Purification And Functions Of A Novel Kazal-type Trypsin Inhibitor From The Leech Of Hirudinaria Manillensis

Background and purpose:Trypsin inhibitors are found in a large number of living organisms and play crucial roles in various biological and physiological processes.In recent years,its anti-cancer effect has become a research hotspot.Therapy with bloodsucking leeches has a long and widespread tradition.In China,a record of medical leeches was first created in “Shen Nong Ben Cao Jing”.With the progress of the modern pharmacology researches,emerging studies have verified the antitumor effects of leeches.For example,leech extract and hirudin isolated from leech have been identified inhibitory effects on tumor growth and proliferation,induction of tumor apoptosis,inhibition of tumor angiogenesis,etc.Till now,most researches about Hirudinaria manillensis focus on anti-coagulation,lowering cholesterol levels,anti-thrombus and thrombolysis,whereas whether H.manillensis has an effect on tumor and which compositions show anti-cancer effects remain unclear.Methods:In this study,by DEAE Sephadex A-50 gel filtration and RP-HPLC,a trypsin inhibitor was isolated from leech H.manillensis.MALDI-TOF-MS analysis gave its observed molecular weight(MW).Specific primers were designed according to its partial N-terminal amino acid sequence identified by automated Edman degradation and c DNA sequence coding the protein was cloned from c DNA library of the head of H.manillensis.The theoretical MW of linear mature bdellin-HM was calculated as 17,438.04 Da by http://web.expasy.org/compute_pi/.The BLAST analysis and Lasergene software were used to analyze its structural features.The inhibitory effect on trypsin,elastase,chymotrypsin,kallikrein,Factor(F)XIIa,FXIa,FXa,thrombin and plasmin were determined by the protease inhibitory activity and enzyme kinetics analysis.Results:1.In this study,by DEAE Sephadex A-50 gel filtration and RP-HPLC,bdellin-HM was isolated from leech H.manillensis.2.The mature peptide is comprised of 149 amino acid residues with six cysteines.The theoretical MW of linear mature bdellin-HM was calculated as 17,438.04 Da.Considering that bdellin-HM contains six cysteines,which are likely to form three intra-molecular disulfide bridges and a typical Kazal domain,its MW should be17,432.04 Da,which matches well with the observed MW 17,432.8Da.3.Bdellin-HM had the sequence similarity with bdellin-KL and bdellin B-3 and belongs to Kazal-type protease inhibitor.4.Under the assay conditions,Bdellin-HM had no inhibitory effect on elastase,chymotrypsin,kallikrein,Factor(F)XIIa,FXIa,FXa,thrombin and plasmin,but it showed a potent ability to inhibit trypsin with an inhibition constant(Ki)of(8.12±0.18)*10-9 M.Conclusion:These results suggest that bdellin-HM from the leech of H.manillensis plays a potent and specific inhibitory role towards trypsin.Further researches must be taken to determine the role of bdellin-HM in tumor progression.