Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.
Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system.
Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education and College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, PR China.
Ye-Qing
YQ
Qian
Ye
Y
Li
Fan
F
Yang
Yan-Qin
YQ
Yu
Jin-Shu
JS
Yang
Wei-Jun
WJ
Yang
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005780:Gelatin / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D033541:Palaemonidae / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects
D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D005780:Gelatin