DOCSLIB.ORG

Short Report: Immunodiagnosis of Human Fascioliasis Using Recombinant Fasciola Hepatica Cathepsin L1 Cysteine Proteinase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Short Report: Immunodiagnosis of Human Fascioliasis Using Recombinant Fasciola Hepatica Cathepsin L1 Cysteine Proteinase Am. J. Trop. Med. Hyg., 60(5), 1999, pp. 749±751 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene SHORT REPORT: IMMUNODIAGNOSIS OF HUMAN FASCIOLIASIS USING RECOMBINANT FASCIOLA HEPATICA CATHEPSIN L1 CYSTEINE PROTEINASE SANDRA M. O'NEILL, MICHAEL PARKINSON, ANDREW J. DOWD, WILMA STRAUSS, RENE ANGLES, AND JOHN P. DALTON School of Biological Sciences, Dublin City University, Dublin, Ireland; Unidad de Parasitologia, Instituto Nacional de Laboratorios de Salud, Secretaria Nacional de Salud, La Paz, Bolivia Abstract. Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detec- tion of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1). In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this im- munodiagnostic test and compared its performance with native CL1. Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA. A highly statistically signi®cant correlation (r2 5 0.751, P , 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins. These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F. hepatica eggs. All seronegative patients were also coprologically negative. Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1. Therefore, recombinant CL1 shows excellent potential for the development of the ®rst standardized assay for the sensitive and speci®c diagnosis of human fascioliasis. Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade. Fascioliasis is caused by liver ¯ukes of the genus Fasci- 6 SD age of 15.23 6 12.46 years. Coprologic analysis for ola. Although the disease is predominantly one of domestic F. hepatica eggs was performed on fecal samples obtained animals such as sheep and cattle, it is now emerging as an from all individuals using the sedimentation method of Rich- important chronic disease of humans.1±3 Recent estimates ie.11 Serum samples obtained from patients infected with suggest that as many as 2.4 million people are infected with schistosomiasis mansoni (20), cysticercosis (15), hydatidosis the parasite worldwide and 2.4 million are at risk.3,4 The (15) and Chagas disease (15) were obtained from the Insti- disease is particularly prevalent among the Aymaran people tuto Nacional de Laboratorios de Salud (INLASA) serum of the northern Altiplano region of Bolivia, with incidences library and used to test for cross-reactivity in the test. Con- of up to 60% being reported.5±8 Our laboratory recently de- trol serum samples were provided by eight volunteers at veloped a simple ELISA for the detection of serum IgG4 Dublin City University. The study was approved by the Hu- antibodies reactive with F. hepatica cathepsin L1 (CL1) pro- man Ethics Committee of INLASA and the Department of teinase.7 The use of puri®ed CL1 antigen in this ELISA pro- Health of Bolivia. vided a more speci®c immunodiagnosis of human fascioli- The ELISAs were carried out as previously described by asis than tests that use parasite somatic or excretory/secre- O'Neill and others.7 Brie¯y, 100 ml of native or recombinant tory (ES) antigen. CL1 antigen (5 mg/ml) was incubated overnight at 378Cin We have also recently reported the expression of func- wells of microtiter plates (Nuclon, Kamstrup, Roskilde, Den- tionally active recombinant F. hepatica CL1 in the yeast mark). The wells were blocked with a solution of 2% bovine Saccharomyces cerevisiae.9 In the present study, we exam- serum albumin/0.1% Tween 20 in phosphate-buffered saline ined the potential of this recombinant protein as a diagnostic for 30 min at 378C and human sera (1:125 dilution) was then reagent for human fascioliasis in the IgG4-ELISA and com- added into triplicate wells. Bound human antibodies were pared the performance of the antigen with native CL1 anti- detected using biotin-conjugated anti-human IgG4 (1:2,000 gen. Recombinant CL1 was puri®ed from yeast culture me- dilution), avidin-conjugated alkaline phosphatase, and the dium by gel ®ltration chromatography on Sephacryl S200 substrate 2,2-azino-bis (3-ethylbenzthiazoline-6-su¯onic HR,9 and native CL1 was puri®ed from ES products by a acid) (all from the Sigma Chemical Co., Poole, Dorset, Unit- combination of gel ®ltration chromatography on Sephacryl ed Kingdom). Plates were read after 15 min on a Titerteck S200 HR and ion exchange chromatography on QAE-Se- (Helsinki, Finland) multiscan ELISA plate reader at an ab- phadex.10 The concentration of each antigen preparation was sorbance of 405 nm. measured using a bicinchoninic acid protein assay kit (Pierce The absorbance readings obtained from the ELISAs using and Warner, Chester, United Kingdom). recombinant and native CL1 were plotted as scattergrams