Intramolecular inhibition of human defensin HNP-1 by its propiece. E V Valore, … , S S Harwig, T Ganz J Clin Invest. 1996;97(7):1624-1629. https://doi.org/10.1172/JCI118588. Research Article 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. Find the latest version: https://jci.me/118588/pdf Intramolecular Inhibition of Human Defensin HNP-1 by Its Propiece Erika V. Valore, Edith Martin, Sylvia S.L. Harwig, and Tomas Ganz Will Rogers Institute Pulmonary Research Laboratory, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90095-1736 Abstract of antimicrobial activity against gram-positive and gram-nega- tive bacteria, fungi, enveloped viruses, and parasites (1, 5, 6), We examined mechanisms that protect host defense cells but are also cytotoxic to various types of mammalian cells (7, from their cytotoxic effector molecules. Human neutrophil 8). Defensins kill bacterial and mammalian cells by permeabi- peptides (HNP) 1–3 are microbicidal and cytotoxic defensins, lization of the cell membrane, followed by additional events initially synthesized as 94–amino acid preproHNP1–94, that result in irreversible progression to cell death. During the cotranslationally proteolyzed to proHNP20–94, then con- initial phase of cytotoxicity, cells can be rescued from death by verted by removal of the anionic propiece to mature HNP65–94 preventing the binding of defensins to cell surface or reversing (HNP-1 and -3) and HNP66–94 (HNP-2). We hypothesized the defensin-mediated permeabilization of the cell membrane that during synthesis and subcellular sorting the anionic (9, 10). propiece inhibits the cytotoxicity of the cationic defensin. Mammalian defensins are abundant in the cytoplasmic We expressed preproHNP-1 cDNA in recombinant baculo- granules of neutrophils, Paneth cells of the small intestine, and virus-infected insect cells that secreted the normally tran- some macrophages. The defensins human neutrophil peptides sient proHNP-120–94 into the medium. Cyanogen bromide (HNP)1 1–3 together with the less abundant HNP-4 constitute cleaved proHNP-120–94 at the fortuitously located Met64 to 30% of the protein of the microbicidal azurophil granules of yield mature recombinant HNP-165–94 and unlinked pro- neutrophils, and two other human defensins, HD-5 and HD-6, piece. Recombinant and native HNP-1 purified from PMN have been localized recently by in situ hybridization to the in- were identical as judged by mass spectrometry, retention testinal Paneth cells. time in reverse-phase high performance liquid chromatog- During the synthesis of defensins HNP-1–3, the initial raphy, migration on acid-urea polyacrylamide gels, and re- translation product is a 94–amino acid (aa) precursor that con- action with a conformation-specific antibody. Recombinant tains a 19-aa endoplasmic reticulum signal sequence, a 45-aa and native HNP-1 had comparable microbicidal activity to- anionic propiece, and a 29–30-aa cationic mature peptide (11). wards Listeria monocytogenes and were similarly potent in The signal sequence is cotranslationally removed and over sev- permeabilizing K562 leukemia cells, but proHNP-120–94 was eral hours the proprotein is sequentially cleaved from its virtually inactive in both assays. Addition of unlinked pro- amino terminus to generate the mature defensin peptide (Fig. → piece (proHNP-120–64 with Met64 homoserine) inhibited the 1). Since the metabolic cost of defensin biosynthesis is approx- bactericidal and cell-permeabilizing activity of mature imately doubled by the relatively large anionic propiece, we HNP-1 in a dose-dependent manner. Linked, and to a lesser hypothesized that the propiece has an important functional extent unlinked, propiece interfered with the binding of role. In previous work we established a model system of mu- HNP-1 to target cells. The propiece thus acts as an efficient rine myeloid 32D cl3 cells transduced with HNP-1 cDNA in a intramolecular inhibitor of defensin HNP-1 cytotoxicity. (J. retroviral vector (12). Deletion of preproHNP-1 residues Clin. Invest. 