Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.
Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an uncommon P(2) amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P(1) Ser and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P(2) amino acid residue from Gly to Pro, mimicking the P(2) residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P(2) residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci.
Donpudsa
Suchao
S
Department of Comparative Physiology, Uppsala University, Norbyvägen 18 A, SE-752 36 Uppsala, Sweden.
Söderhäll
Irene
I
Rimphanitchayakit
Vichien
V
Cerenius
Lage
L
Tassanakajon
Anchalee
A
Söderhäll
Kenneth
K
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D018076:DNA, Complementary / Q000235:genetics; D006434:Hemocytes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009868:Oomycetes / Q000201:enzymology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism*; D020860:Subtilisin / Q000037:antagonists & inhibitors; D014361:Trypsin Inhibitors / Q000378:metabolism
D018076:DNA, Complementary; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D020860:Subtilisin