Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase

Nature Genetics volume 35, pages 90–96 (2003)Cite this article

Abstract

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase1, found in lysosome-like granules, cause cyclic hematopoiesis2 and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity4. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis5, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) β-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mutation of AP3B1 causes canine cyclic hematopoiesis.
Figure 2: Interaction of AP3 and neutrophil elastase.
Figure 3: Mislocalization of neutrophil elastase in mutants lacking the μ3a sorting signal and C-terminal extension (Y199X) or just the C-terminal signal (205ΔC) relative to wild-type (WT).
Figure 4: Mutations of neutrophil elastase that cause cyclic hematopoiesis generally accentuate granule accumulation, whereas those that cause SCN generally accentuate membrane accumulation.
Figure 5: Transmembrane properties of neutrophil elastase identified by protease protection assays in selectively permeabilized cells.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Bieth, J.G. Leukocyte elastase. in Handbook of Proteolytic Enzymes (ed. Woessner, J.F.) 54–60 (Academic, San Diego, 1998).

    Google Scholar 

  2. Horwitz, M., Benson, K.F., Person, R.E., Aprikyan, A.G. & Dale, D.C. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat. Genet. 23, 433–436 (1999).

    Article  CAS  Google Scholar 

  3. Dale, D.C. et al. Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Blood 96, 2317–2322 (2000).

    CAS  PubMed  Google Scholar 

  4. Li, F.Q. & Horwitz, M. Characterization of mutant neutrophil elastase in severe congenital neutropenia. J. Biol. Chem. 276, 14230–14241 (2001).

    Article  CAS  Google Scholar 

  5. Lothrop, C.D. Jr. et al. Cyclic hormonogenesis in gray collie dogs: interactions of hematopoietic and endocrine systems. Endocrinology 120, 1027–1032 (1987).

    Article  CAS  Google Scholar 

  6. Dell'Angelica, E.C., Shotelersuk, V., Aguilar, R.C., Gahl, W.A. & Bonifacino, J.S. Altered trafficking of lysosomal proteins in Hermansky–Pudlak syndrome due to mutations in the β3A subunit of the AP-3 adaptor. Mol. Cell 3, 11–21 (1999).

    Article  CAS  Google Scholar 

  7. Huizing, M. et al. Nonsense mutations in ADTB3A cause complete deficiency of the β3A subunit of adaptor complex-3 and severe Hermansky–Pudlak syndrome type 2. Pediatr. Res. 51, 150–158 (2002).

    Article  CAS  Google Scholar 

  8. Boehm, M. & Bonifacino, J.S. Genetic analyses of adaptin function from yeast to mammals. Gene 286, 175–186 (2002).

    Article  CAS  Google Scholar 

  9. Simpson, F., Peden, A.A., Christopoulou, L. & Robinson, M.S. Characterization of the adaptor-related protein complex, AP-3. J. Cell Biol. 137, 835–845 (1997).

    Article  CAS  Google Scholar 

  10. Bonifacino, J.S. & Dell'Angelica, E.C. Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell Biol. 145, 923–926 (1999).

    Article  CAS  Google Scholar 

  11. Rapoport, I., Chen, Y.C., Cupers, P., Shoelson, S.E. & Kirchhausen, T. Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site. EMBO J. 17, 2148–2155 (1998).

    Article  CAS  Google Scholar 

  12. Feng, L. et al. The β3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky–Pudlak syndrome and night blindness. Hum. Mol. Genet. 8, 323–330 (1999).

    Article  CAS  Google Scholar 

  13. Aguilar, R.C. et al. Signal-binding specificity of the μ4 subunit of the adaptor protein complex AP-4. J. Biol. Chem. 276, 13145–13152 (2001).

    Article  CAS  Google Scholar 

  14. Gullberg, U. et al. Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell line RBL. J. Biol. Chem. 270, 12912–12918 (1995).

    Article  CAS  Google Scholar 

  15. Bainton, D.F. Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy. J. Immunol. Methods 232, 153–168 (1999).

    Article  CAS  Google Scholar 

  16. Horwitz, M. et al. Role of neutrophil elastase in bone marrow failure syndromes: molecular genetic revival of the chalone hypothesis. Curr. Opin. Hematol. 10, 49–54 (2003).

    Article  CAS  Google Scholar 

  17. Yang, W., Li, C., Ward, D.M., Kaplan, J. & Mansour, S.L. Defective organellar membrane protein trafficking in Ap3b1-deficient cells. J. Cell Sci. 113, 4077–4086 (2000).

