Structure-activity determinants in paneth cell alpha-defensins: loss-of-function in mouse cryptdin-4 by charge-reversal at arginine residue positions.
Paneth cells secrete microbicidal enteric alpha-defensins into the small intestinal lumen, and cryptdin-4 (Crp4) is the most bactericidal of the mouse alpha-defensin peptides in vitro. Here, site-directed Arg to Asp mutations in Crp4 have been shown to attenuate or eliminate microbicidal activity against all of the bacterial species tested regardless of the Arg residue position. R31D/R32D charge-reversal mutagenesis at the C terminus and mutations at R16D/R18D, R16D/R24D, and R18D/R24D in the Crp4 polypeptide chain eliminated in vitro bactericidal activity, blocked peptide-membrane interactions, as well as Crp4-mediated membrane vesicle disruption. Lys for Arg charge-neutral substitutions in (R16K/R18K)-Crp4 did not alter the bactericidal activity relative to Crp4, showing that bactericidal activity appears not to require the guanidinium side chain of Arg at those two positions. Partial restoration of (R31D/R32D)-Crp4 bactericidal activity occurred when an electropositive Arg for Gly substitution was introduced at the peptide N terminus and the (G1R/R31D/R32D)-Crp4 peptide exhibited intermediate membrane binding capability. Also, the loss of peptide bactericidal activity in (G1D/R31D/R32D)-Crp4 and (R16D/R24D)-Crp4 mutants corresponded with diminished phospholipid vesicle disruptive activity. Fluorophore leakage from anionic phospholipid vesicles induced by the charge-reversal variants was negligible relative to Crp4 and lower than that induced by pro-Crp4, the inactive Crp4 precursor. Thus, Arg residues function as determinants of Crp4 bactericidal activity by facilitating or enabling target cell membrane disruption. The role of the Arg residues, however, was surprisingly independent of their position in the polypeptide chain.
Tanabe
Hiroki
H
Departments of Pathology, College of Medicine, University of California, Irvine, California 92697-4800, USA.
Qu
Xiaoqing
X
Weeks
Colby S
CS
Cummings
Jason E
JE
Kolusheva
Sofiya
S
Walsh
Kevin B
KB
Jelinek
Raz
R
Vanderlick
T Kyle
TK
Selsted
Michael E
ME
Ouellette
Andre J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers; D051379:Mice; D008969:Molecular Sequence Data; D019879:Paneth Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D023084:alpha-Defensins / Q000737:chemistry / Q000502:physiology*
D017931:DNA Primers; D011994:Recombinant Proteins; D023084:alpha-Defensins; C401847:cryptdin 4, mouse; D001120:Arginine