The nature of the study was explained to each individual, (Figure 1). Hierarchical agglomerative cluster analysis re- and prior to volunteering to provide blood and feces, each vealed that the scatter of points divided into two clusters, person signed a document of consent that also carried their one compact cluster of low absorbance readings and a dif- name and personal details. Blood samples were obtained in fuse cluster of high absorbance readings (Figure 1). Cut-off 1986 from 64 native Aymara living in the village of Cutu- points between the clusters were set at 3.09 standard devi- suma (approximately 50 km north of La Paz, Bolivia). The ations from the mean of the low absorbance cluster and were serum was separated and stored at 2208C. The age of the 0.117 and 0.121 for the recombinant and native proteins, volunteers ranged between seven and 76 years, with a mean respectively. Using the cut-off points, 38 individuals (59%) 749 750 O'NEILL AND OTHERS TABLE 1 Summary of the serologic and coprologic data obtained from 64 individuals in the village of Cutusuma, Bolivia (1986)* Group Recombinant CL1 Native CL1 Egg1 Seropositive 26 26 Egg1 Seronegative 0 0 Egg2 Seropositive 12 12 Egg2 Seronegative 26 26 Total 64 64 * CL1 5 cathespin L1. cercosis, hydatidosis, and Chagas disease were negative by IgG4-ELISAs that used either recombinant or native CL1. Although schistosomes also express cathepsin L proteinas- es,12 we have shown that antibodies prepared against F. he- FIGURE 1. Scattergram of the data obtained with the IgG4- patica cathepsin L proteinases do not cross-react with these ELISA using recombinant cathespsin L1 (CL1) and native CL1 an- (Brady C, Dalton JP, unpublished data). tigens. Hierarchical agglomerative cluster analysis using the com- Human fascioliasis is becoming increasingly recognized bined data obtained for both proteins separated the population into as a serious public health problem, particularly in regions of low absorbance (seronegative) and high absorbance (seropositive) subpopulations. The vertical and horizontal dashed lines indicate the Bolivia and Peru. Regional climatic factors coupled with the calculated cut-off points for each antigen. The closed circles repre- rural lifestyle and dietary habits of the indigenous population sent individuals who were coprologically positive and the open cir- contribute to transmission of the disease year round.6,8,13 cles represent those who were coprologically negative. The diagonal Clinical manifestations of human fascioliasis include fever, line represents the regression of seropositive individuals for recom- binant CL1 against native CL1. right hypochondrial pain, anorexia, weight loss, persistent diarrhea, and vomiting. However, many individuals are asymptomatic or present vague symptoms rendering clinical were seropositive and 26 were seronegative by the IgG4- diagnosis problematic.1 Accordingly, the development of a ELISA using both the recombinant and native CL1 (Figure simple, sensitive, and cost-effective diagnostic assay is im- 1 and Table 1). Chi-square analysis on whether individuals perative. While coprologic analysis may ful®ll some of these were seronegative or seropositive showed a highly signi®- requirements, it is clear that it provides a de®nitive parasi- cant relationship between the two proteins. Regression and tologic diagnosis only when eggs are found in the patient's correlation analysis of the data for the seropositive individ- feces, and that early infections cannot be detected using this uals showed that there was a highly linear relationship be- method. tween the two antigens (r2 5 0.751, P , 0.001) (Figure 1). Cathepsin L1 was considered a potential candidate for se- All patients who were coprologically positive (26) were rologic diagnosis of fascioliasis because it was secreted by also seropositive in the IgG4-ELISA using the recombinant all stages of the parasite that develop in the mammalian host or native proteins. Chi-square analysis showed that it was and was highly immunogenic in infected animals (antibodies highly unlikely (P , 0.001) that the coprologic and serolog- can be detected within two weeks after infection).10 O'Neill ically positive individuals would group together by chance and others7 demonstrated that this de®ned antigen discrimi- and thus reinforced the designation of the high absorbance nated between seropositive and seronegative individuals for cluster as the infected subpopulation. However, 12 copro- fascioliasis markedly better than crude antigens such as logically negative patients were identi®ed as seropositive. whole worm extracts of total ES products. Both the present These individuals may harbor early (prepatent) infections, study and that of O'Neill and others7 have shown that all since the appearance of eggs in the feces only follows the patients who were coprologically positive for F.
