Novel Isoforms of Neutrophil Elastase Produced by Neutropenia-Associated Mutations of the Initiation Codon and an Internal Ribosomal Entry Site (IRES) of ELANE

Abstract 9

Severe congenital neutropenia is most commonly caused by mutations in the gene, ELANE, encoding neutrophil elastase, through a mechanism that is not fully understood. Two prevalent hypotheses point to mislocalization of neutrophil elastase or, alternatively, endoplasmic reticulum (ER) stress resulting from protein misfolding. We have recently identified disease-causing mutations in the “Kozak” ribosome-binding sequence and the initiation codon that do not easily fit with either theory. In cell culture models of neutrophil elastase expression, we found that when the ribosome binding site of ELANE is mutated, at least four polypeptides are expressed, all shorter than the wild type. All of the isoforms are reactive to a carboxyl-terminal antibody, indicating that they are likely produced from alternate start sites or represent amino-terminal truncations. ELANE contains three downstream in-frame initiation codons (ATG) that potentially could be utilized as alternate translation start sites, and mutations in the vicinity of these sequences predictably correlate with production of each of the shorter isoforms. Furthermore, a protein-coding region of ELANE mRNA is complementary to the 18s ribosomal RNA subunit and demonstrates internal ribosome entry site (IRES) activity, when tested using a bicistronic luciferase assay. Neutropenia-associated mutations within the identified IRES also affect expression of the shorter isoforms of neutrophil elastase. These findings suggest that some ELANE mutations could cause neutropenia, not through disturbing the primary sequence of full-length neutrophil elastase protein, but instead by altering mRNA-ribosome interactions, thus generating shorter isoforms of normal sequence that are initiated from internal translational start sites. We have therefore biochemically characterized the shorter isoforms. We found that they do not demonstrate proteolytic activity. Because they lack signal sequences and cannot be trafficked through the ER, it is also not surprising that they fail to induce the ER stress response. They do not trigger apoptosis, as measured in several assays. Nevertheless, they do reduce myeloid cell viability in a colony forming test. We propose that these shortened isoforms contribute to the pathogenesis of ELANE-associated neutropenia by impairing myeloid cell development. Because a diversity of ELANE mutations from different patients seem to have identical effects, these shorter neutrophil elastase polypeptides, common to multiple patients regardless of mutation, may offer promise as targets for drug development.