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. 1988 Dec 1;168(6):2295–2308. doi: 10.1084/jem.168.6.2295

Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein

PMCID: PMC2189145  PMID: 3199069

Abstract

A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2- terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2- terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic.

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Selected References

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  1. Ackerman S. J., Kephart G. M., Habermann T. M., Greipp P. R., Gleich G. J. Localization of eosinophil granule major basic protein in human basophils. J Exp Med. 1983 Sep 1;158(3):946–961. doi: 10.1084/jem.158.3.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ackerman S. J., Loegering D. A., Venge P., Olsson I., Harley J. B., Fauci A. S., Gleich G. J. Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin. J Immunol. 1983 Dec;131(6):2977–2982. [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barker R. L., Gleich G. J., Pease L. R. Acidic precursor revealed in human eosinophil granule major basic protein cDNA. J Exp Med. 1988 Oct 1;168(4):1493–1498. doi: 10.1084/jem.168.4.1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berger S. L., Birkenmeier C. S. Inhibition of intractable nucleases with ribonucleoside--vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry. 1979 Nov 13;18(23):5143–5149. doi: 10.1021/bi00590a018. [DOI] [PubMed] [Google Scholar]
  6. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  7. Butterworth A. E., Wassom D. L., Gleich G. J., Loegering D. A., David J. R. Damage to schistosomula of Schistosoma mansoni induced directly by eosinophil major basic protein. J Immunol. 1979 Jan;122(1):221–229. [PubMed] [Google Scholar]
  8. Filley W. V., Ackerman S. J., Gleich G. J. An immunofluorescent method for specific staining of eosinophil granule major basic protein. J Immunol Methods. 1981;47(2):227–238. doi: 10.1016/0022-1759(81)90123-x. [DOI] [PubMed] [Google Scholar]
  9. Frigas E., Gleich G. J. The eosinophil and the pathophysiology of asthma. J Allergy Clin Immunol. 1986 Apr;77(4):527–537. doi: 10.1016/0091-6749(86)90341-6. [DOI] [PubMed] [Google Scholar]
  10. Frigas E., Loegering D. A., Solley G. O., Farrow G. M., Gleich G. J. Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Mayo Clin Proc. 1981 Jun;56(6):345–353. [PubMed] [Google Scholar]
  11. Gabay J. E., Heiple J. M., Cohn Z. A., Nathan C. F. Subcellular location and properties of bactericidal factors from human neutrophils. J Exp Med. 1986 Nov 1;164(5):1407–1421. doi: 10.1084/jem.164.5.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ganz T., Selsted M. E., Szklarek D., Harwig S. S., Daher K., Bainton D. F., Lehrer R. I. Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest. 1985 Oct;76(4):1427–1435. doi: 10.1172/JCI112120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gleich G. J., Adolphson C. R. The eosinophilic leukocyte: structure and function. Adv Immunol. 1986;39:177–253. doi: 10.1016/s0065-2776(08)60351-x. [DOI] [PubMed] [Google Scholar]
  14. Gleich G. J., Frigas E., Loegering D. A., Wassom D. L., Steinmuller D. Cytotoxic properties of the eosinophil major basic protein. J Immunol. 1979 Dec;123(6):2925–2927. [PubMed] [Google Scholar]
  15. Gleich G. J., Loegering D. A., Bell M. P., Checkel J. L., Ackerman S. J., McKean D. J. Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease. Proc Natl Acad Sci U S A. 1986 May;83(10):3146–3150. doi: 10.1073/pnas.83.10.3146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gleich G. J., Loegering D. A., Kueppers F., Bajaj S. P., Mann K. G. Physiochemical and biological properties of the major basic protein from guinea pig eosinophil granules. J Exp Med. 1974 Aug 1;140(2):313–332. doi: 10.1084/jem.140.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gleich G. J., Loegering D. A., Mann K. G., Maldonado J. E. Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils. J Clin Invest. 1976 Mar;57(3):633–640. doi: 10.1172/JCI108319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  20. Kierszenbaum F., Ackerman S. J., Gleich G. J. Destruction of bloodstream forms of Trypanosoma cruzi by eosinophil granule major basic protein. Am J Trop Med Hyg. 1981 Jul;30(4):775–779. doi: 10.4269/ajtmh.1981.30.775. [DOI] [PubMed] [Google Scholar]
  21. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  22. Kozak M. Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 1981 Oct 24;9(20):5233–5252. doi: 10.1093/nar/9.20.5233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  24. Lehrer R. I., Ganz T., Selsted M. E. Oxygen-independent bactericidal systems. Mechanisms and disorders. Hematol Oncol Clin North Am. 1988 Mar;2(1):159–169. [PubMed] [Google Scholar]
  25. Maddox D. E., Kephart G. M., Coulam C. B., Butterfield J. H., Benirschke K., Gleich G. J. Localization of a molecule immunochemically similar to eosinophil major basic protein in human placenta. J Exp Med. 1984 Jul 1;160(1):29–41. doi: 10.1084/jem.160.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  27. Nauseef W. M. Posttranslational processing of a human myeloid lysosomal protein, myeloperoxidase. Blood. 1987 Oct;70(4):1143–1150. [PubMed] [Google Scholar]
  28. Nishio C., Komura S., Kurahashi K. Peptide antibiotic subtilin is synthesized via precursor proteins. Biochem Biophys Res Commun. 1983 Oct 31;116(2):751–758. doi: 10.1016/0006-291x(83)90588-0. [DOI] [PubMed] [Google Scholar]
  29. Olsson I., Persson A. M., Winqvist I. Biochemical properties of the eosinophil cationic protein and demonstration of its biosynthesis in vitro in marrow cells from patients with an eosinophilia. Blood. 1986 Feb;67(2):498–503. [PubMed] [Google Scholar]
  30. Olsson I., Venge P. Cationic proteins of human granulocytes. II. Separation of the cationic proteins of the granules of leukemic myeloid cells. Blood. 1974 Aug;44(2):235–246. [PubMed] [Google Scholar]
  31. Ooi C. E., Weiss J., Elsbach P., Frangione B., Mannion B. A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein. J Biol Chem. 1987 Nov 5;262(31):14891–14894. [PubMed] [Google Scholar]
  32. Peters M. S., Rodriguez M., Gleich G. J. Localization of human eosinophil granule major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin by immunoelectron microscopy. Lab Invest. 1986 Jun;54(6):656–662. [PubMed] [Google Scholar]
  33. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Rosenberg S. A., Grimm E. A., McGrogan M., Doyle M., Kawasaki E., Koths K., Mark D. F. Biological activity of recombinant human interleukin-2 produced in Escherichia coli. Science. 1984 Mar 30;223(4643):1412–1414. doi: 10.1126/science.6367046. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schnell N., Entian K. D., Schneider U., Götz F., Zähner H., Kellner R., Jung G. Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature. 1988 May 19;333(6170):276–278. doi: 10.1038/333276a0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wasmoen T. L., Bell M. P., Loegering D. A., Gleich G. J., Prendergast F. G., McKean D. J. Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein. J Biol Chem. 1988 Sep 5;263(25):12559–12563. [PubMed] [Google Scholar]
  41. Weiss J., Elsbach P., Olsson I., Odeberg H. Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J Biol Chem. 1978 Apr 25;253(8):2664–2672. [PubMed] [Google Scholar]
  42. Weller P. F., Ackerman S. J., Smith J. A. Eosinophil granule cationic proteins: major basic protein is distinct from the smaller subunit of eosinophil peroxidase. J Leukoc Biol. 1988 Jan;43(1):1–4. doi: 10.1002/jlb.43.1.1. [DOI] [PubMed] [Google Scholar]
  43. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5449–5453. doi: 10.1073/pnas.84.15.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. van Hofsten P., Faye I., Kockum K., Lee J. Y., Xanthopoulos K. G., Boman I. A., Boman H. G., Engström A., Andreu D., Merrifield R. B. Molecular cloning, cDNA sequencing, and chemical synthesis of cecropin B from Hyalophora cecropia. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2240–2243. doi: 10.1073/pnas.82.8.2240. [DOI] [PMC free article] [PubMed] [Google Scholar]

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