1996. 97:1624–1629.) Key words: neutrophil • 21–39, at the amino-terminal end of the propiece, had only mi- antimicrobial peptide • posttranslational processing • cyto- nor effects on defensin synthesis, processing, and subcellular toxicity • inflammation sorting, but all of these processes were dramatically impaired by deletions in the carboxy-terminal segment of the propiece Introduction (aa 40–64). In addition to the essential role of the propiece in defensin biosynthesis, we earlier proposed that the anionic Members of the defensin family are small, variably cationic, propiece may neutralize the cytotoxicity of the cationic mature cysteine- and arginine-rich mammalian peptides, defined by peptide during its biosynthesis, posttranslational processing, their conserved disulfide pattern (1). Peptides of similar size, and transport through subcellular compartments (13). How- composition, and function have also been detected in insects ever, due to the transient nature of prodefensin in myeloid and even plants (2–4). In vitro, defensins exhibit a wide range cells, we were unable to isolate sufficient amounts from natu- ral sources to test this conjecture. In this study, we biosynthe- sized preproHNP-11–94 using a baculovirusրinsect cell system wherein the insect cells cleaved the signal (pre) sequence and Address correspondence to Tomas Ganz, Ph.D., M.D., Department released the 75-aa proHNP-1 into the culture medium. of Medicine, CHS 37-055, UCLA School of Medicine, Los Angeles, 20–94 CA 90095-1736. Phone: 310-825-6112; FAX: 310-206-8766; E-mail: Comparison of the biological activities of proHNP-1 and [email protected] HNP-1 and the analysis of the effect of the propiece on the ac- Received for publication 6 July 1995 and accepted in revised form tivity of mature HNP-1 were facilitated by the serendipitous 11 January 1996. location of the lone methionine (Met64) residue at the carboxy- J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: aa, amino acid: CNBr, cyanogen 0021-9738/96/04/1624/06 $2.00 bromide; HNP, human neutrophil peptide; RP-HPLC, reverse-phase Volume 97, Number 7, April 1996, 1624–1629 HPLC; TSB, trypticase soy broth. 1624 Valore et al. terminal end of the proprotein. ProHNP-1 molecule could be 491 Prep Cell; Bio-Rad Laboratories, Richmond, CA) in a 15.8% cleaved at this residue with cyanogen bromide (indicated by acid-urea polyacrylamide gel (16). Fractions containing proHNP-1 the large arrow in Fig. 1) to yield the carboxy-terminally modi- were assayed by Coomassie-stained acid-urea or SDS-tricine (17) fied (methionine to homoserineրhomoserine lactone) anionic PAGE and further purified by reverse-phase (RP)-HPLC on a 4.6 ϫ propiece and the mature defensin HNP-1. 250 mm Vydac C18 column (Separations Group, Hesperia, CA) using a 1% acetonitrile increment per minute in 0.1% trifluoroacetic acid. Amino acid sequencing. Purified proHNP-1 was analyzed by Methods SDS-Tricine PAGE and then electroblotted to polyvinylidenedifluo- ride membranes (PVDF; Bio-Rad Laboratories). Electrophoretic Construction of HNP-1-baculovirus. The baculovirus expression sys- transfer (Transblot; Bio-Rad Laboratories) was performed in 0.05 M tem was used to overexpress preprodefensin in insect Spodoptera fur- sodium borate, pH 9, 20% methanol, 0.05% SDS at 20 V (0.18 A) for giperda (Sf21) cells as described by the manufacturer (CLONTECH, at least 2 h. The PVDF membrane was stained with 0.4% napthol Palo Alto, CA). Briefly, HNP-1 cDNA (12) fragment containing the blue black dye, 25% isopropanol, 10% methanol and destained in wa- ribosomal binding site, the translation start codon ATG, the entire ter. Amino acid sequence of the predominant protein band (8 kD) coding region of preproHNP-1, a stop codon, and BamHI restriction was determined by the UCLA Peptide Sequencing Facility by Edman