Homology search of Defensins among different

species

HT05 Fall Research Training Project of MBB Program

XIA Han

The Purpose of My Project

Database of Defensins 10~40 amino acids length OS: Plant, Invertebrate and Vertebrate Literatures related to this sequences

Search for conversed sequence patterns Connect sequence’s features with structur

e

What is DEFENSIN?

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

Structural Features

4~8 cysteine (mostly) 2~4 disulfide bridges Lineal or cyclic molecule Cationic Amphipathic

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

Construction of Database

Stage 1: Search for sequences

Protocol for search

Literature Search

Database Search

Data Refine and Validation

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

Data Refinement and Validation

Refinement Strategy 10~40 amino acids 2~4 disulfide bridges

Validation Strategy Consistent with corresponding entry in NCBI

Construction of Database

Stage 2: Accession of Data

Classification : Based on Taxonomy

Format : FASTA

Additional information : References of Sequences

Summary of Database

Plant MusselArachni

-daInsect

Vertebr

-ateTotal

AMSDb

The Number

of Sequences

9 3 2 8 23 44

APD 5 1 0 2 23 31

BLAST 235

Final Data Set

11 3 4 22 77 117

Sequence Alignment

ClustalX Multiple alignment && Profile alignment Alignment based on evolutionary tree More interactive More Protein Weight Matrices

BLOSUM (Henikoff) PAM (Dayhoff) GONNET

Test Runs

Parameters Default settings GONNET series as Protein Weight Matrices

Pairwise alignment: GONNET 250 Multiple Alignment: GONNET series

However……

Test Runs Results

Test Runs Results

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

Profile Alignment

Profile Alignment in a designed order : 1. Profile alignment multiple-aligned insect sequences with multi

ple-aligned Arachnida sequences, get P1.aln (26 sequences) ; 2. P1.aln with multiple-aligned Mussel sequences, get P2.aln (29

sequences) 3. P2.aln with multiple-aligned vertebrate sequences, get P3.aln

(106 sequences)

4. P2.aln with multiple-aligned plant sequences, get P4.aln (40 s

equences)

Profile Alignment Result: P2.aln

Structural Alignment

Secondary Structural Prediction JPRED(http://www.compbio.dundee.ac.uk/~www-jpred/

)

Sequence-based Structural Alignment BLAST MSDlite(http://www.ebi.ac.uk/msd-srv/msdlite

/index.html)

Profile Alignment Result: P2.aln

Superposition : 1L4V and 1ICA

Acknowledgements:

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

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).

Tack sä mycket!

God Jul och

Gott Nytt Är!