A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated α-defensins

Science

Volumn 286, Issue 5439, 1999, Pages 498-502

  Tang, Yi Quan ^a   Yuan, Jun ^a   Ösapay, George ^a   Ösapay, Klara ^a   Tran, Dat ^a   Miller, Christopher J ^b   Ouellette, Andre J ^a   Selsted, Michael E ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIINFECTIVE AGENT; DEFENSIN; RHESUS THETA DEFENSIN 1; SODIUM CHLORIDE; UNCLASSIFIED DRUG;

ANIMAL CELL; ANTIBACTERIAL ACTIVITY; ARTICLE; LEUKOCYTE; NONHUMAN; NUCLEOTIDE SEQUENCE; PEPTIDE SYNTHESIS; PRIORITY JOURNAL; PROTEIN CONFORMATION; RHESUS MONKEY;

AMINO ACID SEQUENCE; ANIMALS; ANTI-BACTERIAL AGENTS; ANTI-INFECTIVE AGENTS; BACTERIA; CLONING, MOLECULAR; DEFENSINS; DISULFIDES; FUNGI; HUMANS; LEUKOPOIESIS; MACACA MULATTA; MOLECULAR SEQUENCE DATA; MONOCYTES; NEUTROPHILS; OLIGOPEPTIDES; OSMOLAR CONCENTRATION; PEPTIDES, CYCLIC; PROTEIN BIOSYNTHESIS; PROTEIN CONFORMATION; PROTEIN PRECURSORS; PROTEIN PROCESSING, POST-TRANSLATIONAL; PROTEINS;

ANIMALIA; FUNGI; MACACA MULATTA; PRIMATES; TRIXIS;

EID: 0033569410     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5439.498     Document Type: Article

References (49)

