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


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.

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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.

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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.).

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Correspondence to Marshall Horwitz.

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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).

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