RESUMEN
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.
Asunto(s)
Variación Genética , Inmunoglobulina A/química , Inmunoglobulina A/genética , Ornitorrinco/genética , Ornitorrinco/inmunología , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Clonación Molecular , Evolución Molecular , Regulación de la Expresión Génica , Inmunoglobulina A/metabolismo , Inmunoglobulina E/genética , Inmunoglobulina E/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Inmunoglobulina M/genética , Inmunoglobulina M/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Bazo/inmunología , Tachyglossidae/inmunologíaRESUMEN
To trace the emergence of the modern post-switch immunoglobulin (Ig) isotypes in vertebrate evolution we have studied Ig expression in mammals distantly related to eutherians. We here present an analysis of the Ig expression in an egg-laying mammal, a monotreme, the duck-billed platypus (Ornithorhynchus anatinus). Fragments of platypus IgG and IgE cDNA were obtained by a PCR-based screening using degenerate primers. The fragments obtained were used as probes to isolate full-length cDNA clones of three platypus post-switch isotypes, IgG1, IgG2, and IgE. Comparative amino acid sequence analysis against IgY, IgE and IgG from various animal species revealed that platypus IgE and IgG form branches that are clearly separated from those of their eutherian (placental) counterparts. However, the platypus IgE and IgG still conform to the general structure displayed by the respective Ig isotypes of eutherian and marsupial mammals. According to our findings, all of the major evolutionary changes in the expression array and basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the separation of monotremes from marsupial and placental mammals. Hence, our results indicate that the modern post-switch isotypes appeared very early in the mammalian lineage, possibly already 310-330 million years ago.
Asunto(s)
Evolución Biológica , Cambio de Clase de Inmunoglobulina , Inmunoglobulina E/genética , Inmunoglobulina G/genética , Ornitorrinco/inmunología , Secuencia de Aminoácidos , Animales , Northern Blotting , Inmunoglobulina E/química , Inmunoglobulina G/química , Inmunoglobulina G/clasificación , Datos de Secuencia Molecular , Alineación de Secuencia , Bazo/inmunologíaRESUMEN
As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the epsilon chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the epsilon chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian epsilon-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE epsilon chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 x His-CH2-CH3-CH4 protein product.
