Prasanth Kumar Nadh Dehydrogenase Subunit 1


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Prasanth Kumar Nadh Dehydrogenase Subunit 1

  1. 1. Characterization of the NADH dehydrogenase subunit I of the Fasciola gigantica using Computational tools Introduction: Fasciola gigantica, a parasitic worm belonging to class Trematoda (Phylum: Platyhelminthes).This parasite causes fascioliasis in cattle, buffalo, goats, sheep, donkeys and human. The infection of ruminants with the parasite Fasciola gigantica is a cause of important economic loss throughout Asia and Africa. Infected ruminants showed weight loss, reduced productivity and poor milk production. It is regarded as one of the prominent platyhelminth infections of ruminants in Asia and Africa [1]. Human Fascioliasis (HF) has now been found in almost all governorates of the Delta region, in the city of Alex, in some governorates of Upper Egypt, and in the reclaimed desert land. The population at risk in Egypt is considered to be 27 million [2]. Infection cycle: The flowchart shown below reveals the different stages of F.gigantica life cycle. It has been reported that the most important intermediate hosts of F. gigantica are L. auricularia; however, L. rufescens and L. acuminata are the host snails in the Indian Subcontinent [3][4]. Infection strategy across globe: Human Fascioliasis is increasingly recognized as causing significant human disease, with 2.4 million people infected across the globe [5] [6].Being endemic in 61 countries, the infection prevalence is estimated to be high in the areas where extensive sheep and cattle raising occurs and where dietary practices include the consumption of raw aquatic vegetables.
  2. 2. Figure 1: Flowchart depicting the different stages in the life-cycle of F.gigantica. Selecting NADH dehydrogenase subunit 1 for Computational studies: The recent studies made in inferring the taxonomic relationship between Parasitic helminthes (includes, Fasciola sp.) and Facultative Anaerobic Eukaryotes based on Multiple Lineages of the Mitochondrial Gene NADH dehydrogenase subunit 1 (ND1) by Lynne van Herwerden, David Blair and Takeshi Agatsuma [7].This study invoked us to employ computational aspects of Bioinformatics to characterize the protein NADH dehydrogenase subunit 1.
  3. 3. Extracting informations from the Protein sequence: The protein sequence for NADH dehydrogenase subunit 1 was retrieved from the NCBI (National Centre for Biotechnological Informations) Accession Number: FLNFLYLGFSSFFAFVMIMVFVAFFILGERKVLGYMQIRKGPNKVGLL GLLQSFADLMKLVIKFKFVFFQNRSWLSWWGIYLLVLLACGYCLLFF LSFGGVSSVNFMLWFLVVTSMTGYSLLSVGWGCYNKFALVSCVRS AFGSVSFEACFMCIVVLVALVWGSYGAFSLFSGFGGM Motif Analysis: The identification of motif from the protein sequence is computationally possible but in many cases, it will lead to random matches which will be biologically insignificant. Hence, we proceeded the scanning of motif from multiple approaches such as PeroxiBase profiles, PROSITE patterns, PROSITE profiles, Pfam HMMs (both local and global models) through the program MotifScan [8]. Figure 2: Result of MotifScan program.
  4. 4. Table 1: The list of motifs generated by MotifScan program for the F.gigantica NADH dehydrogenase subunit1 protein sequence. Motif No Residue Residue Types of Motif Computational position position Method (start) (end) 1 71 74 N-glycosylation site freq_pat 2 53 56 Casein kinase II freq_pat phosphorylation site 3 49 54 N-myristoylation freq_pat 3 99 104 N-myristoylation freq_pat 3 163 168 N-myristoylation freq_pat 4 1 5 Big-1 (bacterial Ig– prf like domain 1) 5 1 25 Phenylalanine rich prf region profile pfam_fs 6 63 NADH pfam_fs dehydrogenase 6 84 106 CD47 pfam_fs immunoglobulin-like domain pfam_ls 6 177 NADH pfam_ls dehydrogenase Many motifs were identified and we had safely ignored the matches produced by different methods to make our proceedings sensible relying on the fact that it belongs to the NADH dehydrogenase family of proteins and we had concentrated on NADH dehydrogenase motif. Pair wise Comparisons: To study the phylogenetical analysis of this protein, we compared the NADH dehydrogenase subunit 1 of Fasciola gigantica with that of Fasciola hepatica and the results showed that they are found to be Orthologous proteins because these two proteins perform the same function.