Load more

Recommended publications
  • Novel Binding Partners of PBF in Thyroid Tumourigenesis
    NOVEL BINDING PARTNERS OF PBF IN THYROID TUMOURIGENESIS By Neil Sharma A thesis presented to the College of Medical and Dental Sciences at the University of Birmingham for the Degree of Doctor of Philosophy Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine August 2013 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. SUMMARY Thyroid cancer is the most common endocrine cancer, with a rising incidence. The proto-oncogene PBF is over-expressed in thyroid tumours, and the degree of over-expression is directly linked to patient survival. PBF causes transformation in vitro and tumourigenesis in vivo, with PBF-transgenic mice developing large, macro-follicular goitres, effects partly mediated by the internalisation and repression of the membrane-bound transporters NIS and MCT8. NIS repression leads to a reduction in iodide uptake, which may negatively affect the efficacy of radioiodine treatment, and therefore prognosis. Work within this thesis describes the use of tandem mass spectrometry to produce a list of potential binding partners of PBF. This will aid further research into the pathophysiology of PBF, not just in relation to thyroid cancer but also other malignancies.
    [Show full text]
  • Fasciola Hepatica
    Fasciola hepatica Robinson, M., Dalton, J., O'Brien, B., & Donnelly, S. (2013). Fasciola hepatica: The therapeutic potential of a worm secretome. International Journal for Parasitology, 43(3-4), 283–291. https://doi.org/10.1016/j.ijpara.2012.11.004 Published in: International Journal for Parasitology Document Version: Peer reviewed version Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights NOTICE: this is the author’s version of a work that was accepted for publication in International Journal for Parasitology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in International Journal for Parasitology, [VOL 43, ISSUE 2-3, 2013] General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact [email protected].
    [Show full text]
  • Proteomics and Phylogenetic Analysis of the Cathepsin L
    Research Proteomics and Phylogenetic Analysis of the Cathepsin L Protease Family of the Helminth Pathogen Fasciola hepatica EXPANSION OF A REPERTOIRE OF VIRULENCE-ASSOCIATED FACTORS* Mark W. Robinson‡§¶, Jose F. Tort‡ʈ, Jonathan Lowther‡, Sheila M. Donnelly‡, Emily Wong‡, Weibo Xu‡, Colin M. Stack‡**, Matthew Padula‡‡, Ben Herbert‡‡, and John P. Dalton‡§§ Cathepsin L proteases secreted by the helminth pathogen zymes. The prosegment region was highly conserved Fasciola hepatica have functions in parasite virulence in- between the clades except at the boundary of prosegment cluding tissue invasion and suppression of host immune and mature enzyme. Despite the lack of conservation at this Downloaded from responses. Using proteomics methods alongside phylo- section, sites for exogenous cleavage by asparaginyl en- genetic studies we characterized the profile of cathepsin dopeptidases and a Leu-Ser2His motif for autocatalytic L proteases secreted by adult F. hepatica and hence iden- cleavage by cathepsin Ls were preserved. Molecular & tified those involved in host-pathogen interaction. Phylo- Cellular Proteomics 7:1111–1123, 2008. genetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications https://www.mcponline.org followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) The helminth pathogens Fasciola hepatica and Fasciola and two clades specific to infective juvenile stages gigantica are the causative agents of liver fluke disease (fas- (Clades 3 and 4). Consistent with these observations our ciolosis) in sheep and cattle. Although infections of F. hepat- proteomics studies identified representatives from ica occur predominantly in regions with temperate climates, Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F.
    [Show full text]
  • Spatiotemporal Proteomics Uncovers Cathepsin-Dependent Host Cell
    bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Spatiotemporal proteomics uncovers cathepsin-dependent host 2 cell death during bacterial infection 3 Joel Selkrig1, Nan Li1,2,3, Jacob Bobonis1,4, Annika Hausmann5, Anna Sueki1,4, Haruna 4 Imamura6, Bachir El Debs1, Gianluca Sigismondo3, Bogdan I. Florea7, Herman S. 5 Overkleeft7, Pedro Beltrao6, Wolf-Dietrich Hardt5, Jeroen Krijgsveld3‡, Athanasios Typas1‡. 6 7 1 European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 8 Heidelberg, Germany. 9 2 Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology (SIAT), 10 Chinese Academy of Sciences (CAS), 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, 11 China. 12 3 German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. 13 4 Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences 14 5 Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland. 15 6 European Bioinformatics Institute, European Molecular Biology Laboratory, Wellcome Trust Genome 16 Campus, Hinxton, Cambridge, CB10 1SD 17 7 Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 18 55, 2333 CC Leiden, the Netherlands. 19 20 21 ‡ to whom correspondence should be addressed: 22 [email protected] & [email protected] 23 24 SUMMARY 25 Immune cells need to swiftly and effectively respond to invading pathogens.
