Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells.

The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream (Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5'-UTR region of 84 bp, an ORF of 729 bp and a 3'-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, alpha1, alpha2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the alpha1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA+LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.

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