SPAG11/isoform HE2C, an atypical anionic beta-defensin-like peptide.
A human caput epididymidal cDNA, HE2C, was cloned based on its homology to the known chimpanzee counterpart, suggesting that the encoded beta-defensin-like peptide represented a conserved component of the innate epididymidal epithelial defense system in primates. An approximately 6kDa HE2- related peptide was co-purified together with other HE2 isoforms from human seminal plasma by affinity chromatography. By its antibody reactivity as shown by Western blot analysis, this peptide was distinct from the more abundant HE2 isoforms and was concluded to correspond to HE2C. Similar to other HE2-encoded isoforms, the endogenous HE2C was proteolytically processed from a larger precursor by a furin-like prohormone convertase. This was confirmed by N-terminal sequencing. In order to study the structural and functional properties of HE2C it was recombinantly expressed in insect cells. Post-translational processing also occurred within these cells, yielding the mature processed HE2C peptide. Correct disulfide bonding of the recHE2C peptide was shown by p-aminophenylarsineoxide(PAPAO)-agarose binding assay. Purified recHE2C strongly bound to Escherichia coli DH5alpha and Bacillus subtilis; however, it did not exhibit microbicidal activity when tested in a radial diffusion assay against these bacteria. Different from the previously described beta-defensins, the mature HE2C peptide has an anionic pI and an algebraic net charge of -1. Also, it lacks the amphipathic transitions, which, according to the Shai-Matzusaki-Huang model, are prerequisite for the membranolytic activity of antimicrobial peptides.
von Horsten
Hans Henning
HH
IHF Institute for Hormone and Fertility Research at the University of Hamburg, Hamburg, Germany. hans.vonhorsten@ttuhsc.edu
Schäfer
Bettina
B
Kirchhoff
Christiane
C
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D023083:beta-Defensins / Q000737:chemistry* / Q000378:metabolism
D000954:Antigens, Surface; D018076:DNA, Complementary; D006020:Glycopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C087044:SPAG11A protein, human; D023083:beta-Defensins; D012313:RNA