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Investigation of the interaction between human cathelicidin LL‐37 and CpG DNA (978.2)
Author(s) -
Radford Michelle,
Avelsgard Irmamarie,
Craig Maria
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.978.2
Subject(s) - cathelicidin , isothermal titration calorimetry , dna , electrophoretic mobility shift assay , microbiology and biotechnology , chemistry , blot , dna binding domain , biophysics , peptide , biochemistry , biology , gene , gene expression , antimicrobial peptides , transcription factor
The human cathelicidin LL‐37, an antimicrobial peptide of innate immunity, forms complexes with extracellular DNA and transports DNA across cell membranes. Complex formation between LL‐37 and DNA is the first in a series of events that upregulate an autoimmune response to self‐DNA. In order to characterize LL‐37/DNA binding, we have performed electrophoretic mobility shift assays (EMSA), western blotting, and isothermal titration calorimetry (ITC). By EMSA, we observe a shift in DNA mobility with increasing LL‐37 concentration over the range 5‐50 uM, and quantitative analysis of gel images was performed to derive binding curves for the association of LL‐37 with DNA. Western blots in this concentration range indicate that LL‐37 self‐associates in the absence of DNA, forming complexes that increase in size as LL‐37 concentration increases. These LL‐37 self‐complexes decrease in size in the presence of DNA. The ITC results support a two‐site model for LL‐37/DNA association with a 20:1 peptide: DNA ratio and association binding constants on the order of 10 8 to 10 9 M ‐1 . These data suggest that LL‐37 self‐associates at concentrations sufficient to bind DNA, and that DNA binding to LL‐37 displaces LL‐37 self ‐contacts. This mode of binding may allow efficient incorporation of DNA into complexes at LL‐37 concentrations above a specific threshold level. This work was supported by the Mary Baldwin College Summer Research Fellows Program.