Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1993 May;61(5):1900–1908. doi: 10.1128/iai.61.5.1900-1908.1993

Synthetic peptides of human lysosomal cathepsin G with potent antipseudomonal activity.

W M Shafer 1, M E Shepherd 1, B Boltin 1, L Wells 1, J Pohl 1
PMCID: PMC280782  PMID: 8478079

Abstract

Enzymatically active and inactive (diisopropylfluorophosphate-treated) cathepsin G exerted antibacterial action in vitro against Staphylococcus aureus, whereas only enzymatically active cathepsin G displayed bactericidal action against Pseudomonas aeruginosa. In order to further test the requirement for protease activity for the antipseudomonal action of cathepsin G, synthetic peptides spanning the full-length mature protein were prepared and examined for antibacterial action. Surprisingly, three structurally distinct peptides that correspond to residues 61 to 80, 117 to 136, and 198 to 223 within the full-length protein were found to exert potent antipseudomonal action (> 4.5 logs of killing at 500 micrograms/ml) against P. aeruginosa ATCC 27853 and four mucoid clinical isolates. Only the peptide (CG117-136) corresponding to residues 117 to 136 (117-RPGTLCTVAGWGRVSMRRGT-136) within cathepsin G exerted antibacterial action against the gram-positive pathogen S. aureus. The antipseudomonal action of CG117-136 was rapid and could be inhibited either by increasing concentrations of NaCl or by 0.5 mM MgCl2 plus 0.5 mM CaCl2, and these conditions appeared to reduce binding of the peptide to whole bacteria. Variants of peptide CG117-136 lacking either a hydrophobic N-terminal domain or a positively charged C-terminal domain were found to have significantly less antipseudomonal action than CG117-136. The antibacterial capacity of the all-D-enantiomeric form of peptide CG117-136 was found to be identical to that of the all-L-peptide, suggesting that the mechanism of killing does not require the recognition of a target site possessing a chiral center.

