Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (<i>Apis cerana</i>) venom

Yang, Jie,Lee, Kwang Sik,Kim, Bo Yeon,Choi, Yong Soo,Yoon, Hyung Joo,Jia, Jingming,Jin, Byung Rae PERGAMON PRESS 2017 COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C Vol.201 No.-

<P><B>Abstract</B></P> <P>Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (<I>Apis mellifera</I>) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (<I>A</I>. <I>cerana</I>) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Apis cerana</I> venom serine protease inhibitor (AcVSPI) inhibits trypsin, but not chymotrypsin. </LI> <LI> AcVSPI is a low-molecular-weight serine protease inhibitor Api m 6-like peptide. </LI> <LI> AcVSPI inhibits plasmin and microbial serine proteases. </LI> <LI> AcVSPI functions as an anti-fibrinolytic factor. </LI> <LI> AcVSPI functions as an anti-microbial agent. </LI> </UL> </P>

Review : The Therapeutic Approaches for Hepatitis C Virus: Protease Inhibitors and Polymerase Inhibitors

( Paul Y. Kwo ),( Rakesh Vinayek ) 대한간학회 2011 Gut and Liver Vol.5 No.4

The current standard of care for hepatitis C infection is peginterferon/ ribavirin (PegIFN/RBV). We are entering the era where direct-acting antiviral agents (DAAs) will be added to PegIFN/RBV, leading to higher sustained response rates in genotype 1 infected individuals. Currently DAAs are directed toward specific proteins involved in hepatitis C replication with NS3/NS4A protease inhibitors furthest in development. Telaprevir and boceprevir are both NS3/NS4a inhibitors that significantly improve sustained response when added to PegIFN and RBV. The hepatitis C virus (HCV) polymerase inhibitors are another promising DAA class. These molecules are divided into nucleoside/nucleotide polymerase inhibitors and nonnucleotide/nucleoside polymerase inhibitors. Nucleoside/ nucleotide polymerase inhibitors have a high barrier to resistance and appear to be effective across a broad range of genotypes. Nonnucleoside polymerase inhibitors have a lower barrier of resistance and appear to be genotype specifi c. Preliminary data with these compounds are also promising. A third class, NS5A inhibitors, has also shown potent HCV RNA suppression in preliminary studies as monotherapy and with PegIFN and RBV. Combinations of these agents are also entering clinical trials and indeed a preliminary report has demonstrated that the combination of an NS3/4A protease inhibitor and NS5B polymerase inhibitor can effectively suppress virus in genotype 1 individuals. Future studies will concentrate on combinations of direct-acting antiviral agents without and with PegIFN and RBV. Clinicians will need to be familiar with managing side effects as well as resistance as we enter this new era. (Gut Liver 2011;5:406-417)

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom

Kwang Sik Lee,Jie Yang,Bo Yeon Kim,Yong Soo Choi,Hyung Joo Yoon,Jingming Jia,Byung Rae Jin 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.10

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putativelow-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identifiedfrom honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated.In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shownto act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-likedomain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putativelow-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide.Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin,but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however,it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPIinhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial andfungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. Thesefindings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

Protease Inhibitor Production using Streptomyces sp. SMF13

Kim, In Seop,Kim, Hyoung Tae,Lee, Hyun Sook,Lee, Kye Joon 한국미생물 · 생명공학회 1991 Journal of microbiology and biotechnology Vol.1 No.4

The aim of the current study is to evaluate the effects of medium compositions on the production of protease inhibitor in Streptomyces sp. SMF13. The production of protease inhibitor was countercurrently linked to extra-cellular protease, which were regulated by the culture conditions. Nitrogen source was the most critical ingredient affecting the production of protease inhibitor and protease. Carbon source was an important factor to determine the culture pH which affected very clearly the formation of protease and protease inhibitor. Inorganic phosphate inhibited the protease inhibitor production which was linked to the cell growth rate, although the optimal conditions for the production of protease inhibitor were not favouring to the cell growth.

어류 알의 Protease Inhibitor 활성 분포

지성준,이지선,신준호,박권현,김진수,김경섭,허민수 한국수산과학회 2011 한국수산과학회지 Vol.44 No.1

To identify and examine the distribution of proteolytic inhibitory activity in crude extracts from fish eggs, and to determine the applicability of these protease inhibitors as anti-degradation agents in surimi-based products and fish meat, we compared the inhibitory activities of various extracts from fish eggs to those of commercial proteases, such as trypsin and papain. We used the optimal conditions for the screening of trypsin activity: 30 ug/uL of 0.1% trypsin and 0.6 mM Na-benzoyl-L-arginine-p-nitroanilide (BAPNA) with a pH of 8.0 at 40℃ for 60 min. The activities of papain and four commercial proteases were investigated after mixing with 100 ug/uL enzymes and 0.3% casein with a pH of 8.0 at 40℃ for 60 min. We performed a screening assay to detect the inhibitory activity (%) of crude extracts from eight species of fish eggs against the target proteases trypsin and papain. The assay revealed a wide distribution of trypsin and papain inhibitors in fish eggs. The specific inhibitory activities (11.6–28.6 U/mg) of crude extracts from fish eggs against trypsin and BAPNA substrate were higher than that (0.64 U/mg) of egg whites, used as a commercial inhibitor. The inhibitory activities of crude extracts from fish eggs against trypsin, and of egg whites against casein substrate (1.94–4.51 U/mg), were higher than those of papain (0.24–1.57 U/mg) and commercial protease (0.04–0.32 U/mg). The extracts from fish eggs were rich in protease inhibitors that exhibited strong inhibitory activity against trypsin, a serine protease, and papain, a cysteine protease.

넙치( Paralichthys olivaceus ) 알로부터 Serine Protease Inhibitors의 분획 특성

김형준,이현지,박성환,전유진,김진수,허민수 한국수산과학회 2015 한국수산과학회지 Vol.48 No.2

Protease inhibitors (PI) of trypsin and papain as target proteases from the roe of bastard halibut Paralichthys olivaceus were fractionated out using ammonium sulfate precipitation (A), DEAE 650M anion exchange chromatography (D), and Sephacryl S-300 gel filtration (S). The recovery percentages of the fractions with the strongest inhibitory activity for each fractionation method were 13% for the A4 fraction, 21.2% for the D3 fraction, and 21.3% for the S2 fraction, with specific inhibitory activities of the fractions toward trypsin and casein of 168, 139, and 218 U/mg, respectively, while no inhibition of papain was observed. The IC50 for the trypsin-specific substrate Nα-benzoyl-l-arginine-pnitroanilide (BAPNA) was 0.65, 1.55, 2.26, and 2.85 mg/mL for the A4, S2, A3, and D3 fractions, respectively. These results suggest that chromatographic fractionation methods (D and S) based on the molecular mass and charge of the protein were more effective at fractionating PI than was ammonium sulfate precipitation based on protein solubility, and that the bastard halibut roe extract acts as a serine protease inhibitor. Therefore, the PI fraction from fish roe might be useful for inhibiting proteases in foodstuffs, and could constitute an alternative food-grade inhibitor for the surimi industry.

Protease-Activated Receptor-2 (PAR-2) 억제제 Pal-KTTKS 펩타이드 탐색 및 이를 함유한 국소도포제에 의한 아토피피부염의 임상적 호전

이윤희 ( Yoon Hee Lee ),김민정 ( Min Jung Kim ),공인덕 ( In Duck Kong ),류종석 ( Jong Sung Ryu ),장민열 ( Min Yeol Jang ),이천구 ( Cheon Gu Lee ),최응호 ( Eung Ho Choi ) 대한피부과학회 2010 대한피부과학회지 Vol.48 No.11

Background: Serine protease promotes desquamatation of the stratum corneum and this is controlled by serine protease inhibitors (SPI). After disruption of the skin barrier, signals for barrier recovery are started with the activation of cytokines and a migration of calcium ions. On the other hand, the protease-activated receptor-2 (PAR-2) pathway is initiated as a negative signal. As the pH of the stratum corneum become neutral, activated serine protease and PAR-2 inhibit the secretion of lamellar bodies and the formation of the lamellar structure. Objective: We wanted to screen noble synthetic peptides and identify the efficacy of a selected peptide, Palmitic acid-Lysine Threonine Threonine Lysine Serine (Pal-KTTKS), on PAR-2 in vitro and in vivo, and a clinical study was performed. Methods: In vitro: Changes of the intracellular calcium ion concentration were measured in cultured HaCaT cells by fluorescence imaging according to treatment with sample peptides and trypsin. In vivo animal study: The efficacy of 2% Pal-KTTKS cream as a selected noble peptide was evaluated in an oxazolone-induced atopic dermatitis animal model. Clinical study: A total of twenty three atopic dermatitis patients applied 2.5% Pal-KTTKS peptide-containing cream on the one side of their extremities and pseudo-ceramide containing moisturizer on the other side of the extremities as a control twice a day for 4 weeks. Clinical improvement was evaluated by the Eczema Area Severity Index (EASI) score, a subject questionnaire and comparison of photographs. Results: Suppression of the intracellular calcium concentration via PAR-2 inhibition was noted in the Pal-KTTKS peptide treated cultured HaCaT cells. In the oxazolone-induced atopic dermatitis hairless mice model, 2% Pal-KTTKS peptide containing lotion was more effective than vehicle lotion only. In the atopic dermatitis patients, the sites treated with 2.5% Pal-KTTKS peptide-containing cream showed better improvement for the EASI score, the subject questionnaire and the clinical photographs as compared to that of the control sites. There were no remarkable side effects related to the treatment. Conclusion: A PAR-2 inhibitor-containing topical agent would be an effective and safe modality for treating atopic dermatitis. (Korean J Dermatol 2010;48(11):966∼974)

Antiproliferative Activity of Lavatera cashmeriana- Protease Inhibitors towards Human Cancer Cells

Rakashanda, Syed,Qazi, Asif Khurshid,Majeed, Rabiya,Rafiq, Shaista,Dar, Ishaq Mohammad,Masood, Akbar,Hamid, Abid,Amin, Shajrul Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.6

Background: Proteases play a regulatory role in a variety of pathologies including cancer, pancreatitis, thromboembolic disorders, viral infections and many others. One of the possible strategies to combat these pathologies seems to be the use of protease inhibitors. LC-pi I, II, III and IV (Lavatera cashmerian-protease inhibitors) have been found in vitro to strongly inhibit trypsin, chymotrypsin and elastase, proteases contributing to tumour invasion and metastasis, indicated possible anticancer effects. The purpose of this study was to check in vitro anticancer activity of these four inhibitors on human lung cancer cell lines. Material and Methods: In order to assess whether these inhibitors induced in vitro cytoxicity, SRB assay was conducted with THP-1 (leukemia), NCIH322 (lung) and Colo205, HCT-116 (colon) lines. Results: LC-pi I significantly inhibited the cell proliferation of all cells tested and also LC-pi II was active in all except HCT-116. Inhibition of cell growth by LC-pi III and IV was negligible. $IC_{50}$ values of LC-pi I and II for NCIH322, were less compared to other cell lines suggesting that lung cancer cells are more inhibited. Conclusion: These investigations might point to future preventive as well as curative solutions using plant protease inhibitors for various cancers, especially in the lung, hence warranting their further investigation.

Alkaline protease inhibitor를 생산하는 해양유래 방선균의 탐색 및 동정

강성일(Sung-Il Kang),공재열(Jai-Yul Kong),최영준(Yeung Joon Choi),김민용(Min-Yong Kim),손홍주(Hong-Joo Son) 한국생명과학회 2008 생명과학회지 Vol.18 No.4

Alkaline protease는 식품 가공 공정에 있어 가공 원료의 선도 저하를 초래하거나 맛살류 제품 원료인 어류의 근육조직을 파괴하여 gel 구조를 파괴시키는 것으로 알려져 있다. 본 연구는 해양으로부터 alkaline protease에 대한 저해력을 가지는 방선균들을 분리하여 그 중 저해력이 가장 높은 C12 균주를 최종 선정하였다. 본 균주의 형태학적, 배양적 및 생리학적 특성을 조사한 결과, 포자의 크기는 2.0 ㎛로 외형은 원통형이고, 편모가 없으며, 포자형태는 smooth하였다. ISP 9 배지를 제외한 대부분의 배지에서 잘 성장하였다. 또한 15~50oC에서 잘 성장하였으며, 9% (w/v) NaCl이 포함된 배지에서도 성장하는 것으로 확인되었다. Gram 양성, citrate 음성, catalase 양성이었으며, melanin 색소를 생성하지 않았다. Starch, casein 및 gelatin 분해능이 있었으며, glucose, galactose, maltose, lactose, fructose 및 mannse 등은 잘 이용하였지만, sorbitol과 sucrose는 이용하지 않았다. 이러한 특성을 토대로 본 균주는 Streptomyces sp.로 확인되었다. 보다 정확한 균주 동정을 위하여 16S rDNA 염기서열 분석을 수행하였으며, 그 결과 C12 균주는 S. thermocarboxydus와 계통진화학적으로 가장 유연관계가 높았다. In this study, we screened and identified the bacterial strain showing high alkaline protease inhibitor activity from marine environment. Nine bacterial strains with alkaline protease inhibitor activity were isolated from marine sediments. Among them, strain C12 had the highest alkaline protease inhibitor activity and was selected for further taxonomical study. On the basis of morphological and physiological characteristics, strain C12 was identified as the genus Streptomyces. A phylogenetic analysis of the 16S rDNA showed that the isolated strain was actually a member of the genus Streptomyces, because the determined sequence exhibited a higher homology with Streptomyces thermocarboxydus. Morphological characteristics showed cylindrical spore chain and smooth spore surface by scanning electron microscope. Strain C12 was grown on all media except for ISP 9 agar. This strain could be grown in the medium containing up to 9% NaCl.

어류 알로부터 Protease Inhibitors의 크로마토그래피법에 의한 분획

김진수,김기현,김현정,김민지,박성환,이현지,허민수 한국수산과학회 2013 한국수산과학회지 Vol.46 No.4

A protease inhibitor from fish eggs was fractionated using chromatographic methods. The fractionation efficiency was evaluated in terms of specific inhibitory activity (SIA, U/mg), purity (fold), total inhibitory activity (TIA, U), and recovery (%). The protease inhibitor (PI) from egg extracts of skipjack tuna (ST Katsuwonus pelamis), yellowfin tuna (YT Thunnus albacares) and Alaska pollock (AP Theragra chalcogramma) was fractionated using Sephadex G-50 gel filtration and DEAE-Sepharose CL-6B anion exchange chromatography based on protein size exclusion and net charge, respectively. Fractions exhibiting strong inhibitory activity were contained in the 30-50 kDa fraction on gel filtration and in the range of 0.4-0.7 M NaCl gradient fraction on anion exchange chromatography. The respective TIA and percent recovery of the fraction obtained with gel filtration toward trypsin and Nα-benzoyl-L-arginine-pnitroanilide (BAPNA) were 2,758.7 U and 29.6% for ST, 1,005.5 U and 25.6% for YT, and 1,267.5 U and 26.0% for AP. Gel filtration chromatography was more effective at fractionating PI than using ion exchange chromatography. These results suggest that fish eggs act as serine protease inhibitors and might be useful for protease inhibition in foodstuffs.