Characterization and functional analyses of a novel chicken CD8 alpha variant X1 (CD8 alpha 1)

Abstract

We provide the first description of cloning and of structural and functional analysis of a novel variant in the chicken cluster of differentiation 8 alpha (CD8 alpha) family, termed the CD8 alpha X1 (CD8 alpha 1) gene. Multiple alignments of CD8a1 with known CD8 alpha and CD8 beta sequences of other species revealed relatively low conservation of AA residues involved in the specific and unique structural domains among CD8 alpha genes. For example, cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, the O-linked glycosylation sites (XPXX motif) are not found in the chicken CD8 alpha 1 sequence, and the A beta strand and complementarity-determining region 1 and 2 sequences are poorly conserved between chicken CD8 alpha 1 and avian CD8 alpha. Furthermore, the alignment showed that the transmembrane regions show relatively high sequence similarity, whereas the cytoplasmic regions show relatively low similarity, indicating poor conservation. Moreover, the motif (CXCP) that is thought to be responsible for binding the p56 lymphocyte cell kinase subunit (p56(lck)) is missing in the CD8 alpha 1 sequence. The chicken CD8 alpha 1 genomic structure is similar to that of chicken CD8 alpha, but their protein structures differ. Phylogenetic analysis showed that chicken CD8 alpha 1 grouped with known avian CD8 alpha sequences but was somewhat distantly related to the CD8 alpha molecules of other species. Moreover, we analyzed the signal transduction and cytokine response to CD8 alpha 1 treatment to determine the specific biological functions of chicken CD8 alpha 1 in immune cells. The results showed that chicken CD8 alpha 1 is a key regulator of the expression of genes that are associated and cooperate with transcription factors in the major histocompatibility complex class I and II promoter regions and activates Janus kinase (JAK) 1/2, signal transducer and activator of transcription (STAT), and suppressor of cytokine signaling (SOCS) 1 signaling-related genes. Immune cells that express functional CD8 alpha 1 induce proinflammatory cytokines as well as innate immune responses. Therefore, our data indicate that CD8 alpha 1 may have immunoregulatory activity by regulating the expression of proinflammatory or anti-inflammatory cytokines via its effect on immune cells.