  5. 5. We used the program EMBOSS (European Molecular Biology Open- Software) Suite hosted by EBI-EMBL (European Bioinformatics Institute-European Molecular Biology Laboratory).Blosum 62 amino acid substitution matrix was utilized to facilitate global sequence alignment using Needleman-Wunsch Algorithm and the parameters are briefly listed here. Matrix Blosum62 Open Gap penalty 10.0 Extension Gap penalty 0.5 Table 2: Parameters used in the EMBOSS program Figure 3: Pair wise sequence comparison of F.hepatica and F.gigantica Identity: 91.5 % (162/177) Similarity: 93.2 % (165/177) Gaps: 0.0 % (0/177) Score: 856.0 (This result shows that this pair wise alignment is significant.)
  6. 6. Prediction of the 3 Dimensional Structures: We searched through all the template structural databases to identify a potential template for our protein sequence to build a 3-D model using Homology modeling as our protein doesn’t have a structure(s) in any of the Primary databases like PDB, MMDB, SCOP and CATH. No template(s) was identified by programs like Geno3D,SWISS-MODEL and EsyPred [9]. Secondary Structure Prediction: Figure 4: Secondary Structure Prediction using QuickPhyre server. (Helices and Coils were identified using Consensus). Therefore, we decided to perform secondary structure prediction and to build a 3-D model using folds from different families. This fold represents different protein families and it is accomplished by QuickPhyre Server, Imperial College, London [10].
  7. 7. Fold Recognition:
  8. 8. Fold Recognition (continued ): Figure 5: Fold recognized from different protein families using QuickPhyre server. These individual folds helped us to build a 3-D model irrespective of its protein families, but by the sequence comparison of our protein sequence with that of each individual fold. Modeling of 3-D Protein Structure: Due to the absence of template for building a model, we utilized the above folds to conceptually build a protein model using QuickPhyre server. We compared the secondary structure prediction analysis results (Figure 4) with that of our modeled protein. Helices and coils shown in the consensus are typically true because the NADH dehydrogenase complex (of human) contains 60 trans-membrane helices and some coils if we hope that this protein is conserved for its functions through generations and maintained its structure.
  9. 9. Predicted 3-D model of the NADH dehydrogenase subunit1 : Figure 6: The picture depicts the coiled region (red) of the protein NADH dehydrogenase subunit1. Results & Discussions: Our procedure to model a protein is based on the fold recognition from different families. The drawback of our procedure is that helical regions were not built as the bond between hydrogen is far apart or fewer and due to the lack of structural template to assist the hydrogen bonding. The potential identification is the Coils which can play a vital role in protein complex formation as well as other functions like translocation of protons in the respiratory chain. How far the F.gigantica NADH dehydrogenase is similar in function to human? With the identification of known redox centers and NADH binding site in human enzyme, the ability of complex formation and other unidentified information can help
  10. 10. us to study this particular protein, its affinity for binding proteins and to prepare a more effective drug than Triclabendazole in future. References: [1] Analysis of potential vaccine candidate molecules in Fasciola gigantica -Krai Meemon, Prasert Sobhon, Rudi Grams, Annemarie Hofmann, and Günter Korge [2] Lotfy WM and Hillyer GV (2003) Fasciola species in Egypt. Exp Pathol Parasitol 6(11) 9-22 [3] Maurice J (1994) Is something lurking in your liver? New Scientist 1917 26-31. [4] WHO (1995) Control of foodborne trematode infections. Technical sheet no. 849, Geneva. [5] Malek EA (1980) Snail-transmitted parasitic disease. Library of Congress cataloging in publication data, USA II, 131-170. [6] Souslby EJ (1982) Helminths, arthropods and domesticated animals. 7th Ed., Bailliere, Tindall and Cassell, London. pp. 40-52. [7] Multiple Lineages of the Mitochondrial Gene NADH Dehydrogenase Subunit 1 (ND1) in Parasitic Helminths: Implications for Molecular Evolutionary Studies of Facultatively Anaerobic Eukaryotes Lynne van Herwerden, David Blair, Takeshi Agatsuma 1 [8] Hulo N,Bairoch A,Builiard V,Cerutti L,Cuche BA,de Castro E,Lachaize C,Langendijk-Genevaux PS,Sigrist CJ.” The 20 years of PROSITE “ Nucleic Acids Res.2008;36 (Database issue):D245-9.URL: [9] ExPasy tools: [10] Protein structure prediction on the web: a case study using the Phyre server. Kelley LA & Sternberg MJE Nature Protocols. 4, 363 - 371 (2009)