Molecular cloning and tissue transcriptional analysis of a novel gene encoding proteasome activator PA28-α from common carp (Cyprinus carpio L.)

Abstract

To identify the homolog of the PA28-a subunit, a leukocyte cDNA library of common carp was screened and the full-length cDNA sequence of the PSME1 gene coding for the PA28-a was obtained. It encompasses 1097 nucleotides (nt), with an open reading frame encoding 249 amino acids. The deduced protein sequence exhibits identities of 92, 78, 78, 77, 75, 56, 54, 53 and 54% with zebrafish, Atlantic salmon, northern pike, rainbow trout, rainbow smelt, mouse, pig, cattle and human PA28-a subunits, respectively. Multiple sequence alignment of the PA28-a protein of common carp and other known PA28 subunits indicates that it contains a PA28-a subunit-specific insert corresponding to the KEKE motif of the known PA28-a (Region B), a characteristic proline-rich motif (Region A), a potential protein kinase C recognition site (Region D), a conserved activation loop (Region C), and a highly homologous C-terminal region (Region E). Phylogenetic analysis reveals that the PA28-c subunit is related to the presumed ancestor of the PA28-a and PA28-b subunits. In addition, the genomic DNA obtained by PCR is composed of eleven exons and ten introns with 3583 nt, typical of the PA28-a gene organization. Compared with the other known PA28-a genomic DNA, in spite of structural conservation through evolution, the fish PA28-a genes showed a much greater sequence divergence than their counterparts among mammals. Tissue transcriptional analysis of carp PA28-a indicated that it was visibly enhanced in tissues associated with immunity (gill, spleen, kidney and liver), and weakly transcribed in muscle, brain and heart.