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-terminal sequence was identified as a non-classical Kazal-type. A cDNA clone encoding the inhibitor was isolated by reverse transcription-PCR and 5' rapid 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 HHDD. The bdellin-KL sequence, from the constructed genomic library of Korean leech, was determined for the 2109 bases comprising the open reading frame and flanking regions (3' and 5'). The promoter region contains potential regulatory sequence motifs, including TATA, CAAT, and GC boxes. To characterize the properties of each domain, an N-terminal fragment was prepared by limited proteolysis of the intact protein. The inhibitory activity of the region was as potent as that of the intact protein. This suggests that the compact domain plays an important part in the inhibitory action of bdellin-KL. The C-terminal domain was revealed to have binding affinity to ions such as Ca(2+), Zn(2+), Fe(3+), and Fe(2+) without an influence on the inhibitory activity. This study demonstrates that bdellin-KL may be a novel bifunctional protein with two distinct domains.
Kim
Y H
YH
Bioresource Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Kusong-dong, Yusong-gu, Taejon, 305-701, Korea.
Choi
J G
JG
Lee
G M
GM
Kang
K W
KW
D000818:Animals; D000933:Antifibrinolytic Agents / Q000378:metabolism*; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007865:Leeches / Q000737:chemistry*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009930:Organic Chemicals; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014361:Trypsin Inhibitors / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D000933:Antifibrinolytic Agents; D018076:DNA, Complementary; D009930:Organic Chemicals; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; C107927:bdellin; D007501:Iron; D015032:Zinc; D002118:Calcium