Cytidine monophospho-N-acetylneuraminic acid hydroxylase itself upregulates the inflammatory responses through phosphorylation of c-Jun and NF-κB

Cytidine monophospho (CMP)-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) is an enzyme synthesizing CMP-N-glycolylneuraminic acid (Neu5Gc) from CMP-Neu5Ac. In human tissues, the lack of Neu5Gc is associated with the loss of specific exon 6 of the human CMAH gene (cmah). Surprisingly, immune related function and signal transducing capacity of CMAH protein lacking for hydroxylase activity have been observed in inflammatory response. Therefore, we aimed to understand pig cmah function in the protein level, but not Neu5Gc level. Levels of the expressed COX-2, iNOS, IL-1β, IL-6 and IFN-γ, known as inflammatory mediators or cytokines were significantly reduced at the mRNA levels in cmah shRNA-transfected RAW 264.7 cells. Interestingly, activation levels of signaling molecules including NF-κB, c-Jun and mitogen-activated protein kinases (MAPK) were also reduced in cmah knock out (KO) cell line. Neu5Gc-synthesizing pig CMAH expression activates the NF-κB and c-Jun function. Furthermore, in the pig CMAH, its 3’variant-2 and -3 overexpressing cells, the pig CMAH and its variant proteins activated NF-κB, c-Jun and MAPK in Neu5Gc independent manners. In addition, it was found that pig CMAH and its variants interact with the α-actinin-4. Thus, this is the first demonstration for the truncated CMAH proteins activate the inflammatory responses with emphasis to the crucial roles for rapidly evolving sialic acid synthetic proteins for animal to primate such as human evolution in immune system.