Domain and genomic sequence analysis of bdellin-KL, a leech-derived trypsin-plasmin inhibitor

Bdellin-KL is a trypsin-plasmin inhibitor from Hirudo nipponia, whose N-ter minal sequence was identified as a non-classical Kazal-type. A cDNA clone e ncoding the inhibitor was isolated by reverse transcription-PCR and 5' rapi d amplification of cDNA ends. The cDNA showed an open reading frame of 155 amino acids comprising one signal peptide and two separated domains. The C- terminal domain consists of distinct internal repeats, including HHEE and H HDD. The bdellin-KL sequence, from the constructed genomic library of Korea n leech, was determined for the 2109 bases comprising the open reading fram e and flanking regions (3' and 5'). The promoter region contains potential regulatory sequence motifs, including TATA, CAAT, and GC boxes. To characte rize the properties of each domain, an N-terminal fragment was prepared by limited proteolysis of the intact protein. The inhibitory activity of the r egion was as potent as that of the intact protein. This suggests that the c ompact domain plays an important part in the inhibitory action of bdellin-K L. The C-terminal domain was revealed to have binding affinity to ions such as Ca2+, Zn2+, Fe3+, and Fe2+ without an influence on the inhibitory activ ity This study demonstrates that bdellin-KL may be a novel bifunctional pro tein with two distinct domains.