Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1989 Mar 1;169(3):1071–1086. doi: 10.1084/jem.169.3.1071

Elastase inhibitor. Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils

PMCID: PMC2189272  PMID: 2926322

Abstract

A fast-acting inhibitor of serine elastase has been detected at high levels in human neutrophils, fresh monocytes, matured monocytes, and macrophages. The elastase inhibitor was isolated from large scale cultures of the monocyte-like cell line U937 by DNase chromatography, disulfide exchange, Phenyl-Sepharose, Red A-agarose, and DEAE HPLC chromatography with an average yield of 480 micrograms from 1.8 x 10(10) cells. The isolated polypeptide was verified as elastase inhibitor by its ability to (a) form a covalent complex with elastase; and (b) inhibit the elastinolytic activity of elastase. The purified elastase inhibitor molecule is unique, i.e., physiochemical and/or functional properties distinguish it from all other serine proteinase inhibitors. Treatment with iodoacetamide abrogates the ability of the molecule to form a complex with elastase, thereby providing evidence for the presence of an essential cysteine residue. Based on functional criteria, this elastase inhibitor has been grouped with the proteinase inhibitors of the serpin superfamily. The purified elastase inhibitor is a single polypeptide of Mr approximately 42,000. The NH2 terminus appears to be blocked. Compositional analyses indicates five cysteine residues per molecule of approximately 360 amino acid residues. Negligible levels of carbohydrate were detected on gas-liquid chromatography. This finding and the insensitivity of the molecule to peptide N-glycosidase F treatment strongly indicate that the elastase inhibitor is a nonglycosylated protein.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Astedt B., Lecander I., Brodin T., Lundblad A., Löw K. Purification of a specific placental plasminogen activator inhibitor by monoclonal antibody and its complex formation with plasminogen activator. Thromb Haemost. 1985 Feb 18;53(1):122–125. [PubMed] [Google Scholar]
  2. Babior B. M. The respiratory burst of phagocytes. J Clin Invest. 1984 Mar;73(3):599–601. doi: 10.1172/JCI111249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blondin J., Rosenberg R., Janoff A. An inhibitor in human lung macrophages active against human neutrophil elastase. Am Rev Respir Dis. 1972 Sep;106(3):477–479. doi: 10.1164/arrd.1972.106.3.477. [DOI] [PubMed] [Google Scholar]
  4. Campbell E. J., Senior R. M., McDonald J. A., Cox D. L. Proteolysis by neutrophils. Relative importance of cell-substrate contact and oxidative inactivation of proteinase inhibitors in vitro. J Clin Invest. 1982 Oct;70(4):845–852. doi: 10.1172/JCI110681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carrell R. W., Jeppsson J. O., Laurell C. B., Brennan S. O., Owen M. C., Vaughan L., Boswell D. R. Structure and variation of human alpha 1-antitrypsin. Nature. 1982 Jul 22;298(5872):329–334. doi: 10.1038/298329a0. [DOI] [PubMed] [Google Scholar]
  6. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  7. Davis G. S., Giancola M. S., Costanza M. C., Low R. B. Analyses of sequential bronchoalveolar lavage samples from healthy human volunteers. Am Rev Respir Dis. 1982 Oct;126(4):611–616. doi: 10.1164/arrd.1982.126.4.611. [DOI] [PubMed] [Google Scholar]
  8. Dewald B., Rindler-Ludwig R., Bretz U., Baggiolini M. Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes. J Exp Med. 1975 Apr 1;141(4):709–723. doi: 10.1084/jem.141.4.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doolittle R. F. Angiotensinogen is related to the antitrypsin-antithrombin-ovalbumin family. Science. 1983 Oct 28;222(4622):417–419. doi: 10.1126/science.6604942. [DOI] [PubMed] [Google Scholar]
  10. Dubin A. A polyvalent proteinase inhibitor from horse-blood-leucocyte cytosol. Isolation, purification and some molecular parameters. Eur J Biochem. 1977 Mar 1;73(2):429–435. doi: 10.1111/j.1432-1033.1977.tb11334.x. [DOI] [PubMed] [Google Scholar]
  11. FOLEY G. E., LAZARUS H., FARBER S., UZMAN B. G., BOONE B. A., MCCARTHY R. E. CONTINUOUS CULTURE OF HUMAN LYMPHOBLASTS FROM PERIPHERAL BLOOD OF A CHILD WITH ACUTE LEUKEMIA. Cancer. 1965 Apr;18:522–529. doi: 10.1002/1097-0142(196504)18:4<522::aid-cncr2820180418>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  12. Gadek J. E., Fells G. A., Wright D. G., Crystal R. G. Human neutrophil elastase functions as a type III collagen "collagenase". Biochem Biophys Res Commun. 1980 Aug 29;95(4):1815–1822. doi: 10.1016/s0006-291x(80)80110-0. [DOI] [PubMed] [Google Scholar]
