Characterization of a leech-derived trypsin-plasmin inhibitor, bdellin-KL

Abstract

Bdellin-KL is a trypsin-plasmin inhibitor from Hirudo nipponia, whose N-terminal sequence was identified as a non-classical Kazal-type. A cDNA clone encoding the inhibitor was isolated by reverse transcription-PCR (RT-PCR) and 5`` rapid amplification of cDNA end (RACE). The cDNA showed an open reading frame of 155 amino acids comprising one signal peptide and two separated domains. While various amino acids and all disulfide bonds are at the N-terminal domain, the remaining region, more than 65 % of total sequence, consists of distinct internal repeats such as HHEE and HHDD. To characterize the structure and function of each domain, we prepared an N-terminal fragment (amino acid residue 19-66) by limited proteolysis of the intact protein. The inhibitory activity of the region was as potent as the intact protein. This suggests that the compact domain plays an important part in inhibition action of bdellin-KL. The C-terminal domain was revealed to have a binding affinity to ions such as $Ca^{2+}$, $Zn^{2+}$, $Fe^{2+}$, and $Fe^{3+}$ without influence on the inhibitory activity. This study demonstrates that bdellin-KL can be a novel bifunctional protein with two distinct domains. Bdellin-KL sequence, from the constructed genomic library of Korean leech, was determined for the 2109 bases. The sequence contains the open reading frame (ORF) composed of 3 exons and 2 introns. The consensus TATA box (TATAAA) was found at -26 bp from the transcription start point (tsp). The promoter region contains potential regulatory sequence motifs including CAAT boxes and GC boxes. There are also putative transcription factor binding sites for AP-1, AP-3, E box, Pit-1, and GATA-1 in the 5``-flanking region and the first intron. In the 3``-flanking region, a polyadenylation signal (AATAAA) has been identified. The information will provide a clue to the structure-function relationship of bdellin and may helpful to understand the multifunctional proteins in vivo.