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Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-l-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato metalloprotease Produces a Truncated but Active Enzyme

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Journal of Integrative Plant Biology 2005年第11期47卷 1352-1363页

作者： Jian-Feng LI Robert QI Liang-Hu QU Autar K Mattoo Ning LI Department of Biology The Hong Kong University of Science and Technology Hong Kong SAR China Key Laboratory of Gene Engineering of the Ministry of Education Zhongshan University Guangzhou 510275 China Department of Biochemistry The Hong Kong University of Science and Technology Hong Kong SAR China Sustainable Agricultural Systems Laboratory United States Department of Agriculture-Agriculture Research Service Beltsville Maryland 20705-2350 USA

1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.

关键词： 1-aminocyclopropane-1-carboxylic acid synthase cleavage sites metalloprotease proteolytic processing.

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The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

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Journal of Integrative Agriculture 2020年第8期19卷 2044-2055页

作者： PAN Li-jun LU Lin LIU Yu-ping WEN Sheng-xian ZHANG Zeng-yan College of Agriculture Hunan Agricultural UniversityChangsha 410128P.R.China National Key Facility for Crop Gene Resources and Genetic Improvement Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijing 10080P.R.China Institute for Cereal and Oil Crops Hebei Academy of Agriculture and Forestry SciencesShijiazhuang 050035P.R.China

Wheat(Triticum aestivum L.)is an important staple crop for global *** necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot,a devastating disease of ***,we identified RcMEP1,a zinc metalloproteaseencoding gene from *** genomic sequences,and characterized its pathogenesis ***1 expressed at markedly-high levels during *** infection process to *** predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH).The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death,and that the predicted signal peptide functions and is required for secretion and cell *** purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation,and to inhibit expression of host chitinases when infiltrated into wheat and *** leaves,while the M43 domain-deleting peptide and negative control lacked the ***,compared with the control pretreatment,the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat,whereas the M43 domain-deleting peptide *** results suggest that RcMEP1 acted as an important pathogenicity factor during *** infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of *** study provides insights into pathogenesis role of M43 domain-containing metalloproteases during *** infection to wheat.

关键词： cell death metalloprotease Rhizoctonia cerealis pathogenicity wheat

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Cloning of catalase gene and antioxidant genes in Scophthalmus maximus response to metalloprotease of Vibrio anguillarum stress

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Journal of Oceanology and Limnology 2022年第1期40卷 322-335页

作者： Hai REN Jian LI Ping LIU Xianyun REN Tao SONG Guisheng GAO Duwen LI Shuaiting LIU Hebei Key Laboratory of Preventive Veterinary Medicine Hebei Normal University of Science and TechnologyQinhuangdao 066004China Key Laboratory of Sustainable Development of Marine Fisheries Ministry of AgricultureYellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdao 266071China

metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from oxidative damage due to the production of excessive reactive oxygen species(ROS).Catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx)are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in *** evaluate the eff ects of metalloprotease-induced ROS on the antioxidation defense mechanism of *** head kidney cells,the cDNA of CAT gene(designated as SmCAT)was cloned and *** comprises a 1584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A)*** analysis revealed an active site signature sequence,a heme-ligand signature sequence,and three catalytic amino acid *** deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%-92.4%with those of other *** analysis revealed that SmCAT is classifi ed with CAT of other fi *** real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues,especially in *** expression of SmCAT,SmMnSOD,and SmGPx were inhibited signifi cantly in head kidney cells treated with metalloprotease from 12 to 24 *** 6 to 24 h metalloprotease-treated groups compared to that of the untreated group,it was found that the production of ROS was markedly increased,and the mitochondrial membrane potential was decreased *** 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0μg/mL metalloprotease for 12 and 24 ***,the toxic eff ects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels,which trigger the activation of apoptosis in the head kidney cells of *** fi ndings provide a better under

关键词： gene cloning expression reactive oxygen species metalloprotease head kidney cells

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Identification and Characterization of Peptides Mimicking the Epitopes of metalloprotease of Schistosoma Japonicum

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Cellular & Molecular Immunology 2005年第3期2卷 219-223页

作者： Lianfei Tang Yuxiao Chen Linqian Wang Shunke Zhang Xianfang Zeng Xinyuan Yi Department of Parasitology Xiangya School of Medicine Central SouthUniversity Changsha 410008 China Technology Centre Hunan Entry-Exit Inspection and Quarantine BureauChangsha China

In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum （S. japonicum）, polyclonal anti-metalloprotease sera was prepared to screen a 12-mer random peptide library to isolate phages binding specially to antisera IgG. Then, phage ELISA, animal immunization, DNA sequencing, Western blotting and enzymatic activity neutralizing analysis were used to characterize the selected phage clones. All of ten randomly picked clones were shown to be positive. Five peptides of different amino acid sequences deduced from DNA sequences were obtained and two of them （peptides 2 and 3） could induce significant reduction （31.0% and 31.8%, respectively） in worm burden and high reduction （52.6% and 54.9%, respectively） in liver eggs per gram （LEPG）, while, unexpectedly, others （peptides 1, 4 and 5） could not elicit enough protection against infection of S. japonicum. Peptides 2 and 3 could be recognized by S. japonicum infected mouse sera （IMS） and could elicit neutralizing Abs. The results show that peptides 2 and 3 are antigenic and immunogenic. They are true mimics of epitopes of metalloprotease and useful as novel vaccine candidates against ***. Cellular ＆ Molecular Immunology. 2005;2（3）：219-223.