  13. Garver R. I., Jr, Mornex J. F., Nukiwa T., Brantly M., Courtney M., LeCocq J. P., Crystal R. G. Alpha 1-antitrypsin deficiency and emphysema caused by homozygous inheritance of non-expressing alpha 1-antitrypsin genes. N Engl J Med. 1986 Mar 20;314(12):762–766. doi: 10.1056/NEJM198603203141207. [DOI] [PubMed] [Google Scholar]
  14. Ginsburg D., Zeheb R., Yang A. Y., Rafferty U. M., Andreasen P. A., Nielsen L., Dano K., Lebo R. V., Gelehrter T. D. cDNA cloning of human plasminogen activator-inhibitor from endothelial cells. J Clin Invest. 1986 Dec;78(6):1673–1680. doi: 10.1172/JCI112761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunt L. T., Dayhoff M. O. A surprising new protein superfamily containing ovalbumin, antithrombin-III, and alpha 1-proteinase inhibitor. Biochem Biophys Res Commun. 1980 Jul 31;95(2):864–871. doi: 10.1016/0006-291x(80)90867-0. [DOI] [PubMed] [Google Scholar]
  16. Janoff A., Blondin J. Inhibition of the elastase-like esterase in human leukocyte granules by human leukocyte cell sap. Proc Soc Exp Biol Med. 1971 Apr;136(4):1050–1053. doi: 10.3181/00379727-136-35425. [DOI] [PubMed] [Google Scholar]
  17. Janoff A., Feinstein G., Malemud C. J., Elias J. M. Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. I. Penetration of enzyme into rabbit articular cartilage and release of 35SO4-labeled material from the tissue. J Clin Invest. 1976 Mar;57(3):615–624. doi: 10.1172/JCI108317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Janoff A., Pryor W. A., Bengali Z. H. NHLBI workshop summary. Effects of tobacco smoke components on cellular and biochemical processes in the lung. Am Rev Respir Dis. 1987 Oct;136(4):1058–1064. doi: 10.1164/ajrccm/136.4.1058. [DOI] [PubMed] [Google Scholar]
  19. Kim K. C., Wasano K., Niles R. M., Schuster J. E., Stone P. J., Brody J. S. Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9304–9308. doi: 10.1073/pnas.84.24.9304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Lazarides E., Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. doi: 10.1073/pnas.71.12.4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mainardi C. L., Dixit S. N., Kang A. H. Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules. J Biol Chem. 1980 Jun 10;255(11):5435–5441. [PubMed] [Google Scholar]
  23. Martin J. P. Further examples confirming the existence of Pi null (Pi-). Pathol Biol (Paris) 1975 Sep;23(7):521–524. [PubMed] [Google Scholar]
  24. McDonald J. A., Kelley D. G. Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments. J Biol Chem. 1980 Sep 25;255(18):8848–8858. [PubMed] [Google Scholar]
  25. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  26. Ny T., Sawdey M., Lawrence D., Millan J. L., Loskutoff D. J. Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6776–6780. doi: 10.1073/pnas.83.18.6776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ohlsson K., Olsson I. The neutral proteases of human granulocytes. Isolation and partial characterization of granulocyte elastases. Eur J Biochem. 1974 Mar 1;42(2):519–527. doi: 10.1111/j.1432-1033.1974.tb03367.x. [DOI] [PubMed] [Google Scholar]
  28. Ossanna P. J., Test S. T., Matheson N. R., Regiani S., Weiss S. J. Oxidative regulation of neutrophil elastase-alpha-1-proteinase inhibitor interactions. J Clin Invest. 1986 Jun;77(6):1939–1951. doi: 10.1172/JCI112523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Plow E. F. Leukocyte elastase release during blood coagulation. A potential mechanism for activation of the alternative fibrinolytic pathway. J Clin Invest. 1982 Mar;69(3):564–572. doi: 10.1172/JCI110482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Potempa J., Dubin A., Watorek W., Travis J. An elastase inhibitor from equine leukocyte cytosol belongs to the serpin superfamily. Further characterization and amino acid sequence of the reactive center. J Biol Chem. 1988 May 25;263(15):7364–7369. [PubMed] [Google Scholar]
  31. Reeck G. R., Fisher L. A statistical analysis of the amino acid compositions of proteins. Int J Pept Protein Res. 1973;5(2):109–117. doi: 10.1111/j.1399-3011.1973.tb02325.x. [DOI] [PubMed] [Google Scholar]
  32. Remold-O'Donnell E. A fast-acting elastase inhibitor in human monocytes. J Exp Med. 1985 Dec 1;162(6):2142–2155. doi: 10.1084/jem.162.6.2142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Remold-O'Donnell E., Lewandrowski K. Two proteinase inhibitors associated with peritoneal macrophages. J Biol Chem. 1983 Mar 10;258(5):3251–3257. [PubMed] [Google Scholar]