1. W. F. Broekaert et al., Crit. Rev. Plant Sci. 16, 297 (1997); H. Boman and W. F. Broekaert, Immunologist 6, 234 (1998); J. A. Hoffmann, F. C. Kafatos, C. A. Janeway Jr., R. A. B. Ezekowitz, Science 284, 1313 (1999).
2. W. F. Broekaert et al., Crit. Rev. Plant Sci. 16, 297 (1997); H. Boman and W. F. Broekaert, Immunologist 6, 234 (1998); J. A. Hoffmann, F. C. Kafatos, C. A. Janeway Jr., R. A. B. Ezekowitz, Science 284, 1313 (1999).
3. W. F. Broekaert et al., Crit. Rev. Plant Sci. 16, 297 (1997); H. Boman and W. F. Broekaert, Immunologist 6, 234 (1998); J. A. Hoffmann, F. C. Kafatos, C. A. Janeway Jr., R. A. B. Ezekowitz, Science 284, 1313 (1999).
4. A. J. Ouellette and M. E. Selsted, FASEB J. 10, 1280 (1996); T. Ganz and R. I. Lehrer, Curr. Opin. Hematol. 4, 53 (1997); G. Diamond and C. L. Bevins, Clin. Immunol. Immunopathol. 88, 221 (1998).
5. A. J. Ouellette and M. E. Selsted, FASEB J. 10, 1280 (1996); T. Ganz and R. I. Lehrer, Curr. Opin. Hematol. 4, 53 (1997); G. Diamond and C. L. Bevins, Clin. Immunol. Immunopathol. 88, 221 (1998).
6. A. J. Ouellette and M. E. Selsted, FASEB J. 10, 1280 (1996); T. Ganz and R. I. Lehrer, Curr. Opin. Hematol. 4, 53 (1997); G. Diamond and C. L. Bevins, Clin. Immunol. Immunopathol. 88, 221 (1998).
7. E. D. Stolzenberg, G. M. Anderson, M. R. Ackermann, R. H. Whitlock, M. E. Zasloff, Proc. Natl. Acad. Sci. U.S.A. 94, 8686 (1997); P. K. Singh et al., ibid. 95, 14961 (1998).
8. E. D. Stolzenberg, G. M. Anderson, M. R. Ackermann, R. H. Whitlock, M. E. Zasloff, Proc. Natl. Acad. Sci. U.S.A. 94, 8686 (1997); P. K. Singh et al., ibid. 95, 14961 (1998).
9. R. I. Lehrer et al., J. Clin. Invest. 84, 553 (1989).
10. 6 cells; 91% neutrophils, 5% mononuclear cells, 4% eosinophils) was snap frozen, suspended in 0.5 ml ice-cold 30% acetic acid, and stirred on melting ice for 18 hours. The suspension was clarified by centrifugation at 4°C, and the supernatant was lyophilized and then dissolved in 0.5 ml methanol: water (80:20). After 6 to 8 hours of stirring at 8°C, the sample was clarified by centrifugation, and the supernatant was lyophilized. The dry powder was dissolved in 0.5 ml 5% acetic acid before RP-HPLC.
11. Antibacterial activity of HPLC fractions, lyophilized and dissolved in 0.01% acetic acid, was determined using an agar diffusion assay [R. I. Lehrer, M. Rosenman, S. S. L. Harwig, R. Jackson, P. Eisenhauer, J. Immunol. Methods 137, 167 (1991)].
12. Sequence analysis was performed by automated Edman degradation with online phenylthiohydantoin amino acid analysis.
13. Y.-Q. Tang, J. Yuan, C. J. Miller, M. E. Selsted, Infect. Immun., in press.
14. Mass spectroscopy (MS) was performed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) on a PE Biosystems Voyager RP mass spectrometer in the linear mode. Samples (1 to 10 pmol) were dissolved in watenacetonitrile (1:1) containing 0.1% trifluoroacetic acid (TFA). All values are reported as the average mass of the nonprotonated species.
15. A. H. Henschen, in Advanced Methods in Protein Microsequence Analysis, B. Wittmann-Liebold, J. Salnikow, V. A. Erdman, Eds. (Springer-Verlag, Berlin, 1986), p. 244.
16. Peptide (1 nmol) was dissolved in 24 μl of 1 M methanolic HCI for 48 hours at room temperature. Edman sequencing of two separate reaction mixtures disclosed the sequence shown in Fig. 2A.
17. A 2-nmol sample of S-pyridylethylated peptide was digested at 37°C for 10 min with 0.4 μg TPCK trypsin or TLCK α-chymotrypsin in 50 μl of 1% ammonium bicarbonate, pH 8.0. Peptide fragments were purified by C-18 RP-HPLC and characterized by amino acid analysis, MALDI-TOF MS, and automated sequencing.
18. 2) for 2 hours at 37°C. The reaction was terminated by addition of 5 μl of 0.1% TFA/acetonitrile. One-microliter aliquots of the mixture were analyzed by MALDI-TOF MS as described above. In a separate experiment, ∼3 nmol of the 17-mer was digested with thermolysin under similar conditions, and the thermolytic fragments were isolated by HPLC. MALDI-TOF MS analysis of individual peaks confirmed the fragment pattern obtained by MS analysis of the unfractionated digestion mixture.
19. Y.-Q. Tang and M. E. Selsted, J. Biol. Chem. 268, 6649 (1993).
20. Supplemental material can be found at www. sciencemag.org/feature/data/1041865.shl
21. S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997).
22. V. N. Kokryakov et al., FEBS Lett. 327, 231 (1993); C. Q. Zhao, T. Ganz, R. I. Lehrer, FEBS Lett. 368, 197 (1995); R. L. Fahrner et al., Chem. Biol. 3, 543 (1996).
23. V. N. Kokryakov et al., FEBS Lett. 327, 231 (1993); C. Q. Zhao, T. Ganz, R. I. Lehrer, FEBS Lett. 368, 197 (1995); R. L. Fahrner et al., Chem. Biol. 3, 543 (1996).
24. V. N. Kokryakov et al., FEBS Lett. 327, 231 (1993); C. Q. Zhao, T. Ganz, R. I. Lehrer, FEBS Lett. 368, 197 (1995); R. L. Fahrner et al., Chem. Biol. 3, 543 (1996).
25. M. Zanetti, R. Gennaro, D. Romeo, FEBS Lett. 374, 1 (1995).
26. A three-dimensional model of RTD-1 was constructed using the Insight II program with the consistent valence force field based on the backbone conformation of residues 6 to 15 found in the solution structure of PG-1 (entry 1pg1 in the Brookhaven Protein Database). The molecule was placed into a 25.0 Å radius sphere of water. After energy minimization, a 250-ps molecular dynamics simulation was carried out at 300 K. The backbone atom root-mean-square fluctuation around the mean structure reached a steady state in the last 200 ps. Subsequent energy minimization of the average structure resulted in the structure shown in Fig. 2E.
27. R. Derua, K. R. Gustafson, L. K. Pannell, Biochem. Biophys. Res. Commun. 228, 632 (1996).
28. K. R. Gustafson et al., J. Am. Chem. Soc. 116, 9337 (1994).
29. J. P. Tam, Y. A. Lu, J. L. Yang, K. W. Chiu, Proc. Natl. Acad. Sci. U.S.A. 96, 8913 (1999).
30. A. Galvez, G. Gimenez-Gallego, M. Maqueda, E. Valdivia, Antimicrob. Agents Chemother. 33, 437 (1989); Martinez-Bueno et al., J. Bacteriol. 176, 6334 (1994).
31. A. Galvez, G. Gimenez-Gallego, M. Maqueda, E. Valdivia, Antimicrob. Agents Chemother. 33, 437 (1989); Martinez-Bueno et al., J. Bacteriol. 176, 6334 (1994).
32. A. Blond et al., Eur. J. Biochem. 259, 747 (1999).
33. 10VCRCIC of RTD-1 was one of several sense primers used for 3' RACE (35). Amplification products were subcloned and sequenced. Two of the amplified products contained sequences that encoded TRGFCRLL(stop) and RRGVCQLL(stop), corresponding to the COOH-termini of RTD1a and RTD1b, respectively. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M. Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Sen T, Thr; V, Val; W, Trp; and Y, Tyr.
34. 6 clones screened, 15 were purified and sequenced.
35. N. Y. Yount et al., J. Immunol. 155, 4476 (1995); T. Ganz and R. I. Lehrer, Curr. Opin. Hematol 4, 53 (1997).
36. N. Y. Yount et al., J. Immunol. 155, 4476 (1995); T. Ganz and R. I. Lehrer, Curr. Opin. Hematol 4, 53 (1997).
37. S. H. White, W. C. Wimley, M. E. Selsted, Curr. Opin. Struct. Biol. 5, 521 (1995).
38. 6 plaque-forming units were screened with a combined RTD1a and RTD1b cDNA probe by standard procedures. Of the >50 double-positive clones, 10 were selected at random for further purification and analysis. Genomic clones coding for RTD1a and RTD1b were distinguished by Southern (DNA) blot hybridization of polymerase chain reaction (PCR) products using sequence specific primers. Restriction analysis demonstrated that all 10 clones were unique. Appropriate PCR products were inserted in pCR 2.1 vector (Invitrogen) and sequenced.
39. M. E. Selsted and A. J. Ouellette, Trends Cell Biol. 5, 114 (1995).
40. RNA was extracted from snap-frozen tissues, and 40 μg of each sample was resolved on a 1.2% agarose-formaldehyde gel, and transferred to a membrane. The membrane was hybridized with probes specific for RTD1a (nucleotides 241-325) and RTD1b (nucleotides 236-320) generated by PCR. The specificity of the probes for RTD-1 was demonstrated by their ability to hybridize selectively RTD-1 sequences and their lack of hybridization to rhesus myeloid α-defensin cDNAs.
41. A 1,2-mg sample of acyclic RTD-1 was conjugated to 1.2 mg of ovalbumin in 2.4 ml of 0.1 M sodium phosphate, pH 7.4, containing 0.1% glutaraldehyde with stirring for 18 hours. The reaction was quenched by addition of 0.3 M glycine hydrochloride, dialyzed against water, and split for immunization of two New Zealand White rabbits. The antiserum from both rabbits was ∼1:1000, as determined by ELISA (enzyme-linked immunosorbent assay) using acyclic RTD-1 conjugated to goat γ-globulin as the target antigen. Cytospin preparations of peripheral blood leukocytes and bone marrow cells, fixed with 4% paraformaldehyde, were preincubated with avidin biotin, and Fc receptor blocker, then incubated with 1:100 rabbit anti-RTD-1 antiserum and developed with biotinylated goat anti-rabbit immunoglobulin-G, washed and incubated with avidin/biotin/glucose oxidase complex that was visualized with nitroblue tetrazolium. Negative control incubations were performed with anti-RTD-1 antiserum that was preabsorbed with 1 mg of synthetic acyclic RTD-1 per milliliter of antiserum.
42. M. E. Selsted, D. Szklarek, R. I. Lehrer, Infect. Immun. 45, 150 (1984); R. Bals, M. T. Goldman, J. M. Wilson, ibid. 66, 1225 (1998); E. V. Valore et al., J. Clin. Invest. 101, 1633 (1998).
43. M. E. Selsted, D. Szklarek, R. I. Lehrer, Infect. Immun. 45, 150 (1984); R. Bals, M. T. Goldman, J. M. Wilson, ibid. 66, 1225 (1998); E. V. Valore et al., J. Clin. Invest. 101, 1633 (1998).
44. M. E. Selsted, D. Szklarek, R. I. Lehrer, Infect. Immun. 45, 150 (1984); R. Bals, M. T. Goldman, J. M. Wilson, ibid. 66, 1225 (1998); E. V. Valore et al., J. Clin. Invest. 101, 1633 (1998).
45. J. J. Smith, S. M. Travis, E. P. Greenberg, M. J. Welsh, Cell 85, 229 (1996); M. J. Goldman et al., Cell 88, 553 (1997).
46. J. J. Smith, S. M. Travis, E. P. Greenberg, M. J. Welsh, Cell 85, 229 (1996); M. J. Goldman et al., Cell 88, 553 (1997).
47. M. A. Frohman, in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, San Diego, CA, 1990), p. 28.
48. M. E. Selsted, in Genetic Engineering: Principles and Methods, vol. 15, J. K. Setlow, Ed. (Plenum, New York, 1993), p. 131.
49. Supported in part by NIH grant AI22931 and Biosource Technologies, Inc (M.E.S.), NIH grants DK44632 and DK33506 (A.J.O.), and NIH grant RR00169 (C.J.M.). We thank H. Truong, B. Hoover, P. Tran, D. Lu, and Y. Wang for expert technical assistance, as well as A. Fowler at the University of California Los Angeles Microsequencing Core Facility and A. Henschen, Director of the University of California Irvine Microchemical Core Facility.