关键词： phage displayed peptide library epitope Schistosomajaponicum metalloprotease

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Detection of Vibrio anguillarum and Its Virulent metalloprotease Using the Method of ELISA

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High Technology Letters 2002年第4期8卷 18-20页

作者：张振冬 Zhang Peijun Mo Zhaolan Chen Shiyong Graduate School of the Chinese Academy of Sciences Beiing 100039P.R China Key Laboratory of Experimental Marine Biology Institute of Oceanology Chinese Academy of Sciences Qingdao 266071 P.R China

A method detecting pathogenic vibrio anguillarum and its virulent metalloprotease is reported. The metalloprotease is isolated from extracellular product of vibrio anguillarum by the cellophane plate technique and purified by gel filtration and ion-exchange chromatography. Anti-sera are prepared by injecting vibrio anguillarum cells and metalloprotease into the rabbits. Slide agglutination assay is used to detect V. anguillarum in the infection experiment and enzyme linked immunosorbent assay (ELISA) is carried out to detect concentration of metalloprotease. The results show that bacterium strain M3 is able to diffuse into the viscera of infected fish through the blood circulating system 10 hours after intramuscular infection, and E-LASA is a sensitive method to detect the metalloprotease with detectable amount of 7.8 ng. The aim of this study is to establish a sensitive and specific method to observe the infection of vibrio anguillarum in the host.

关键词： metalloprotease Vibrio anguillarum ELISA Paralichthys Olivaceus

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Purification,Characterization and in vitro Anthelmintic Activity of a Neutral metalloprotease from Laccocephalum mylittae

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Chinese Journal of Chemical Engineering 2010年第1期18卷 122-128页

作者：周立华许勤勤张一琼周振兴管文军李永泉 College of Life Sciences Zhejiang University Hangzhou 310058 China

A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic *** protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,*** protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a *** Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, *** vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris *** electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of ***.

关键词： metalloprotease Laccocephalum mylittae purification characterization anthelmintic activity

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An in vitro and in silico study of anti-dermatophytic activity of gossypol from fruits of Thespesia populnea(L.)*** Correa

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Asian Pacific Journal of Tropical Biomedicine 2021年第12期11卷 543-552页

作者： Anuthara R Sebastian Jose Midhun Jyothis Mathew Microbiology Research Lab School of BiosciencesMahatma Gandhi UniversityKottayamKeralaIndia

Objective:To isolate,purify,and characterize gossypol from the fruits of Thespesia populnea(L)*** Correa,test its antidermatophytic activity,identify its targets on the dermatophyte,and confirm the binding of gossypol with the fungal target by molecular docking ***:Gossypol from Thespesia populnea was characterized by high performance liquid chromatography,liquid chromatographmass spectrometry,Fourier transform infrared spectroscopy,and nuclear magnetic *** anti-dermatophytic activity of gossypol was tested against four different dermatophytes,*** mentagrophytes,Trichophyton rubrum,Microsporum canis,and Microsporum *** mentagrophytes was selected for further *** inhibitory mode of action of gossypol on Trichophyton mentagrophytes was determined by analyzing the modulation of gene expression in various pathways of the ***:Gossypol inhibited all the *** minimum inhibitory concentrations were 12.5μg/mL for Trichophyton mentagrophytes and Microsporum canis and 25μg/mL for Trichophyton rubrum and Microsporum *** minimum fungicidal concentrations were 50μg/mL for Trichophyton mentagrophytes,100μg/mL for Microsporum canis and Trichophyton rubrum,and 200μg/mL for Microsporum *** inhibited the mRNA expression of metalloprotease(MEP4)and isocitrate lyase(ICL).The binding of gossypol with the enzymes was confirmed by molecular docking *** best docking poses were found and the low binding energies were recorded with the two target ***:Gossypol is a potential antifungal agent and can be further explored as an anti-dermatophytic drug.

关键词： Thespesia populnea Gossypol Anti-dermatophytic activity Trichophyton mentagrophytes Mechanism of action Gene expression Molecular docking metalloprotease MEP4 Isocitrate lyase ICL

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ADAM9特异性siRNA对肾透明细胞癌786-0细胞ADAM9基因表达及体外侵袭能力的影响

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郑州大学学报（医学版） 2014年第1期49卷 59-62页

作者：张新恒马曜辉单中杰新乡医学院研究生处新乡473003 郑州人民医院泌尿外科郑州450003

目的:探讨去整合素金属蛋白酶9(ADAM9)特异性siRNA对人肾透明细胞癌(RCCC)786-0细胞中ADAM9基因表达及体外侵袭能力的影响。方法:设计合成ADAM9特异性siRNA。将786-0细胞分4组处理,分别为正常对照组(正常培养786-0细胞)、空脂质体转染... 详细信息

目的:探讨去整合素金属蛋白酶9(ADAM9)特异性siRNA对人肾透明细胞癌(RCCC)786-0细胞中ADAM9基因表达及体外侵袭能力的影响。方法:设计合成ADAM9特异性siRNA。将786-0细胞分4组处理,分别为正常对照组(正常培养786-0细胞)、空脂质体转染组、阴性转染组(转染无义siRNA)和ADAM9 siRNA转染组。转染24、48、72 h后,采用RT-PCR法检测细胞ADAM9 mRNA的表达;转染48 h后,采用Western blot法检测细胞ADAM9蛋白的表达,并用Transwell法检测细胞侵袭能力。结果:转染24、48、72 h后,ADAM9 siRNA转染组786-0细胞ADAM9 mRNA的表达显著低于其他3组(F=47.945、180.456、60.978,P均<0.001);转染48 h后,ADAM9 siRNA转染组786-0细胞ADAM9蛋白表达显著降低(F=142.816,P<0.001),穿膜细胞数显著减少(F=45.389,P<0.001)。结论:ADAM9特异性siRNA能够有效沉默786-0细胞中ADAM9的表达并降低其体外侵袭能力。

关键词：肾透明细胞癌去整合素金属蛋白酶9 侵袭 a disintegrin and metalloprotease 9

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Snake Serum Albumin as an Inhibitor of Venom Zinc Proteases:Purification,Characterization,and cDNA Cloning of Trimeresurus flavoviridis Serum Albumin

Snake Serum Albumin as an Inhibitor of Venom Zinc Proteases:...

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“基因、进化与生理功能多样性”海内外学术研讨会暨中国生理学会第七届比较生理学学术会议

作者： Xin-Rong Xing,Zhi-Kui Cheng,Wen-Dong Liu,Yong-De Hong,Hui-Jian Wu~* Department of Bioscience and Biotechnology,Dalian University of Technology,Dalian 116024

The protein inhibitor of metalloproteases in the venom of Habu snake Trimeresurus flavovirids was purified from the serum. This protein inhibited both proteolytic and fibrinolytic activies of zinc *** molecular weight was calculated to be approximately 67 kDa and 62 kDa with and without carbohydrates,*** ration of amino acid composition of the inhibitor showed a certain similarity to metalloprotease inhibitor designated HSF isolated from the serum of Habu snake Trimeresurus *** determination of the amino acid sequences for several peptide fragments,cDNA cloning was performed to determine a full sequence of the *** inhibitor precursor was found to consist of the 584 amino acid residues,in which the inhibitor is with 558 residues and the signal peptide with 26 *** protein was judged to be a member of serum albumin, and the elucidated sequence was found to be very much similar to serum albumin of Naja naja *** inhibitor,namely,albumin, completely inhibited the lysis of fibrin, while other ordinary albumins from various sources exhibited no *** analysis suggested that the serum albumin of this particular toxic snake has evolved or co-evolved for detoxification.

关键词： Inhibitor metalloprotease Purification cDNA Cloning Albumin

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Renal thrombotic microangiopathies induced by severe hypertension

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南京医科大学学报(自然科学版) 2008年第7期 956-956页

作者： Zhang,B. Xing,C.Y. Yu,X.B. Sun,B. Zhao,X.F. Qian,J.Nanjing Med Univ,Affiliated Hosp 1,Dept Nephrol,Nanjing 210029,Peoples R China.

Renal thrombotic microangiopathy(TMA) is an uncommon vascular complication of severe *** now,its clinical-pathological characteristics and renal survival have been ***-one patients with biopsy-proven renal TMA and with severe or malignant hypertension were retrospectively *** the patients exhibited severe hypertension,with systolic blood pressure at 200-280 mmHg and diastolic pressure at 110-180 *** patients had hemorrhagic *** lactate dehydrogenase and thrombocytopenia were found in 6 and 5 patients,*** proteinuria(> 3 g/day) was present in 2 patients and microscopic hematuria in 18 *** patients presented with renal insufficiency(creatinine 3.1 +/-2.1 mg/dL).The level of von Willebrand factor:antigen(vWF:Ag) in patients was not significantly higher than that in the healthy subjects,while the ADAMTS13(a disintegrin and metalloprotease,with thrombospondin-1-like domains) activity was not significantly lower than that in the healthy *** histology showed a TMA involving preglomerular arterioles and/or interlobular arteries character-ized by fibrin deposits and vascular wall *** glomerular microthrombi were not observed in these *** patients required hemodialysis upon admission for severe acute renal *** follow-up,3 patients had recovered normal renal function and 14 had mild renal insufficiency(creatinine 1.8 +/-0.3 mg/dL),while 4 patients still required persistent *** conclusion,compared with patients having hemolytic uremic syndrome/thrombocytopenic purpura,our patients showed a low incidence of throm-bocytopenia and better renal outcome.

关键词： microangiopathy kidney hypertension hemolytic-uremic syndrome von-willebrand-factor systemic-lupus-erythematosus thrombocytopenic purpura disease anemia adults metalloprotease protease

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