  34. Sandhaus R. A., McCarthy K. M., Musson R. A., Henson P. M. Elastolytic proteinases of the human macrophage. Chest. 1983 May;83(5 Suppl):60S–62S. doi: 10.1378/chest.83.5_supplement.60s. [DOI] [PubMed] [Google Scholar]
  35. Schraufstätter I. U., Revak S. D., Cochrane C. G. Proteases and oxidants in experimental pulmonary inflammatory injury. J Clin Invest. 1984 Apr;73(4):1175–1184. doi: 10.1172/JCI111303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scott R. W., Bergman B. L., Bajpai A., Hersh R. T., Rodriguez H., Jones B. N., Barreda C., Watts S., Baker J. B. Protease nexin. Properties and a modified purification procedure. J Biol Chem. 1985 Jun 10;260(11):7029–7034. [PubMed] [Google Scholar]
  37. Senior R. M., Campbell E. J., Landis J. A., Cox F. R., Kuhn C., Koren H. S. Elastase of U-937 monocytelike cells. Comparisons with elastases derived from human monocytes and neutrophils and murine macrophagelike cells. J Clin Invest. 1982 Feb;69(2):384–393. doi: 10.1172/JCI110462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Senior R. M., Huang J. S., Griffin G. L., Deuel T. F. Dissociation of the chemotactic and mitogenic activities of platelet-derived growth factor by human neutrophil elastase. J Cell Biol. 1985 Feb;100(2):351–356. doi: 10.1083/jcb.100.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Senior R. M., Huebner P. F., Pierce J. A. Measurement of elastase activity by elastin agar and its use in the detection of antitrypsin deficiency. J Lab Clin Med. 1971 Mar;77(3):510–516. [PubMed] [Google Scholar]
  40. Sinha S., Watorek W., Karr S., Giles J., Bode W., Travis J. Primary structure of human neutrophil elastase. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2228–2232. doi: 10.1073/pnas.84.8.2228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sundström C., Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer. 1976 May 15;17(5):565–577. doi: 10.1002/ijc.2910170504. [DOI] [PubMed] [Google Scholar]
  42. Tarentino A. L., Gómez C. M., Plummer T. H., Jr Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry. 1985 Aug 13;24(17):4665–4671. doi: 10.1021/bi00338a028. [DOI] [PubMed] [Google Scholar]
  43. Thompson R. C., Ohlsson K. Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6692–6696. doi: 10.1073/pnas.83.18.6692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Travis J., Salvesen G. S. Human plasma proteinase inhibitors. Annu Rev Biochem. 1983;52:655–709. doi: 10.1146/annurev.bi.52.070183.003255. [DOI] [PubMed] [Google Scholar]
  45. Valentine R., Goettlich-Riemann W., Fisher G., Rucker R. B. An elastase inhibitor from isolated bovine pulmonary macrophages. Proc Soc Exp Biol Med. 1981 Nov;168(2):238–244. doi: 10.3181/00379727-168-41267. [DOI] [PubMed] [Google Scholar]
  46. Vassalli J. D., Dayer J. M., Wohlwend A., Belin D. Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages. J Exp Med. 1984 Jun 1;159(6):1653–1668. doi: 10.1084/jem.159.6.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Webb A. C., Collins K. L., Snyder S. E., Alexander S. J., Rosenwasser L. J., Eddy R. L., Shows T. B., Auron P. E. Human monocyte Arg-Serpin cDNA. Sequence, chromosomal assignment, and homology to plasminogen activator-inhibitor. J Exp Med. 1987 Jul 1;166(1):77–94. doi: 10.1084/jem.166.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  49. Weiss S. J., Regiani S. Neutrophils degrade subendothelial matrices in the presence of alpha-1-proteinase inhibitor. Cooperative use of lysosomal proteinases and oxygen metabolites. J Clin Invest. 1984 May;73(5):1297–1303. doi: 10.1172/JCI111332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wohlwend A., Belin D., Vassalli J. D. Plasminogen activator-specific inhibitors produced by human monocytes/macrophages. J Exp Med. 1987 Feb 1;165(2):320–339. doi: 10.1084/jem.165.2.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wun T. C., Reich E. An inhibitor of plasminogen activation from human placenta. Purification and characterization. J Biol Chem. 1987 Mar 15;262(8):3646–3653. [PubMed] [Google Scholar]
  52. Ye R. D., Wun T. C., Sadler J. E. cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta. J Biol Chem. 1987 Mar 15;262(8):3718–3725. [PubMed] [Google Scholar]
  53. van Mourik J. A., Lawrence D. A., Loskutoff D. J. Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J Biol Chem. 1984 Dec 10;259(23):14914–14921. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES