The first kazal-type serine proteinase inhibitor in the swimming crab Portunus trituberculatus involved in immune response to bacteria and fungi

Abstract

A cDNA clone, namely PtKPI, coding for a one-kazal domain serine proteinase inhibitor (SPI) was identified from haemocyte cDNA library of the swimming crab Portunus trituberculatus. It is the first kazal-type SPI reported from crabs, also the first one-kazal domain SPI in crustaceans. The full-length cDNA sequence was 683bp and contained an open reading frame of 444bp, 36bp of 5′-untranslated region (UTR), and 203bp of 3′-UTR with a poly(A) tail. It encoded a polypeptide of 147 amino acids with an estimated molecular mass of 15.85kDa and a theoretical pI of 8.99. A signal peptide was defined at N-terminus with a putative cleavage site located after position 16 (GNA-YN). The domain of PtKPI with C-X3-C-X7-C-X6-Y-X3-C-X7-C-X12-C was similar to the consensus pattern of the kazal motif. One α-helix surrounded by an adjacent three-stranded β-sheet constructed the classical three-dimensional structure of kazal domain. PtKPI transcript was detected at a high level in the eyestalk and haemocytes that are involved in immune function. Upon challenge with three different microorganisms Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris, the temporal expression of PtKPI in haemocytes showed different activation times against bacteria and fungi within an experimental period of 72h. These results indicate that PtKPI is potentially involved in acute response against invading bacteria and fungi in P. trituberculatus.