    [Show full text]
  • Felipe Jun Fuzita Molecular Physiology of Digestion In
    FELIPE JUN FUZITA MOLECULAR PHYSIOLOGY OF DIGESTION IN ARACHNIDA: FUNCTIONAL AND COMPARATIVE-EVOLUTIONARY APPROACHES Thesis presented to the Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, to obtain the Title of Doctor in Biotechnology. São Paulo 2014 FELIPE JUN FUZITA MOLECULAR PHYSIOLOGY OF DIGESTION IN ARACHNIDA: FUNCTIONAL AND COMPARATIVE-EVOLUTIONARY APPROACHES Thesis presented to the Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, to obtain the Title of Doctor in Biotechnology. Concentration area: Biotechnology Advisor: Dr. Adriana Rios Lopes Rocha Corrected version. The original electronic version is available either in the library of the Institute of Biomedical Sciences and in the Digital Library of Theses and Dissertations of the University of Sao Paulo (BDTD). São Paulo 2014 DADOS DE CATALOGAÇÃO NA PUBLICAÇÃO (CIP) Serviço de Biblioteca e Informação Biomédica do Instituto de Ciências Biomédicas da Universidade de São Paulo © reprodução total Fuzita, Felipe Jun. Molecular physiology of digestion in Arachnida: functional and comparative-evolutionary approaches / Felipe Jun Fuzita. -- São Paulo, 2014. Orientador: Profa. Dra. Adriana Rios Lopes Rocha. Tese (Doutorado) – Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan. Área de concentração: Biotecnologia. Linha de pesquisa: Bioquímica, biologia molecular, espectrometria de massa. Versão do título para o português: Fisiologia molecular da digestão em Arachnida: abordagens funcional e comparativo-evolutiva. 1. Digestão 2. Aranha 3. Escorpião 4. Enzimologia 5. Proteoma 6. Transcriptoma I. Rocha, Profa. Dra. Adriana Rios Lopes I. Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan III.
    [Show full text]
  • Fasciola Gigantica
    MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TYPE 1 CYSTATIN STEFIN-2 OF THE LIVER FLUKE FASCIOLA GIGANTICA BY MISS SINEE SIRICOON A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY (BIOMEDICAL SCIENCES) GRADUATE PROGRAM IN BIOMEDICAL SCIENCES FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2015 COPYRIGHT OF THAMMASAT UNIVERSITY MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TYPE 1 CYSTATIN STEFIN-2 OF THE LIVER FLUKE FASCIOLA GIGANTICA BY MISS SINEE SIRICOON A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY (BIOMEDICAL SCIENCES) GRADUATE PROGRAM IN BIOMEDICAL SCIENCES FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2015 COPYRIGHT OF THAMMASAT UNIVERSITY (1) Dissertation Title Molecular and biochemical characterization of type 1 cystatin stefin-2 of the liver fluke Fasciola gigantica Author Miss Sinee Siricoon Degree Doctor of Philosophy (Biomedical Sciences) Department/Faculty/University Graduate Program in Biomedical Sciences Faculty of Allied Health Sciences Thammasat University Dissertation Advisor Associate Professor Hans Rudi Grams, Dr. rer. nat. Dissertation Co-Advisor Professor Vithoon Viyanant, Ph.D. Dissertation Co-Advisor Professor Peter Smooker, Ph.D. Dissertation Co-Advisor Assistant Professor Suksiri Vichasri Grams, Dr. rer. nat. Dissertation Co-Advisor Assistant Professor Amornrat Geadkaew, Ph.D. Academic Years 2015 ABSTRACT Cysteine proteases including cathepsin B and cathepsin L are involved in physiological and biological processes in all living organisms and imbalanced activity of these proteases may cause several diseases. They are also important antigens in the trematode genus Fasciola and have vital roles in parasite survival including protection, infection and nutrition of the liver fluke.