    CAS  PubMed  Google Scholar 

  18. Moller, S., Croning, M.D. & Apweiler, R. Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17, 646–653 (2001).

    Article  CAS  Google Scholar 

  19. Owen, C.A., Campbell, M.A., Sannes, P.L., Boukedes, S.S. & Campbell, E.J. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J. Cell Biol. 131, 775–789 (1995).

    Article  CAS  Google Scholar 

  20. Fuks, A., Zucker-Franklin, D. & Franklin, E.C. Identification of elastases associated with purified plasma membranes isolated from human monocytes and lymphocytes. Biochim. Biophys. Acta 755, 195–203 (1983).

    Article  CAS  Google Scholar 

  21. Allen, D.H. & Tracy, P.B. Human coagulation factor V is activated to the functional cofactor by elastase and cathepsin G expressed at the monocyte surface. J. Biol. Chem. 270, 1408–1415 (1995).

    Article  CAS  Google Scholar 

  22. Kolkenbrock, H., Zimmermann, J., Burmester, G.R. & Ulbrich, N. Activation of progelatinase B by membranes of human polymorphonuclear granulocytes. Biol. Chem. 381, 49–55 (2000).

    Article  CAS  Google Scholar 

  23. Kaup, M. et al. Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G. Biol. Chem. 383, 1011–1020 (2002).

    Article  CAS  Google Scholar 

  24. Clark, J.M., Vaughan, D.W., Aiken, B.M. & Kagan, H.M. Elastase-like enzymes in human neutrophils localized by ultrastructural cytochemistry. J. Cell Biol. 84, 102–119 (1980).

    Article  CAS  Google Scholar 

  25. Mardones, G. & Gonzalez, A. Selective plasma membrane permeabilization by freeze-thawing and immunofluorescence epitope access to determine the topology of intracellular membrane proteins. J. Immunol. Methods 275, 169–177 (2003).

    Article  CAS  Google Scholar 

  26. Vita, F., Soranzo, M.R., Borelli, V., Bertoncin, P. & Zabucchi, G. Subcellular localization of the small GTPase Rab5a in resting and stimulated human neutrophils. Exp. Cell Res. 227, 367–373 (1996).

    Article  CAS  Google Scholar 

  27. Kjeldsen, L., Bainton, D.F., Sengelov, H. & Borregaard, N. Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identification of a distinct gelatinase-containing granule subset by combined immunocytochemistry and subcellular fractionation. Blood 82, 3183–3191 (1993).

    CAS  PubMed  Google Scholar 

  28. Danielson, P.B. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr. Drug Metab. 3, 561–597 (2002).

    Article  CAS  Google Scholar 

  29. Paigen, K. Mammalian β-glucuronidase: genetics, molecular biology, and cell biology. Prog. Nucleic Acid Res. Mol. Biol. 37, 155–205 (1989).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Miller for help with microscopy, E. Ostrander for sample conveyance, G. Cuneo for collie photo, X. Zhu and J. Bonifacino for reagents and advice and M. Gelb, E. Davie and the late H. Neurath for critical discussion. This work was supported by grants from the US National Institutes of Health, Doris Duke Foundation (in vitro studies only) and Burroughs-Wellcome (to M.H.).

Author information

Author notes

  1. Dalila Albani

    Present address: Cabinet Beau de Loménie, 59800, Lille, France

  2. Jeremy Wechsler

    Present address: Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana, 59840, USA

  3. Kimberly Meade-White

    Present address: University of Miami School of Medicine, Miami, Florida, 33101, USA

Authors and Affiliations

  1. Division of Medical Genetics / Department of Medicine, University of Washington School of Medicine, Box 357720, 1705 NE Pacific Street, HSB-K236B, Seattle, 98195, Washington, USA

    Kathleen F Benson, Feng-Qian Li, Dalila Albani, Zhijun Duan, Jeremy Wechsler, Kimberly Meade-White, Kayleen Williams & Marshall Horwitz

  2. Department of Pathology, University of Washington School of Medicine, Box 357720, 1705 NE Pacific Street, HSB-K236B, Seattle, 98195, Washington, USA

    Richard E Person

  3. College of Veterinary Medicine, Cornell University, Ithaca, 14853, New York, USA

    Gregory M Acland

  4. College of Veterinary Medicine, Auburn University, Auburn, 36849, Alabama, USA

    Glenn Niemeyer & Clinton D Lothrop

Authors
  1. Kathleen F Benson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng-Qian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Richard E Person
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dalila Albani
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhijun Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeremy Wechsler
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kimberly Meade-White
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kayleen Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gregory M Acland
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Glenn Niemeyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Clinton D Lothrop
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Marshall Horwitz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marshall Horwitz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Benson, K., Li, FQ., Person, R. et al. Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase. Nat Genet 35, 90–96 (2003). https://doi.org/10.1038/ng1224

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng1224

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing