic DNA and, based on their structure, are likely independently derived in the platypus and echidna. When all the monotreme sequences were included in phylogenetic analyses, we found no apparent orthologous relationships between the platypus and echidna Mhc-I. Analyses that included a large number of Mhc-I sequences from other taxa support a separate monotreme Mhc-I clade, basal to a therian Mhc-I clade that is comprised of sequences from marsupial and placental mammals. The phylogenies also support the hypothesis that Mhc-I genes of placental mammals, marsupials, and monotremes are derived from three separate lineages of Mhc-I genes, best explained by two rounds of duplications and deletions. The first round would have occurred prior to the divergence of monotremes and therians, and the second prior to the divergence of marsupials and placental mammals. The sequences described here represent the first reported functional monotreme Mhc-I, as well as the first processed pseudogenes of any type from monotremes.

Characterization of immunoglobulin gamma 1 from a monotreme, Tachyglossus aculeatus.

A full-length immunoglobulin gamma clone from the echidna (Tachyglossus aculeatus) was isolated from a spleen cDNA library. The clone was 1,664 base pairs long and encoded the entire open reading frame, incorporating the V, D, J and C regions. The echidna clone had approximately 41% identity and 67% similarity at the amino acid level with both marsupial and eutherian IgG molecules. The presence of IgG in the monotremes confirms that the appearance of IgG occurred prior to the separation of the three extant mammalian lineages, but after their separation from the reptilian lineage, pinpointing the date to between 310 and 170 million years ago. Phylogenetic analyses using the immunoglobulin sequence data strongly support the 'Theria' hypothesis, with the monotreme lineage diverging prior to the separation of the marsupial and eutherian lineages.

Immunization of equines with phospholipase A2 protects against the lethal effects of Crotalus durissus terrificus venom

Equines (2 horses and 2 donkeyes) immunized with whole Crotalus durissus terrificus venom or its phospholipase A2 component either presented an increased survival time determined 3 days after challenge or were totally resistant to a challenging lethal dose of 200 mg crude venom 270 days after the initial immunization or 90 days after the last booster injection. the resistance was demonstrable on the basis of a good correlation with antibody titers determined by the ELISA method but not with the flocculation and neutralization assays. Since phospholipase A2 is essentially montoxic, it can be used as a substitute for whole venom for the production of commercial antisera ad as an immuniaing agent in prophylalctic progams

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.