    [Show full text]
  • Investigation of the Underlying Hub Genes and Molexular Pathogensis in Gastric Cancer by Integrated Bioinformatic Analyses
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Investigation of the underlying hub genes and molexular pathogensis in gastric cancer by integrated bioinformatic analyses Basavaraj Vastrad1, Chanabasayya Vastrad*2 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract The high mortality rate of gastric cancer (GC) is in part due to the absence of initial disclosure of its biomarkers. The recognition of important genes associated in GC is therefore recommended to advance clinical prognosis, diagnosis and and treatment outcomes. The current investigation used the microarray dataset GSE113255 RNA seq data from the Gene Expression Omnibus database to diagnose differentially expressed genes (DEGs). Pathway and gene ontology enrichment analyses were performed, and a proteinprotein interaction network, modules, target genes - miRNA regulatory network and target genes - TF regulatory network were constructed and analyzed. Finally, validation of hub genes was performed. The 1008 DEGs identified consisted of 505 up regulated genes and 503 down regulated genes.
    [Show full text]
  • Structural Dynamics Investigation of Human Family 1 & 2 Cystatin
    RESEARCH ARTICLE Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes Suman Kumar Nandy, Alpana Seal* Department of Biochemistry & Biophysics, University of Kalyani, Kalyani, West Bengal, India * [email protected] Abstract a11111 Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through OPEN ACCESS protein docking & comparative molecular dynamics. An overall stabilization effect is Citation: Nandy SK, Seal A (2016) Structural Dynamics Investigation of Human Family 1 & 2 observed in all cystatins on complex formation. Complexes are mostly dominated by van Cystatin-Cathepsin L1 Interaction: A Comparison of der Waals interaction but the relative participation of the conserved regions varied exten- Binding Modes. PLoS ONE 11(10): e0164970. sively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly doi:10.1371/journal.pone.0164970 acts as electrostatic interaction site. In fact the comparative dynamics study points towards Editor: Claudio M Soares, Universidade Nova de the instrumental role of L1 loop in directing the total interaction profile of the complex either Lisboa Instituto de Tecnologia Quimica e Biologica, towards electrostatic or van der Waals contacts.
    [Show full text]
  • Supplementary Information (2.608Mb)
    Dynamic pigmentary and structural coloration within cephalopod chromatophore organs Thomas L. Williams1*, Stephen L. Senft2*, Jingjie Yeo3,4,5*, Francisco J. Martín-Martínez4, Alan M. Kuzirian2, Camille A. Martin1, Christopher W. DiBona1, Chun-Teh Chen4, Sean R. Dinneen,1 Hieu T. Nguyen,6 Conor M. Gomes1, Joshua J. C. Rosenthal2, Matthew D. MacManes6, Feixia Chu6, Markus J. Buehler4, Roger T. Hanlon2#, Leila F. Deravi1# 1 Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, United States. 2 The Marine Biological Laboratory, Woods Hole, MA 02543, United States. 3 Department of Biomedical Engineering, Tufts University, Medford, MA 02155, United States. 4 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States. 5 Institute of High Performance Computing, A*STAR, Singapore 138632, Singapore. 6 Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH 03824, United States. These authors contributed equally: Thomas L. Williams, Stephen L. Senft, Jingjie Yeo Correspondence and requests for materials should be addressed to L.F.D. (email: [email protected]) or to R.T.H. (email: [email protected]) 1 Figure S1. QSpec analysis showing both scale and significance of differences in protein expression, as measured by spectral count MS/MS data. The three chromatophores are compared pairwise (Brown vs. Red, Brown vs. Yellow, and Red vs. Yellow) to show how protein abundance differs between brown, red, and yellow chromatophores. For each protein detected in more than one chromatophore, significance is expressed as the negative log of the false discovery rate (- log10(FDR)), where a value greater than 1.301 signifies an FDR of less than 5%, while the magnitude of the differential expression between the two types of chromatophore is shown as the log base two of the fold change (log2((Fold Change)), where a value of 1 or -1 means that the protein is twice as abundant in the indicated chromatophore color as compared to the other.
