A macrophage migration inhibitory factor like oxidoreductase from pearl oyster Pinctada fucata involved in innate immune responses

Abstract

Macrophage migration inhibitory factor (MIF) is an important cytokine and plays a crucial role as a pivotal regulator of innate immunity. In this study, a MIF cDNA was identified and characterized from the pearl oyster Pinctada fucata (designated as PoMIF). The full-length of PoMIF was 1544 bp and consisted of a 5′-untranslated region (UTR) of 45 bp, a 3′-UTR of 1139 bp with a polyadenylation signal (AATAAA) at 12 nucleotides upstream of the poly (A) tail. The open reading frame (ORF) of PoMIF was 360 bp which encoded a polypeptide of 120 amino acids with an estimated molecular mass of 13.3 kDa and a predicted pI of 6.1. SMART analysis showed that PoMIF contained the catalytic-sites P 2 and K 33 for tautomerase activity, a motif C 57GSV 60 for oxidoreductase activity and a MIF family signature D 55PCGSVEVYSIGALG 69. Homology analysis revealed that the PoMIF shared 40.3–65.5% similarity and 26.9–45.0% identity to other known MIF sequences. PoMIF mRNA was constitutively expressed in seven selected tissues of healthy pearl oysters, with the highest expression level in digestive gland. Eight hours after P. fucata was injected with Vibrio alginolyticus, the expression of PoMIF mRNA was significantly up-regulated in digestive gland, gills, hemocytes and intestine. The cDNA fragment encoding mature protein of PoMIF was subcloned to expression vector pRSET and transformed into Escherichia coli BL21 (DE3). The recombinant PoMIF (rPoMIF) was expressed and purified under optimized conditions. Function analysis showed that rPoMIF had oxidoreductase activity and could utilize dithiothreitol (DTT) as reductant to reduce insulin.