Intramolecular inhibition of human defensin HNP-1 by its propiece

J Clin Invest. 1996 Apr 1;97(7):1624-9. doi: 10.1172/JCI118588.


We examined mechanisms that protect host defense cells from their cytotoxic effector molecules. Human neutrophil peptides (HNP) 1-3 are microbicidal and cytotoxic defensins, initially synthesized as 94-amino acid preproHNP(1-94), cotranslationally proteolyzed to proHNP(20-94), then converted by removal of the anionic propiece to mature HNP(65-94)(HNP-1 and -3) and HNP(66-94) (HNP-2). We hypothesized that during synthesis and subcellular sorting the anionic propiece inhibits the cytotoxicity of the cationic defensin. We expressed preproHNP-1 cDNA in recombinant baculovirus-infected insect cells that secreted the normally transient proHNP-1(20-94) into the medium. Cyanogen bromide cleaved proHNP-1(20-94) at the fortuitously located Met64 to yield mature recombinant HNP-1(65-94) and unlinked propiece. Recombinant and native HNP-1 purified from PMN were identical as judged by mass spectrometry, retention time in reverse-phase high performance liquid chromatography, migration on acid-urea polyacrylamide gels, and reaction with a conformation-specific antibody. Recombinant and native HNP-1 had comparable microbicidal activity towards Listeria monocytogenes and were similarly potent in permeabilizing K562 leukemia cells, but proHNP-1(20-94) was virtually inactive in both assays. Addition of unlinked propiece (proHNP-1(20-64) with Met64-->homoserine) inhibited the bactericidal and cell-permeabilizing activity of mature HNP-1 in a dose-dependent manner. Linked, and to a lesser extent unlinked, propiece interfered with the binding of HNP-1 to target cells. The propiece thus acts as an efficient intramolecular inhibitor of defensin HNP-1 cytotoxicity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blood Bactericidal Activity
  • Blood Proteins / antagonists & inhibitors*
  • Blood Proteins / genetics
  • Blood Proteins / metabolism
  • Cell Line
  • Cytotoxicity, Immunologic
  • DNA, Complementary / genetics
  • Defensins
  • Humans
  • Listeria monocytogenes / drug effects
  • Molecular Sequence Data
  • Neutrophils / immunology
  • Neutrophils / metabolism
  • Nucleopolyhedroviruses / genetics
  • Protein Precursors / genetics
  • Protein Precursors / metabolism
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spodoptera
  • Tumor Cells, Cultured
  • alpha-Defensins*


  • Blood Proteins
  • DNA, Complementary
  • Defensins
  • Protein Precursors
  • Recombinant Proteins
  • alpha-Defensins
  • human neutrophil peptide 1
  • human neutrophil peptide 2