Homology search of Defensins among different species

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Homology search of Defensins among different
species

• HT05 Fall Research Training Project of MBB
  Program
• XIA Han

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The Purpose of My Project

• Database of Defensins
• 1040 amino acids length
• OS Plant, Invertebrate and Vertebrate
• Literatures related to this sequences
• Search for conversed sequence patterns
• Connect sequences features with structure

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What is DEFENSIN?

• Antimicrobial Peptide (AMP)
• Germline-encoded
• Widespread
• Cysteine-rich
• Wide antimicrobial spectrum

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Structural Features

• 48 cysteine (mostly)
• 24 disulfide bridges
• Lineal or cyclic molecule
• Cationic
• Amphipathic

Tang, Y. Q. et al. Science 286, 498-502 (1999)
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Construction of Database

• Stage 1 Search for sequences
• Protocol for search
• Literature Search
• Database Search
• Data Refine and Validation

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Database Search

• AMSDb
• Antimicrobial Sequences Database
• http//www.bbcm.units.it/tossi/amsdb.html
• APD
• Antimicrobial Peptide Database
• http//aps.unmc.edu/AP/main.php
• NCBI

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Data Refinement and Validation

• Refinement Strategy
• 1040 amino acids
• 24 disulfide bridges
• Validation Strategy
• Consistent with corresponding entry in NCBI

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Construction of Database

• Stage 2 Accession of Data
• Classification Based on Taxonomy
• Format FASTA
• Additional information References of Sequences

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Summary of Database
Plant Plant Mussel Mussel Arachni-da Arachni-da Insect Vertebr -ate Vertebr -ate Total
AMSDb The Number of Sequences 9 9 3 3 2 2 8 23 23 44
APD The Number of Sequences 5 5 1 1 0 0 2 23 23 31
BLAST The Number of Sequences 235 235 235 235 235 235 235 235 235 235
Final Data Set The Number of Sequences 11 3 3 4 4 22 22 77 117 117
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Sequence Alignment

• ClustalX
• Multiple alignment Profile alignment
• Alignment based on evolutionary tree
• More interactive
• More Protein Weight Matrices
• BLOSUM (Henikoff)
• PAM (Dayhoff)
• GONNET

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Test Runs

• Parameters
• Default settings
• GONNET series as Protein Weight Matrices
• Pairwise alignment GONNET 250
• Multiple Alignment GONNET series
• However

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Test Runs Results
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Test Runs Results
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Profile Alignment

• Important Parameters
• End Gap Separation On
• Residue Specific Penalties and Hydrophilic Gap
  Penalties On
• New Gaps before Alignment and Reset All Gaps
  before Alignment Off
• BLOSUM series as Protein Weight Matrices
• Pairwise alignment BLOSUM 30
• Multiple Alignment BLOSUM series

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Profile Alignment

• Profile Alignment in a designed order
• Profile alignment multiple-aligned insect
  sequences with multiple-aligned Arachnida
  sequences, get P1.aln (26 sequences)
• P1.aln with multiple-aligned Mussel sequences,
  get P2.aln (29 sequences)
• P2.aln with multiple-aligned vertebrate
  sequences, get P3.aln (106 sequences)
• P2.aln with multiple-aligned plant sequences, get
  P4.aln (40 sequences)

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Profile Alignment Result P2.aln
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Structural Alignment

• Secondary Structural Prediction
• JPRED(http//www.compbio.dundee.ac.uk/www-jp
  red/ )
• Sequence-based Structural Alignment
• BLAST
• MSDlite(http//www.ebi.ac.uk/msd-srv/msdlite/index
  .html)

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Profile Alignment Result P2.aln
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Superposition 1L4V and 1ICA
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Acknowledgements

• My Supervisor Anders Backlund
• Everyone in Department of Pharmacognosy
• Margareta Krabble
• Mikael Thollesson
• My Supporting, lovely Classmates!

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References

1. Bulet, P., Stocklin, R. Menin, L.
   Anti-microbial peptides from invertebrates to
   vertebrates. Immunol Rev 198, 169-84 (2004).
2. Tang, Y. Q. et al. A cyclic antimicrobial peptide
   produced in primate leukocytes by the ligation of
   two truncated alpha-defensins. Science 286,
   498-502 (1999).
3. Gueguen, Y. et al. Characterization of a defensin
   from the oyster crassostrea gigas Recombinant
   production, folding, solution structure,
   antimicrobial activities and gene expression. J
   Biol Chem (2005).
4. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A.
   Ezekowitz, R. A. Phylogenetic perspectives in
   innate immunity. Science 284, 1313-8 (1999).
5. Mygind, P. H. et al. Plectasin is a peptide
   antibiotic with therapeutic potential from a
   saprophytic fungus. Nature 437, 975-80 (2005).

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Tack sä mycket!God Jul och Gott Nytt Är!