Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes

Abstract

Mutations in the ELA2 gene encoding neutrophil elastase (NE) are present in most patients with severe congenital neutropenia (SCN). However, the mechanisms by which these mutations cause neutropenia remain unknown. To investigate the effects of mutant NE expression on granulopoiesis, we used the HL-60 promyelocytic cell line retrovirally transduced with the G185R NE mutant that is associated with a severe SCN phenotype. We show that the mutant enzyme accelerates apoptosis of differentiating but not of proliferating cells. Using metabolic labeling, confocal immunofluorescence microscopy, and immunoblot analysis of subcellular fractions, we also demonstrate that the G185R mutant is abnormally processed and localizes predominantly to the nuclear and plasma membranes rather than to the cytoplasmic compartment observed with the wildtype (WT) enzyme. Expression of the G185R mutant appeared to alter the subcellular distribution and expression of adaptor protein 3 (AP3), which traffics proteins from the trans-Golgi apparatus to the endosome. These observations provide further insight into potential mechanisms by which NE mutations cause neutropenia and suggest that abnormal protein trafficking and accelerated apoptosis of differentiating myeloid cells contribute to the severe SCN phenotype resulting from the G185R mutation. (Blood. 2005;105:3397-3404)

Digenic mutations in severe congenital neutropenia

“…Effects on proteolytic activity, on cellular trafficking and on unfolded protein response (UPR) have been demonstrated only for subgroups of ELANE mutations, respectively. [11][12][13][14] Furthermore, other ELANE mutations described as causative for congenital neutropenia, e.g. p.Val72Met 1 were also categorized as benign by the prediction algorithms.…”

Neutrophil elastase depends on serglycin proteoglycan for localization in granules

“…[54][55][56] There have been several contradicting reports on disturbances in the subcellular localization of neutrophil elastase caused by mutations associated with CN or SCN. 10,57 It has particularly been suggested that mutations in neutrophil elastase affect the sorting to granules via disorganization of putative transmembrane and cytosolic domains. 58 Our finding that lack of serglycin completely prevents granule localization of elastase argues against a significant role of putative transmembrane and cytosolic domains of neutrophil elastase in granule targeting.…”

Pathogenic mechanisms and clinical implications of congenital neutropenia syndromes

“…This might be due to inappropriate ANC monitoring, G-CSF treatment related cycle modification, incomplete mutation spectrum data, phenotype diversification by modifier genes and additional somatic mutations, especially in the colony stimulating factor 3 receptor gene (CSF3R) [11][12][13][14]15 16,17]. The monomeric serine chymotryptic-type protease neutrophil elastase is a major component of neutrophil granulocyte azurophilic granules and is named after its first known substrate elastin [129].…”