    [Show full text]
  • Proteolysis in Septic Shock Patients: Plasma Peptidomic Patterns Are Associated with Mortality J
    British Journal of Anaesthesia, ▪ (▪): 1e10 (2018) doi: 10.1016/j.bja.2018.05.072 Advance Access Publication Date: xxx Clinical Investigation CLINICAL INVESTIGATION Proteolysis in septic shock patients: plasma peptidomic patterns are associated with mortality J. Bauza-Martinez 1,#, F. Aletti2,#, B. B. Pinto3, V. Ribas4, M. A. Odena1, R. Dı´az1, E. R. Medina5, R. Ferrer6, E. B. Kistler7, G. Tedeschi8,9, G. W. Schmid-Schonbein€ 2, A. Herpain10, K. Bendjelid3,* and E. de Oliveira1,* 1Proteomics Platform, Barcelona Science Park, Barcelona, Spain, 2Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 3Geneva University Hospital, Geneva, Switzerland, 4Eurecat, Technology Centre of Catalonia, Barcelona, Spain, 5University Hospital Mu´ tua Terrassa, Barcelona, Spain, 6Intensive Care Department, Vall d’Hebron University Hospital, Barcelona, Spain, 7Department of Anesthesiology and Critical Care, VA San Diego Healthcare System, San Diego, CA, USA, 8Dipartimento di Medicina Veterinaria, Universita degli Studi di Milano, Milan, Italy, 9Fondazione Filarete Milano, Milan, Italy and 10Universite Libre de Bruxelles, Brussels, Belgium *Corresponding authors. E-mails: [email protected], [email protected] # These authors contributed equally to this study. Abstract Background: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome. Methods: Intensive care patients with septic shock (n¼29) or sepsis (n¼6) and non-hospitalised subjects (n¼9) were recruited as part of the prospective observational trial ‘ShockOmics’ (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects.
    [Show full text]
  • Identification and Characteristics of a Cathepsin L-Like Cysteine Protease from Clonorchis Sinensis
    Parasitology Research (2019) 118:829–835 https://doi.org/10.1007/s00436-019-06223-y HELMINTHOLOGY - ORIGINAL PAPER Identification and characteristics of a cathepsin L-like cysteine protease from Clonorchis sinensis Changling Ma1 & Kai Liang2 & Lili Tang2 & Shanshan He2 & Xiaoquan Liu2 & Mian He2 & Yanwen Li2 Received: 24 June 2018 /Accepted: 17 January 2019 /Published online: 28 January 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Cathepsin L-like protease is an important member of the papain-like cysteine protease and plays numerous indispensable roles in the biology of parasitic organisms. In a previous study, we identified a gene encoding a cathepsin L-like protease of Clonorchis sinensis (CsCPL) that was detected in the cercaria, metacercaria, and adult worm stages by immunolocalization, suggesting that this cysteine protease may be important and involved in the development of C. sinensis. In this study, the mature domain of CsCPL (CsCPL-m) was cloned and expressed in the form of inclusion bodies in Escherichia coli. After refolding, the recom- binant CsCPL-m displayed optimal protease activity towards Z-Phe-Arg-AMC substrates but not towards Z-Arg-Arg-AMC, and the activity of the protease was inhibited completely by the cysteine protease-specific inhibitors E-64 and IAA, which further demonstrated that CsCPL belongs to the cathepsin L-like cysteine protease family. Recombinant CsCPL-m exhibited consider- able activity at temperatures ranging from 28 to 42 °C, with the highest activity observed at 42 °C. Furthermore, recombinant CsCPL-m exhibited activity across a broad range of pH values (pH 4.0–8.0), with an optimal pH of 5.5.
    [Show full text]
  • Characterization of the Cathepsin-Like Cysteine Proteinases of Schistosoma Mansoni JOHN P
    INFECTION AND IMMUNITY, Apr. 1996, p. 1328–1334 Vol. 64, No. 4 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology Characterization of the Cathepsin-Like Cysteine Proteinases of Schistosoma mansoni JOHN P. DALTON,1,2 KAREN A. CLOUGH,1 MALCOLM K. JONES,3 1 AND PAUL J. BRINDLEY * Molecular Parasitology Unit, Queensland Institute of Medical Research, and Australian Centre for International & Tropical Health & Nutrition, Post Office, Royal Brisbane Hospital, Queensland 4029,1 and Centre for Microscopy and Microanalysis, University of Queensland, St. Lucia, Queensland 4072,3 Australia, and School of Biological Sciences, Dublin City University, Dublin 9, Republic of Ireland2 Downloaded from Received 5 September 1995/Returned for modification 29 November 1995/Accepted 15 January 1996 Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into the culture medium. The specific activity of cathepsin L was about double that of cathepsin B when each was assayed at its pH optimum, and moreover, the specific activities of cathepsins L and B in extracts of female schistosomes were 50 to 100% higher than in http://iai.asm.org/ extracts of male schistosomes. Analysis of the primary structure of two cloned S. mansoni cathepsins L, here termed cathepsin L1 and cathepsin L2, revealed that they are only 44% similar and that cathepsin L2 showed more identity (52%) with human cathepsin L than with schistosome cathepsin L1.
    [Show full text]