Full text

PDF
1900

Images in this article

1906Image
on p.1906

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alford C. E., Amaral E., Campbell P. A. Listericidal activity of human neutrophil cathepsin G. J Gen Microbiol. 1990 Jun;136(6):997–100. doi: 10.1099/00221287-136-6-997. [DOI] [PubMed] [Google Scholar]
  2. Bangalore N., Travis J., Onunka V. C., Pohl J., Shafer W. M. Identification of the primary antimicrobial domains in human neutrophil cathepsin G. J Biol Chem. 1990 Aug 15;265(23):13584–13588. [PubMed] [Google Scholar]
  3. Bessalle R., Kapitkovsky A., Gorea A., Shalit I., Fridkin M. All-D-magainin: chirality, antimicrobial activity and proteolytic resistance. FEBS Lett. 1990 Nov 12;274(1-2):151–155. doi: 10.1016/0014-5793(90)81351-n. [DOI] [PubMed] [Google Scholar]
  4. Boman H. G. Antibacterial peptides: key components needed in immunity. Cell. 1991 Apr 19;65(2):205–207. doi: 10.1016/0092-8674(91)90154-q. [DOI] [PubMed] [Google Scholar]
  5. Boomer S., Coulter W. A., Guthrie D. J. Effects on oral bacteria of a peptide derived from human cathepsin G. Biochem Soc Trans. 1992 Feb;20(1):61S–61S. doi: 10.1042/bst020061s. [DOI] [PubMed] [Google Scholar]
  6. Campanelli D., Melchior M., Fu Y., Nakata M., Shuman H., Nathan C., Gabay J. E. Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils. J Exp Med. 1990 Dec 1;172(6):1709–1715. doi: 10.1084/jem.172.6.1709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duvick J. P., Rood T., Rao A. G., Marshak D. R. Purification and characterization of a novel antimicrobial peptide from maize (Zea mays L.) kernels. J Biol Chem. 1992 Sep 15;267(26):18814–18820. [PubMed] [Google Scholar]
  8. Farley M. M., Shafer W. M., Spitznagel J. K. Lipopolysaccharide structure determines ionic and hydrophobic binding of a cationic antimicrobial neutrophil granule protein. Infect Immun. 1988 Jun;56(6):1589–1592. doi: 10.1128/iai.56.6.1589-1592.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frank R. W., Gennaro R., Schneider K., Przybylski M., Romeo D. Amino acid sequences of two proline-rich bactenecins. Antimicrobial peptides of bovine neutrophils. J Biol Chem. 1990 Nov 5;265(31):18871–18874. [PubMed] [Google Scholar]
  10. Gabay J. E., Scott R. W., Campanelli D., Griffith J., Wilde C., Marra M. N., Seeger M., Nathan C. F. Antibiotic proteins of human polymorphonuclear leukocytes. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5610–5614. doi: 10.1073/pnas.86.14.5610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gray P. W., Flaggs G., Leong S. R., Gumina R. J., Weiss J., Ooi C. E., Elsbach P. Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations. J Biol Chem. 1989 Jun 5;264(16):9505–9509. [PubMed] [Google Scholar]
  12. Gudmundsson G. H., Lidholm D. A., Asling B., Gan R., Boman H. G. The cecropin locus. Cloning and expression of a gene cluster encoding three antibacterial peptides in Hyalophora cecropia. J Biol Chem. 1991 Jun 25;266(18):11510–11517. [PubMed] [Google Scholar]
  13. Jenne D., Rey C., Haefliger J. A., Qiao B. Y., Groscurth P., Tschopp J. Identification and sequencing of cDNA clones encoding the granule-associated serine proteases granzymes D, E, and F of cytolytic T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4814–4818. doi: 10.1073/pnas.85.13.4814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lee J. Y., Boman A., Sun C. X., Andersson M., Jörnvall H., Mutt V., Boman H. G. Antibacterial peptides from pig intestine: isolation of a mammalian cecropin. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9159–9162. doi: 10.1073/pnas.86.23.9159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lehrer R. I., Barton A., Daher K. A., Harwig S. S., Ganz T., Selsted M. E. Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity. J Clin Invest. 1989 Aug;84(2):553–561. doi: 10.1172/JCI114198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miyasaki K. T., Bodeau A. L. In vitro killing of oral Capnocytophaga by granule fractions of human neutrophils is associated with cathepsin G activity. J Clin Invest. 1991 May;87(5):1585–1593. doi: 10.1172/JCI115172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Odeberg H., Olsson I. Antibacterial activity of cationic proteins from human granulocytes. J Clin Invest. 1975 Nov;56(5):1118–1124. doi: 10.1172/JCI108186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Odeberg H., Olsson I. Mechanisms for the microbicidal activity of cationic proteins of human granulocytes. Infect Immun. 1976 Dec;14(6):1269–1275. doi: 10.1128/iai.14.6.1269-1275.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pohl J., Pereira H. A., Martin N. M., Spitznagel J. K. Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase. FEBS Lett. 1990 Oct 15;272(1-2):200–204. doi: 10.1016/0014-5793(90)80484-z. [DOI] [PubMed] [Google Scholar]
  22. Romeo D., Skerlavaj B., Bolognesi M., Gennaro R. Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils. J Biol Chem. 1988 Jul 15;263(20):9573–9575. [PubMed] [Google Scholar]
  23. Salvesen G., Farley D., Shuman J., Przybyla A., Reilly C., Travis J. Molecular cloning of human cathepsin G: structural similarity to mast cell and cytotoxic T lymphocyte proteinases. Biochemistry. 1987 Apr 21;26(8):2289–2293. doi: 10.1021/bi00382a032. [DOI] [PubMed] [Google Scholar]
  24. Segal A. W., Geisow M., Garcia R., Harper A., Miller R. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH. Nature. 1981 Apr 2;290(5805):406–409. doi: 10.1038/290406a0. [DOI] [PubMed] [Google Scholar]
  25. Selsted M. E., Harwig S. S., Ganz T., Schilling J. W., Lehrer R. I. Primary structures of three human neutrophil defensins. J Clin Invest. 1985 Oct;76(4):1436–1439. doi: 10.1172/JCI112121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shafer W. M., Martin L. E., Spitznagel J. K. Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate. Infect Immun. 1984 Jul;45(1):29–35. doi: 10.1128/iai.45.1.29-35.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shafer W. M., Onunka V. C., Martin L. E. Antigonococcal activity of human neutrophil cathepsin G. Infect Immun. 1986 Oct;54(1):184–188. doi: 10.1128/iai.54.1.184-188.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shafer W. M., Onunka V. C. Mechanism of staphylococcal resistance to non-oxidative antimicrobial action of neutrophils: importance of pH and ionic strength in determining the bactericidal action of cathepsin G. J Gen Microbiol. 1989 Apr;135(4):825–830. doi: 10.1099/00221287-135-4-825. [DOI] [PubMed] [Google Scholar]
  29. Shafer W. M., Pohl J., Onunka V. C., Bangalore N., Travis J. Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide. J Biol Chem. 1991 Jan 5;266(1):112–116. [PubMed] [Google Scholar]
  30. Skerlavaj B., Romeo D., Gennaro R. Rapid membrane permeabilization and inhibition of vital functions of gram-negative bacteria by bactenecins. Infect Immun. 1990 Nov;58(11):3724–3730. doi: 10.1128/iai.58.11.3724-3730.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Spitznagel J. K. Antibiotic proteins of human neutrophils. J Clin Invest. 1990 Nov;86(5):1381–1386. doi: 10.1172/JCI114851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wasiluk K. R., Skubitz K. M., Gray B. H. Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa. Infect Immun. 1991 Nov;59(11):4193–4200. doi: 10.1128/iai.59.11.4193-4200.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES